Candida Infection Biology – fungal armoury, battlefields ... - FINSysB

FINSysB Conference
Candida Infection
Biology –
fungal armoury,
battlefields
and host defences
10-14 October, 2011
Acquafredda di Maratea, ITALY
1
The FINSysB Marie Curie
Initial Training Network is
supported by the European
Commission.
(grant PITN-GA-2008-214004).

Index
Programme at a glance..................... Pages 4-5
Full Programme.................................
Speaker Abstracts
Pages 7-11
Tuesday, October 11......................... Pages 13-48
Wednesday, October 12.................... Pages 51-70
Thursday, October 13........................
Poster Abstracts
Pages 73-106
The Pathogenic Armoury................... Page 112
The Key Battlefields........................... Page 159
The Defensive Shields....................... Page 177
Defeating the Enemy......................... Page 191
List of Participants............................ Page 203
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MONDAY OCTOBER 10
FINSysB Programme at a glance
Arrivals
TUESDAY OCTOBER 11
08:30 Welcome - Al Brown
08:40 Session I: The Pathogenic Armoury [Fungal virulence factors]
Chair: Bernhard. Hube
Speakers: Malcolm Whiteway, Derek Sullivan, Pedro Miramón
10:15 Coffee and Posters
10:45 Session II: The Pathogenic Armoury [Fungal infection associated genes]
Chair: Joachim Ernst
Speakers: Scott Filler, Joachim Morschhäuser,
Francesco Citiulo, Malcolm McLean,
Ilse Jacobsen
12:30 Lunch and Posters
14:30 Session III: The Pathogenic Armoury [Fungal fitness attributes]
Chair: Mathias Lavie-Richard
Speakers: Janet Quinn, Mike Gustin, Christoph Schüller,
Michael Lorenz, Iryna Bohovych
16:15 Coffee and Posters
16:55 Session IV: The Pathogenic Armoury [Modelling fungal responses]
Chair: Edda Klipp
Speakers: Francesc Posas, Duccio Cavalieri, Katarzyna Tyc,
Iuliana Ene, Quentin Lagadec
18:40 Close
WEDNESDAY OCTOBER 12
08:30 Session V: The Key Battlefields [Candida biofilms]
Chair: Christophe d’Enfert
Speakers: Aaron Mitchell, Carol Kumamoto, Vitor Cabral,
Alice Sorgo
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10:20 Coffee and Posters
11:00 Session VI: The Key Battlefields [Candida cell wall]
Chair: Frans Klis
Speakers: Carol Munro, Peter Lipke, Iaroslava Kos,
Isabel Miranda, Jane Usher, Craig Murdoch
13:00 Lunch and Posters
14:30 Afternoon for networking and sunshine
THURSDAY OCTOBER 13
08:30 Session VII: The Defensive Shields [Host immune responses]
Chair: Mihai Netea
Speakers: Alberto Mantovani, Gordon Brown, Diana Rosentul
10:05 Coffee and Posters
10:45 Session VIII: The Defensive Shields [Host immune responses]
Chair: Anna Vecchiarelli
Speakers: Jürgen Ruland, Julian Naglik, Neelam Pandey,
Shih-Chin Cheng, Eliška Svobodová
12:30 Lunch and Posters
14:30 Session IX: Defeating The Enemy [Novel antifungal and diagnostic targets]
Chair: Andy Porter
Speakers: Antonio Cassone Deborah O’Neil,
Clemens Heilmann, Abhishek Saxena, Kate Dobb
16:15 Coffee and Posters
16:55 Session X: Defeating The Enemy [Novel antifungal and diagnostic targets]
Chair: Jason Oliver
Speakers: Haoping Liu, Inês Correia, Saif Hameed,
Antresh Kumar
18:10 Close
19:30 Conference Dinner
FRIDAY OCTOBER 14
Departures
5

Full Programme
Candida Infection Biology – fungal armoury,
battlefields and host defences
Acquafredda di Maratea, Italy
10-14 October 2011
MONDAY OCTOBER 10 Arrivals
TUESDAY OCTOBER 11
08:30 Welcome - Al Brown
08:40 Session I: The Pathogenic Armoury [Fungal virulence factors]
Chair – Bernhard Hube
8:40-9:20 Keynote Lecture
Malcolm Whiteway [Montreal, Canada]
Transcriptional rewiring of fungal ribosomal gene
9:30-9:55 Derek Sullivan [Dublin, Ireland]
What part of the Pathogenic Armoury is Candida dubliniensis
missing?
10:00-10:10 Pedro Miramón [Jena, Germany]
Dissecting the response of Candida albicans to the attack by
neutrophils
10:15 Coffee and Posters
10:45 Session II: The Pathogenic Armoury [Fungal infection associated
genes]
Chair – Joachim Ernst
10:45-11:10 Scott Filler [Los Angeles, USA]
EGFR and HER2 signalling mediate epithelial cell invasion by
Candida albicans during oropharyngeal infection
11:15-11:40 Joachim Morschhäuser [Würzburg, Germany]
Transcriptional control of white-opaque switching in Candida
albicans and its role in host-pathogen interactions
11:45-11:55 Francesco Citiulo [Jena, Germany]
Characterization of the molecular mechanism used by C. albicans to
acquire zinc from host cells.
12:00-12:10 Malcolm McLean [Revohot, Israel]
Strand asymmetry in Candida genes
7

12:15-12:25 Ilse Jacobsen [Jena, Germany]
Functional analysis of C. albicans infection-associated genes with
unknown function during interaction with host cells and in complex
infection models
12:30 Lunch and Posters
14:30 Session III: The Pathogenic Armoury [Fungal fitness attributes]
Chair - Mathias L. Richard
14:30-14:55 Janet Quinn [Newcastle, UK]
Survival in the host –mechanisms underlying responses to oxidative
stress in the human pathogen Candida albicans
15:00-15:25 Mike Gustin [Houston, USA]
Anticipation in the nitrosative stress response of Candida albicans
15:30-15.40 Christoph Schüller [Vienna, Austria]
Revisiting the Hog pathway of C. glabrata
15:45-15:55 Michael Lorenz [Houston, USA]
Candida albicans alters extracellular pH under host-relevant
conditions of carbon starvation
16:00-16:10 Iryna Bohovych [Aberdeen, UK]
Glucose promotes oxidative stress resistance in Candida albicans via
specific signalling pathways
16:15 Coffee and Posters
16:55 Session IV: The Pathogenic Armoury [Modelling fungal responses]
Chair – Edda Klipp
16:55-17:20 Francesc Posas [Barcelona, Spain]
Stress-responses controlled by the Hog1 stress-activated protein
kinase.
17:25-17:50 Duccio Cavalieri [Florence, Italy]
Friend or Foe: Systems biology approaches to elucidate the
interaction between fungi and their hosts
17:55-18:05 Katarzyna Tyc [Berlin, Germany]
Agent based modelling and host pathogen interactions
18:10-18:20 Iuliana Ene [Aberdeen, UK]
Impact of non-fermentative growth on the C. albicans cell wall and
dynamic responses to osmotic stress
18:25-18:35 Quentin Lagadec [Düsseldorf, Germany]
Target specificity of the Efg1 regulator in Candida albicans
18:40 Close
8

WEDNESDAY OCTOBER 12
08:30 Session V: The Key Battlefields [Candida biofilms]
Chair – Christophe d’Enfert
8:30-9:10 Keynote Lecture
Aaron Mitchell [Pittsburgh, USA]
Genetic control of Candida albicans biofilm formation
9:20-9:45 Carol Kumamoto [Boston, USA]
Candida albicans: sensing the host environment
9:50-10:00 Vitor Cabral [Paris, France]
Identification of Candida albicans genes involved in biofilm formation
by an over-expression approach
10:05-10:15 Alice Sorgo [Amsterdam, The Netherlands]
Cell wall stress elicits a conserved response in Candida albicans
10:20 Coffee and Posters
11:00 Session VI: The Key Battlefields [Candida cell wall]
Chair – Frans Klis
11:00-11:25 Carol Munro [Aberdeen, UK]
Candida albicans cell wall dynamics
11:30-11:55 Peter Lipke [New York, USA]
Force-dependent activation of functional amyloid domains increases
cell adhesion in C. albicans
12:00-12:10 Iaroslava Kos [Thiverval Grignon, France]
Iff2 is a cell surface protein involved in adhesion
12:15-12:25 Isabel Miranda [Porto, Portugal]
Deciphering the role of CUG mistranslation on Candida albicans
adhesion and cell surface variation
12:30-12:40 Jane Usher [Exeter, UK]
Interaction of a Candida glabrata transcription factor knock-out
library with the Drosophila melanogaster innate immune system
12:45-12:55 Craig Murdoch [Sheffield, UK]
Using zebrafish to study Candida albicans-mediated systemic
candidiasis in vivo
13:00 Lunch and Posters
14:30 Afternoon for networking and sunshine
9

THURSDAY OCTOBER 13
08:30 Session VII: The Defensive Shields [Host immune responses]
Chair – Mihai Netea
8:30-9:10 Keynote Lecture
Alberto Mantovani [Milan, Italy]
The long pentraxin PTX3 as a paradigm of humoral pattern
recognition molecules in antimicrobial resistance
9:20-9:45 Gordon Brown [Aberdeen, UK]
Pattern recognition and anti-fungal immunity: The role of C-type
lectins
9:50-10:00 Diana Rosentul [Nijmegen, The Netherlands]
The Tyr238X dectin-1 polymorphism is a predisposing factor for
mucosal Candida albicans infections but not candidemia
10:05 Coffee and Posters
10:45 Session VIII: The Defensive Shields [Host immune responses]
Chair – Anna Vecchiarelli
10:45-11:10 Jürgen Ruland [Munich, Germany]
C-type lectin receptor signaling in innate anti-fungal immunity
11:15-11:40 Julian Naglik [London, UK]
Candida - epithelial interactions and innate immunity
11:45-11:55 Neelam Pandey [Perugia, Italy]
Candida albicans secreted aspartic proteases cause inflammatory
response irrespective of their proteolytic activity
12:00-12:10 Shih-Chin Cheng [Nijmegen, The Netherlands]
A central role of complement for the Candida albicans-induced
cytokine production by human cells
12:15-12:25 Eliška Svobodová [Jena, Germany]
The role of pH-regulated antigen 1 of Candida albicans in the
interaction of the fungus with human neutrophils
12:30 Lunch and Posters
14:30 Session IX: Defeating The Enemy [Novel antifungal and diagnostic
targets]
Chair – Andy Porter
14:30-14:55 Antonio Cassone [Rome, Italy]
Vaccines against opportunistic fungal infections
10

15:00-15:25 Deborah O’Neil [Novabiotics, UK]
Tackling the Candida spp infection spectrum with first-in-class
antimicrobial peptide technology
15:30-15:40 Clemens Heilmann [Amsterdam, The Netherlands]
Wall proteins, absolute quantification and concatenated vaccines:
A new approach for Candida albicans therapy?
15:45-15:55 Abhishek Saxena [Aberdeen, UK]
scFv Phage Display Library against Complex Immunomodulatory
Glycans
16:00-16:10 Kate Dobb [Manchester, UK]
Essential Genes as Broad-Spectrum Antifungal Targets
16:15 Coffee and Posters
16:55 Session X: Defeating The Enemy [Novel antifungal and diagnostic
targets]
Chair – Jason Oliver
16:55-17:20 Haoping Liu [Irvine, USA]
Hyphal development in Candida albicans requires two temporally
linked changes in promoter chromatin for initiation and maintenance
17:25-17:35 Inês Correia [Madrid, Spain]
The transcription factor Sko1 represses the yeast-to-hypha transition
and mediates the oxidative stress response in Candida albicans.
17:40-17:50 Saif Hameed [New Delhi, India]
Calcineurin signaling and membrane lipid homeostasis regulates iron
mediated multidrug resistance mechanisms in Candida albicans
17:55-18:05 Antresh Kumar [New Delhi, India]
Functional analysis of H-loop residues of Cdr1p, an ABC transporter
of human fungal pathogen Candida albicans.
18:10 Close
19:30 Conference Dinner
FRIDAY OCTOBER 14 Departures
11

SPEAKER ABSTRACTS
(In order of presentation)
Tuesday, October 11
13

Transcriptional rewiring of fungal ribosomal gene
M. Whiteway, H. Lavoie, H. Hogues
NRC-BRI 6100 Royalmount Ave, Montreal, and McGill Biology, 1205 Penfield, Montreal
Examination of transcriptional regulatory circuits in fungi have uncovered many
examples of what is termed rewiring - transcription factors dedicated to controlling
a particular regulon in one species are found to control distinct regulons in other
species. A dramatic example of this phenomenon has been observed for the control
of the expression of the genes encoding the proteins that make up the ribosome.
The Tbf1 and Cbf1 proteins are key regulators of ribosomal gene expression in
C. albicans, but these proteins have been replaced in ribosomal control in the
S. cerevisiae lineage by Rap1p, and serve in yeast as telomere binding and
centromere binding proteins respectively. We can trace the evolutionary progression
of this process and speculate on mechanisms driving this exchange of controlling
elements.
14

What part of the Pathogenic Armoury is Candida
dubliniensis missing?
Derek Sullivan and Gary Moran
Division of Oral Biosciences, Dublin Dental University Hospital, Trinity College, Dublin 2,
Ireland.
The species most closely related to Candida albicans is Candida dubliniensis,
which, despite sharing many phenotypic attributes is a far less common cause of
human infection and is significantly less virulent in a wide range of infection models.
One of the most important virulence factors associated with C. albicans is the ability
to produce hyphae. Although both species have the capacity to produce filaments,
C. albicans is able to do so under a wider range of environmental conditions. We
have shown that the presence of nutrients specifically inhibits filamentation by C.
dubliniensis and that this can be alleviated by rapamycin, suggesting that
differences in the Tor1 signalling pathway may play a role in the different ability of
C. albicans and C. dubliniensis to produce hyphae.
Comparison of the genomes of C. albicans and C. dubliniensis has confirmed that
the two species are very closely related, with high levels of synteny and 96% of
genes exhibiting >80% identity. Interestingly, one of the most notable differences
between the two genomes is the absence of hypha-specific virulence genes such
as ALS3, SAP4,5 and HYR1. Comparative transcriptomic analysis has also
identified differences in gene expression in the two species that have led to the
identification of Sfl2, a putative heat shock transcription factor that is divergent in
the two species and which appears to play a role in the control of filamentation in
C. albicans.
These data suggest that C. dubliniensis may be losing the ability to produce hyphae,
and while this may reduce it’s capacity to cause invasive infection it may reflect
adaptation to commensal growth in a specific anatomic niche which has yet to be
identified.
16

Dissecting the response of Candida albicans to the attack
by neutrophils
Pedro Miramón 1 , Iryna Bohovych 4 , Alistair J. P. Brown 4 , Oliver Kurzai 2,3 ,
Bernhard Hube 1,3
1 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product
Research and Infection Biology - Hans Knoell Institute (HKI), Jena, Germany; 2 Department
of Fungal Septomics, Leibniz Institute for Natural Product Research and Infection Biology -
Hans Knoell Institute (HKI), Jena, Germany; 3 Friedrich-Schiller Universität, Jena, Germany;
4 Aberdeen Fungal Group, Institute of Medical Sciences, University of Aberdeen, UK
Candida albicans successfully colonises diverse niches in the human host without causing
any harm thanks to a balance with the microflora and possibly due to a constant surveillance
of patrolling immune cells. However, under certain conditions, this fungus is able to cause
life-threatening deep-seated infections. Although it is know that neutrophils are key players
in controlling and eliminating C. albicans, it is unclear how C. albicans is killed by neutrophils
and how the fungus counteracts these phagocytes.
By means of single-cell expression profiling using GFP reporter strains, we characterised
different responses of C. albicans to three main neutrophil-imposed stresses: nutrient
starvation, oxidative stress and nitrosative stress.
We observed that phagocytosed fungal cells responded to carbohydrate starvation by
upregulating the glyoxylate cycle, but not the glycolytic pathway. Moreover, a mutant lacking
a key gene of the glyoxylate cycle (icl1 ) exhibited decreased resistance in the presence of
neutrophils.
Next, we investigated the contribution of oxidative stress to killing by neutrophil. The catalase
gene CTA1 was induced upon phagocytosis. However, the surface-associated superoxide
dismutase gene SOD5 was induced in yeast cells even before phagocytosis took place,
suggesting that C. albicans can sense and encounter an extracellular oxidative attack. In a
similar fashion, the thioredoxin gene TRX1 was also upregulated upon extracellular contact
with neutrophils. Strikingly, neither cta1 nor trx1 mutants exhibited decreased resistance
to neutrophils, suggesting that they are not essential for normal resistance of C. albicans.
However, sod5 was more sensitive to neutrophils, indicating that Sod5 is an important
detoxifying enzyme to cope with neutrophil-generated oxidative stress. C. albicans also
responded to nitrosative stress generated in the intracellular milieu and this response was
essential for full resistance as a yhb1 mutant, defective in nitric oxide dioxygenase, exhibited
decreased survival.
In summary, our study provides evidence that C. albicans displays specific responses to
overcome either extracellular or intracellular attack by neutrophils.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network (PITN-
GA-2008-214004).
18

EGFR and HER2 signalling mediate epithelial cell invasion
by Candida albicans during oropharyngeal infection
Weidong Zhu, 1 Quynh T. Phan, 1 Pinmanee Boontheung, 2 Norma V. Solis, 1
Joseph A. Loo, 2,3 Scott G. Filler 1,4
1 Department of Medicine, Division of Infectious Diseases, Los Angeles Biomedical Research
Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, USA; 2 Department of
Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, CA 90095,
USA; 3 Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los
Angeles, CA 90095, USA; 4 Department of Medicine, David Geffen School of Medicine at
UCLA, Los Angeles, CA 90024, USA
A key feature of oropharyngeal candidiasis is fungal invasion of oral epithelial cells,
a process that can occur by several mechanisms, including fungal-induced
endocytosis. Two invasins, Als3 and Ssa1, induce epithelial cell endocytosis of C.
albicans, in part by binding to E-cadherin. However, inhibition of E-cadherin function
only partially reduces C. albicans endocytosis, suggesting that there are addition
epithelial cell receptors for this organism. Here, we demonstrate that the epidermal
growth factor receptor (EGFR) and a related receptor, HER2 function cooperatively
to induce the endocytosis of C. albicans hyphae. EGFR and HER2 are bound by
the C. albicans in an Als3- and Ssa1-dependent manner, and this binding induces
receptor autophosphorylation. Signalling through both EGFR and HER2 is required
for maximal epithelial cell endocytosis of C. albicans in vitro. However, heterologous
expression studies in NIH-3T3 cells indicate that HER2 can mediate endocytosis
of C. albicans by itself, whereas EGFR cannot. In the mouse model of oropharyngeal
infection, C. albicans induces phosphorylation of epithelial cell EGFR and HER2.
Importantly, oral treatment of mice with a dual EGFR and HER2 kinase inhibitor
significantly reduces EGFR and HER2 phosphorylation and attenuates the severity
of oropharyngeal candidiasis. These results demonstrate the importance of EGFR
and HER2 signalling in the pathogenesis of oropharyngeal candidiasis and indicate
the feasibility of treating candidal infections by targeting the host cell receptors to
which the fungus binds.
20

Transcriptional control of white-opaque switching in
Candida albicans and its role in host-pathogen
interactions
Michael Weyler, 1 Christoph Sasse, 1 Mike Hasenberg, 2 Matthias Gunzer, 2
Joachim Morschhäuser 1
1 Institut für Molekulare Infektionsbiologie, Universität Würzburg, Germany; 2 Institut für
Molekulare und Klinische Immunologie, Universität Magdeburg, Germany
Candida albicans is a morphologically versatile microorganism that can undergo
various developmental programmes, which in turn determine its interactions with
the mammalian host. The transition from the yeast form to filamentous growth,
which is induced by environmental signals, is associated with tissue invasion during
an infection. In addition, C. albicans strains that are homozygous at the mating type
locus can spontaneously and reversibly switch from the normal yeast morphology
(white) to an elongated cell type (opaque), which is the mating competent form of
the fungus. White-opaque switching also influences the ability of C. albicans to
colonize and proliferate in specific host niches and its susceptibility to host defense
mechanisms. We used live imaging to observe the interaction of white and opaque
cells with host phagocytic cells. When mixed populations of differentially labelled
white and opaque cells were incubated with human polymorphonuclear leucocytes
(PMNs) on a surface, the neutrophils selectively phagocytosed and killed white cells,
despite frequent encounters with opaque cells. White cells were usually attacked
only after they started to form a germ tube, indicating that the suppression of
filamentation in opaque cells saved them from recognition by the PMNs. In contrast
to neutrophils, dendritic cells internalized white and opaque cells without apparent
difference. However, when embedded in a collagen matrix, the PMNs also
phagocytosed both white and opaque cells with equal efficiency. These results
suggest that, depending on the environment, white-opaque switching enables C.
albicans to escape from specific host defense mechanisms. We created a genomewide
inducible transcription factor expression library, which was used to identify
novel transcriptional regulators of the developmental programmes of C. albicans.
These studies provide insight into the regulation of cell fate decisions in C. albicans
and their consequences for the host-pathogen interaction.
22

Characterization of the molecular mechanism used by
C. albicans to acquire zinc from host cells.
Francesco Citiulo1 , Duncan Wilson1 1, 2
and Bernhard Hube
1 Department of Microbial Pathogenicity Mechanisms, Hans-Knöll-Institute (HKI) - Leibniz
Institute for Natural Product Research and Infection Biology, Beutenbergstr. 11a, D-07745
Jena, and 2 Friedrich Schiller University Jena, Germany
Zinc is an essential trace element for several cellular processes of many organisms
ranging from microbes to multicellular animals. Similar to iron up-take systems,
successful microbial pathogens must have specialized zinc acquisition and uptake
systems to gain this essential metal from the host during infection. In this study, we
investigated the molecular mechanisms used by C. albicans to acquire zinc from
the host.
In silico analyses of the C. albicans secretome identified multiple zinc binding
domains in Pra1 (pH-regulated antigen 1), a 35 kDa cell wall associated and
secreted protein, which is expressed by hyphal cells and which has previously been
shown to interact with complement-regulatory proteins and immune cell receptors.
A mutant lacking functional Pra1 (pra1 ) showed growth defects when cultured in
zinc limited conditions suggesting a role for Pra1 in zinc homeostasis.
During interaction with Human Umbilical Vein Endothelial Cells (HUVEC) under zinc
limited conditions, hyphae of the C. albicans pra1 mutant had significantly
decreased abilities to sequester zinc from host cells as compared to wild type cells.
Although pra1 was capable of endothelial invasion, the mutant caused significantly
reduced damage of these cells and this effect was rescued by the addition of
exogenous zinc.
These data suggest that Pra1 plays a role in zinc binding during infection and that
successful acquisition of this metal is critical for C. albicans pathogenicity.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
24

Strand asymmetry in Candida genes
Malcolm McLean
Department of Molecular Genetics, Weizmann Institute of Science, Revohot, Israel
Genes of virtually all organisms have more A than T on the coding strand.
Additionally Candida albicans, in common with many unicellular fungi, shows a
distinct pattern of GC skew. The start of the gene, including the promoter region,
is enriched in C, the end of the gene is enriched in G. However the pattern is much
weaker in the model organisms, Saccharomyces cerevisiae and
Schizosaccharomyces pombe, which is probably why it has not been observed until
now. However similar AT and GC skews have been found in other organisms, and
the standard explanation is mutational pressure. Roughly, the strands are in different
environments when being duplicated or transcribed, and thus accumulate different
mutations.
However it is hard to account for a change of sign of GC skew with the mutational
pressure hypothesis. Furthermore, analysis of recent substitution and indel
mutations, obtained by aligning homologous genes from related species, does not
support mutational pressure as the force maintaining the GC skew. We instead
propose a selective hypothesis. CTP is the nucleotide tri-phosphate with the
smallest pool. We propose that CTP loading is a rate-limiting step in RNA pol II
elongation (the biochemical evidence is ambiguous), and that a gradient of
decreasing C and increasing G in the mRNA transcript reduces the possibility of
RNA pol II traffic jams in highly transcribed genes.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
26

Functional analysis of C. albicans infection-associated
genes with unknown function during interaction with host
cells and in complex infection models
Ilse D. Jacobsen 1 , Duncan Wilson 1 , Francois Mayer 1 , Bernhard Hube 1,2
1 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product
Research and Infection Biology - Hans-Knöll-Institute, Jena, Germany; 2 Friedrich-Schiller
University, Jena
Candida albicans is able to cause a variety of clinical infections. We hypothesized
that C. albicans unknown function genes, which were transcriptionally upregulated
during oral infections, liver invasion or incubation in human blood (“infection
associated genes”) might contribute to the infection process. We selected a subset
of infection-associated genes with unknown function for further analysis.
Twentyfour isogenic deletion mutants were constructed and subsequently analyzed
both in vitro and in infection models of increasing complexity. Sixteen mutants were
impaired in their ability to damage monolayers of human endothelial and/or
epithelial cells. Within this subset, seven mutants additionally displayed decreased
stress resistance in vitro. Only two mutants showed filamentation defects. To
determine whether the deleted genes influenced virulence in more complex
infections models, all 24 mutants were tested in ovo for their ability to kill chicken
embryos infected on the chorio-allantoic membrane. We recently showed that
mortality in this model depends on the fungal ability to invade the membrane and
that the pro-inflammatory host response likely contributes to pathogenesis.
Surprisingly, only seven mutants were attenuated in this model, of which five were
also attenuated in damaging epithelial cells in vitro. Three of the mutants, which
were attenuated in ovo, and one mutant, which was attenuated in damaging
endothelial and epithelial cells but fully virulent in chicken embryos, were
subsequently analyzed in a systemic mouse model. Virulence in mice mirrored the
results obtained in ovo.
Our results demonstrate that genes essential for causing full damage of epithelial
or endothelial cells in vitro are not necessarily crucial for full virulence in complex
infection models. We conclude that either such damage potential is not critical for
full virulence in the complex host situation or that the greater complexity of the
environment in animal models stimulates additional fungal regulatory networks
leading to compensation of the defect.
28

Survival in the host –mechanisms underlying responses to
oxidative stress in the human pathogen Candida albicans
Alessandra da Silva Dantas 1 , Miranda Patterson 1 , Deborah Smith 1 ,
Catherine Bruce 1 , Donna MacCallum 2 , Lars Erwig 3 , Brian Morgan 1 &
Jan Quinn 1
1Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle
University, Newcastle upon Tyne, NE2 4HH, United Kingdom; 2Aberdeen Fungal Group,
Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, United Kingdom;
3Division of Applied Medicine, Institute of Medical Sciences, University of Aberdeen,
Aberdeen, AB25 2ZD, United Kingdom.
The ability of the major systemic fungal pathogen of humans, Candida albicans, to
sense and respond to reactive oxygen species (ROS) such as H 2 O 2 , generated by
the host immune system, is essential for survival in the host. This oxidative stress
response at the transcriptional level is regulated largely by the AP-1-like
transcription factor Cap1 and, to a lesser extent, the Hog1 stress-activated protein
kinase (SAPK). In addition, H 2 O 2 treatment of C. albicans stimulates the formation
of hyperpolarized buds in a mechanism that requires activation of the Rad53
checkpoint kinase. However, despite strong links between filamentous growth,
responses to ROS, and virulence, remarkably little is known about the intracellular
signalling mechanisms that regulate the activation of such H 2 O 2 -responsive
signalling pathways in C. albicans. During this talk I will present recent work from
my group which has investigated the mechanisms underlying oxidative stressmediated
activation of both the Cap1 transcription factor, and the Hog1 and Rad53
stress responsive kinases. Whilst the Saccharomyces cerevisiae paradigm has
provided important insight into oxidative stress signalling mechanisms, our studies
have revealed new circuitry, novel players and unexpected mechanisms in the
medically relevant pathogen C. albicans.
30

Anticipation in the Nitrosative Stress Response of Candida
albicans
Jed Lynn, Erin Stashi, Lisa Sun, Michael Gustin
Dept Biochemistry and Cell Biology, Rice University, Houston, TX 77005
The human-resident fungus Candida albicans is regularly exposed to nitric oxide
(NO) released by immune cells or non-enzymatically generated from nitrite (NO 2 - )
produced by other microbes in the mouth and skin. One focus of our research is
the molecular mechanisms by which C. albicans senses and defends itself against
NO-related nitrosative stress. C. albicans defense against fungistatic NO requires
the YHB1-encoded nitric oxide dioxygenase which converts NO to nontoxic nitrate
(NO 3 ). NO strongly induces expression of YHB1 and this induction plus NO
resistance requires the zinc cluster transcription factor Cta4, bound to the YHB1
promoter. In Saccharomyces cerevisiae, NO induces ScYHB1, but this induction
requires a different class of transcription factor, the zinc finger protein Fzf1. To test
whether Cta4 is sufficient for NO induction of CaYHB1, a S. cerevisiae strain was
constructed containing a CaYHB1 promoter-lacZ reporter gene and a DNA cassette
allowing tetracycline-regulated expression of CaCTA4. Expression of the latter
induced high level activity of the reporter gene, but both basal and Cta4-induced
lacZ expression were unaffected by NO released from nitrite. In contrast, nitrite
strongly induced expression of a ScYHB1 promoter-lacZ reporter gene in S.
cerevisiae. These data show that Cta4 is not sufficient for NO regulation of CaYHB1
expression and suggest NO sensing involves a negative regulator of Cta4 function.
Unexpectedly, C. albicans NO resistance is induced by not just NO or nitrite, but
also by the nontoxic anions nitrate (NO 3 - ) and thiocyanate (SCN - ). Most other
inorganic anions so far tested, e.g., Cl - , HCO 3 - , are without effect. Microarray
analysis of genome-wide mRNA levels show that NO, nitrite, nitrate, and
thiocyanate each induce nearly identical sets of genes. Nitrate and thiocyanate
induction of CaYHB1 each require CTA4. In contrast, the native YHB1 gene in S.
cerevisiae is induced by NO or nitrite, but not by nitrate or thiocyanate. These results
suggest a model in which the non-toxic saliva chemicals nitrate or thiocyanate are
sensed by C. albicans, allowing anticipation and defense against NO-caused
nitrosative stress .
32

Revisiting the Hog Pathway of C. glabrata
Zeljkica Jandric, Wolfgang Reiter, Gustav Ammerer, Christoph Schüller
Max F. Perutz Laboratories, Department of Biochemistry University of Vienna, Dr.Bohr-Gasse
9/5, A-1030 Vienna, Austria
Signal transduction networks mediated by mitogen – activated protein kinases
(MAPK) play essential roles in eukaryotic cells. Yeast cells have developed
osmoadaptation pathways to cope with osmotic changes in the microenvironment.
In Saccharomyces cerevisiae five MAPK pathways are well characterized. One of
these, especially associated with stress response, is the High Osmolarity Glycerol
(HOG) pathway . Stress induced phosphorylation triggers the activation of the key
MAPK, Hog1. After being phosphorylated by the upstream MAPKK Pbs2, Hog1
enters the cell nucleus and modulates the transcription of its target genes.
Orthologues of the all HOG pathway compounds found in Saccharomyces
cerevisiae have been identified in fungi and animals.
The human fungal pathogen Candida glabrata is closely related to Saccharomyces
cerevisiae. It is a common commensal but in immunocompromised cancer and
transplant patients or elder persons it can turn into an opportunistic pathogen.
Depending on the host environment, Candida glabrata has to adapt to
environmental changes during invasion and adaptation steps. We investigated the
role of Hog1 in Candida glabrata. Several stress sources were used to identify HOGspecific
phenotypes and to analyze the role of Hog1 in Candida glabrata. As
expected, the deletion of Hog1 caused osmostress hypersensitivity. Physiological
stress conditions, such as nutrients starvation, were simulated by infection of
macrophages with Candida glabrata and determination of the number of surviving
Candida cells. Cghog1∆ cells showed a much higher sensitivity to macrophages
compared to wild type cells. Further investigations focus on stress induced gene
transcription in Cghog1∆ cells and comparison of the transcription pattern to cells
without Hog1 deletion.
34

Candida albicans alters extracellular pH under
host-relevant conditions of carbon starvation
Slavena Vylkova, Heather Danhof, and Michael C. Lorenz
Dept. of Microbiology and Molecular Genetics, The University of Texas Health Science
Center, 6431 Fannin St., Houston, TX 77030, USA
Phagocytosis by immune cells, such as macrophages, induces a remarkable
change in transcription and physiology in Candida albicans, with ~10% of the
genome affected. A primary aspect of this response is metabolic shift reflecting
deprivation for carbon, and this response is required for full virulence. We developed
an in vitro system to mimic this response, using media that is glucose-poor and
amino acid-rich; surprisingly, in these conditions, we observed a striking change in
the media pH when C. albicans was grown at an initial pH of 4. Over 12-24 hours,
the pH rose to 7-7.5, and this alkalinization was associated with the induction of
hyphal growth – a key virulence trait in this species. This extracellular pH change
was not dependent on factors previously known to regulate pH responses, such as
RIM101 and MNL1. Using a combination of genetic and genomic screens, we have
linked this process to amino acid catabolism: mutation of genes required for amino
acid permease function and breakdown of amino acids as a carbon source are
impaired in pH changes. Transcript profiles confirm that this pH alteration is the
result of a metabolic change to the use of amino acids as a carbon source, similar
to the profile of phagocytosed cells. Alkalinizing cultures release measurable
quantities of ammonia, a strong base, and we propose that catabolism of amino
acids as a carbon source renders the amine group in excess, and this is excreted
to change the pH. This occurs in conditions that mimic those our data suggest exist
after phagocytosis, and we are now examining the intracellular fate of ingested C.
albicans cells. Published reports are contradictory regarding this fate, but the weight
of evidence indicates intracellular trafficking is aberrant. We propose that C.
albicans alters phagolysosomal pH to induce hyphal growth and impair the
fungicidal effects of the immune cell.
36

Glucose promotes oxidative stress resistance in Candida
albicans via specific signalling pathways
Iryna Bohovych 1 , Pedro Miramón 2 , Aaron Mitchell 3 , Bernhard Hube 2 ,
Alistair JP Brown 1
1 Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, UK; 2 Microbial Pathogenicity, Hans Knoell Institute, Beutenbergstraße
11a, Jena 07745, Germany; 3 Department of Biological Sciences, Carnegie Mellon University,
Pittsburgh, Pennsylvania 15213, USA
Candida albicans, a successful human pathogen, displays the phenomenon of
glucose-enhanced oxidative stress resistance (Rodaki et al., 2009), which is not
observed in other yeast species tested. The molecular mechanisms that mediate
glucose-enhanced oxidative stress resistance in C. albicans are not clear. We
reasoned that glucose signalling might play a major role. Therefore we tested the
impact of specific C. albicans mutations to determine which of known signalling
pathways are required for glucose-enhanced oxidative stress resistance.
The Sugar Receptor-Repressor (Hgt4-Rgt1) pathway, homologous to the Rgt2/Snf3
pathway in S. cerevisiae, is not required for the phenotype. In contrast, a snf1
mutant, which lacks a key component of the Glucose Repression Pathway,
displayed high oxidative stress resistance on lactate, not altered by glucose. A kis1
mutant, lacking one of the subunits of Snf1 complex, displayed a related phenotype.
The data indicate that in the absence of glucose the Glucose Repression Pathway
represses glucose-enhanced oxidative stress resistance.
cAMP signalling also appears to play a role in glucose-enhanced oxidative stress
resistance. This phenotype was repressed by exogenous dibutyryl cAMP and by
a pde2 mutation that is thought to increase intracellular cAMP levels. Also the
phenotype was enhanced by inactivation of adenylyl cyclase (cyr1).
Having shown that both cAMP signalling and the Glucose Repression Pathway play
important roles in mediating glucose-enhanced stress resistance, the next step was
to identify targets of these pathways that might contribute to the phenotype.
Comparative analyses of transcriptomic profiles of C. albicans glucose- and lactategrown
cells in response to oxidative stress and glucose treatment correspondingly
revealed a small set of commonly up-regu lated genes (Enjalbert et al., 2006; Rodaki
et al., 2009). The impact of the cAMP and Glucose Repression pathways on these
genes is being determined.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
38

Stress-responses controlled by the Hog1 stress-activated
protein kinase
Sergi Regot, Núria Conde, Mariona Nadal, Carme Solé, Alba Duch, Javier
Jimenez, Alberto Gonzalez, Eulàlia de Nadal and Francesc Posas
Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut. Universitat
Pompeu Fabra (UPF). PRBB. C/ Doctor Aiguader 88. Barcelona E-08003 (Spain).
Eulalia.Nadal@upf.edu; francesc.posas@upf.edu
Exposure of yeast cells to increases in extracellular osmolarity results in the
activation of the Hog1 MAP kinase. Activation of this MAP kinase is required to
generate a set of osmoadaptive responses essential to survive under high
osmolarity conditions. Fast and transient activation of the Hog1 signaling pathway
is required for proper adaptation. Adaptation to osmostress requires the induction
of a large number of genes, which indicates the necessity to regulate several
aspects of the cell physiology. Induction of gene expression is highly dependent
on the presence of the MAP kinase, which suggests a key role for the HOG signaling
pathway in the regulation of gene expression in response to osmostress. In
response to stress, the MAPK controls several mechanisms related to transcription
initiation and elongation as well as chromatin organization. The MAPK also controls
cell cycle. Here, the MAPK is able to modulate cell cycle delay in different phases
which highlight the relevance of cell cycle control in response to stress.
40

Friend or Foe: Systems biology approaches to elucidate
the interaction between fungi and their hosts
Duccio Cavalieri
Department of Preclinic and Clinic Pharmacology, University of Florence, viale Pieraccini 6,
50139 Firenze. duccio.cavalieri@unifi.it, telephone number: +39-055-4271327, fax number:
+39-055-4271280
Modelling the networks subtending the fruitful coexistence of fungi and their
mammalian host is becoming increasingly important to control emerging fungal
pathogens. The goal of Systems Biology is to obtain a superior level of knowledge
of how function arises in dynamic interactions. Systems Biology requires the
acquisition of information on the different levels of regulation of a biological system
and its integration to unravel the complexity and the dynamic nature of the hostfungus
interaction. Immunity is not simply the product of a series of discrete linear
signaling pathways; rather it is comprised of a complex set of integrated responses
arising from a dynamic network of thousands of molecules subject to multiple
influences. In this perspective the fungal microbiota and the host should be
analyzed as an ecosystem, and disease considered as an alteration of the
equilibrium. Our work addresses host-fungus interaction with a special focus on
systems biology approaches to investigate the mechanisms developed by Candida
albicans and Aspergillus fumigatus to circumvent host immune responses during
fungal infections and the phenotypic traits that potentially make S.cerevisiae a
microorganism generally recognized as safe. Such genome wide integrative
approaches hold the promise to significantly improve our ability to understand
which fungal traits can be considered potential threats and the regulatory networks
involved in immune subversion.
42

Agent based modelling and host pathogen interactions
Katarzyna M. Tyc 1 , Clemens Kühn 1 , Edda Klipp 1
1 Theoretische Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany
Mathematical models of pathogen and host interactions provide a tool for medical
applications and potential for pharmaceutical companies for testing novel drug
treatments and consequences of their application. Host pathogen interactions are
highly complex and experimental techniques even when combined with different
computational methods allow only for partial investigation of their mutual
dependences.
Based on the available literature we reconstruct a computational model of a human
oral epithelium invasion by the pathogenic fungus Candida albicans. We investigate
the system’s dynamics using the agent based modelling approach (ABM) where
cells (Candida cells, neutrophils (PMNs)) are represented as individual agents. These
agents interact in different ways: fungal cells form hyphal colonies on the epithelium
and then epithelial cells secrete cytokines upon contact with the hyphae form of
fungi, indicating inflammation. Cytokine secretion is one of the signals stimulating
PMNs recruitment to the site of infection. Mix of different gradients such as lactate
dehydrogenase (LDH) release from damaged epithelium, cytokines, and secreted
hyphal particles influences PMN activity. In our model, we investigate the essential
parameters that establish the system’s dynamics. We also look at the
consequences of an antifungal drug treatment on the Candida albicans survival rate
and its dose dependence. We test the optimal intervals and doses for new
application.
With agent based models, we can investigate the effect of individual dynamics on
global patterns, enabling an intuitive view on host-pathogen interactions that can
provide relevant predictions for further experiments.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
44

Impact of non-fermentative growth on the C. albicans cell
wall and dynamic responses to osmotic stress
Iuliana V. Ene, Alistair J. P. Brown
Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, UK
Candida albicans displays considerable metabolic flexibility and robust stress
responses in vivo. Both contribute to the virulence of this pathogen. C. albicans
assimilates both fermentative and non-fermentative carbon sources in vivo, often
occupying poor glucose niches in its mammalian host. We reasoned that changes
in carbon source might influence stress adaptation, but stress responses are
generally studied during growth on glucose.
We found that the C. albicans cell wall is extremely dynamic and susceptible to
variations in carbon source. Growth on lactate rather than glucose has a major
impact on cell wall structure and flexibility. Although the relative proportions of
chitin, -glucan and mannan are similar within the wall of lactate-grown cells, they
display increased porosity and decreased cell wall biomass. TEM confirmed major
differences in cell wall architecture, underlying the importance of the carbon source
in the cell biology of this pathogen. This in turn influences the responses of this
pathogen to stresses as these differences were reflected in the cell wall-related
phenotypes. Lactate-grown cells displayed higher resistance to Congo Red and
Calcofluor White and showed significantly increased resistance to various doses
of osmotic stress. This change was independent of Hog1 and Mkc1, but correlated
with large changes in cell volume following exposure to NaCl. Growth on lactate
also increased resistance to antifungals such as Caspofungin, Tunicamycin and
Amphotericin B, which is likely to have a major impact upon the behavior of C.
albicans during infection.
The impact of carbon source on stress and drug resistance was confirmed in other
pathogenic Candida species and for other carbon sources, resulting in variations
in resistance when compared to a glucose-grown control. Clearly carbon source
strongly influences cell wall architecture and the resistance of C. albicans to certain
stresses, which is likely to have a major impact on its in vivo behavior.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial
Training Network (PITN-GA-2008-214004).
46

Target specificity of the Efg1 regulator in Candida albicans
Lassak, T., Lagadec, Q., Kurtz, D., Bussmann, M. and Ernst, J. F.
Heinrich-Heine-Universität Düsseldorf, Institut für Mikrobiologie, Molekulare Mykologie
Efg1 is a general transcription factor, which regulates numerous morphogenetic
and metabolic processes in C. albicans. We previously found that overexpression
of EFG1 and activation of Efg1 during hypha formation leads to downregulation of
EFG1 promoter activity and that EFG1 downregulation is necessary to allow the
formation of true hyphae and prevent pseudohyphal growth.
To explore the Efg1 binding specificity its binding to the EFG1 promoter was
analyzed by EMSA and footprint analyses. These experiments revealed binding of
Efg1 to a promoter sequence close to the transcript start site, which contained a
repeated 8-mer motif (referred to as APSES response element, ARE). Surprisingly,
promoter fusions to the RLUC reporter lacking the ARE motif were still able to
downregulate the EFG1 promoter during hypha formation in an Efg1-dependent
manner. Furthermore, extensive deletions of the 10 kb-large 5 untranslated region
of EFG1 still did not prevent regulation suggesting multiple binding sites for Efg1
within the EFG1 promoter. We subsequently analyzed genome-wide occupation of
chromosomal sequences by Efg1 using ChIP chip analysis. These results revealed
the entire 10 kb EFG1 upstream region as a major site of Efg1 occupancy.
Unexpectedly, Efg1 binding to this region was rapidly lost during hypha induction
suggesting a regulatory model, in which Efg1 acts as transcriptional activator
specifically during yeast growth. In further experiments we attempted to reconstitute
the EFG1 autoregulatory circuit in the yeast S. cerevisiae. In this system the
presence of Efg1 was shown to downregulate EFG1 promoter activity, although not
to the same extent as was observed in C. albicans. This finding hints at coregulators
that together with Efg1 mediate autoregulation in C. albicans.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
48

Wednesday, October 12
51

Genetic Control of Candida albicans Biofilm Formation
J.S. Finkel 1 , F. Lanni 1 , J. Nett 2 , D. Huang 1 , J. Suhan 1 , A. Nantel 3 , D. Andes 2 ,
A.P. Mitchell 1
1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA; 2 Department
of Medicine, University of Wisconsin, Madison, WI; 3 Biotechnology Research Institute,
National Research Council of Canada, Montreal, QC
Implanted medical devices, such as venous catheters and artificial heart valves,
are associated with a significant risk of microbial infection. These infections arise
because device surfaces support biofilm formation. We have worked to define the
genetic basis for C. albicans biofilm formation as a means to understand the
underlying mechanisms. We have focused on transcription factors because they
often govern expression of functionally related genes. We have recently identified
transcription factors that are required for adherence to a silicone substrate in vitro.
In vitro assays indicate that many of these transcription factors are not required for
biofilm formation per se, but are required for normal biofilm morphology.
Importantly, several of these newly defined biofilm transcription factors are required
for biofilm formation in vivo, as assayed in a rat venous catheter model. We will
present target gene analysis that points toward the mechanistic basis for these
biofilm defects.
52

Candida albicans: Sensing the Host Environment
Carol A. Kumamoto
Tufts University, Boston, MA, USA
Candida albicans, an opportunistic pathogen and a human commensal, is found
almost exclusively in association with a host. Intestinal tract colonization by this
organism is common in humans and disease is thought to arise due to overgrowth
or escape of organisms from the gut. In the host, the organism’s ability to sense
cues from the environment would be expected to enhance its growth and survival.
C. albicans responds to numerous environmental cues that could be encountered
in the host such as temperature, pH, presence of O 2 , CO 2 , nutrients or antimicrobial
compounds, and stress conditions. Our studies focus on the ability of C. albicans
to sense contact with a surface and to sense the environment within the intestinal
tract. These types of sensing mechanisms promote the organism’s ability to sense
its location within the host, the nature of the tissue that it is encountering and the
status of the host’s immune response. As a result of these sensing mechanisms,
the organism controls its physiology so that it maintains benign colonization in a
healthy host but becomes a destructive pathogen that invades host tissue in a
compromised host.
54

Identification of Candida albicans genes involved in biofilm
formation by an over-expression approach
Vitor Cabral 1,2 , Sadri Znaidi 1,2 , Mélanie Legrand 1,2 , Keunsook Lee 3 , Sophie
Bachelier-Bassi 1,2 , Murielle Chauvel 1,2 , Tristan Rossignol 1,2 , Carol Munro 3 ,
Christophe d’Enfert 1,2
1 Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Département Génomes et
Génétique, F-75015 Paris, France; 2 INRA, USC2019, F-75015 Paris, France; 3 Aberdeen
Fungal Group, University of Aberdeen, School of Medical Sciences, Institute of Medical
Sciences, Aberdeen, AB25 2ZD, United Kingdom
Candida albicans is the major fungal pathogen of humans, being responsible for
benign superficial infections and devastating systemic infections. These latter
infections are often associated with the formation of biofilms on medical devices.
Biofilms are polymicrobial communities adherent to biotic or abiotic surfaces and
are especially resistant to antifungal agents making them difficult to treat. Despite
recent progress, the molecular mechanisms that underlie the formation of biofilms
and the basis for their tolerance to antifungals remain to be fully understood.
Here, we have undertaken to identify and characterize C. albicans genes whose
over-expression alters biofilm formation. To this aim we have developed through
the Gateway cloning methodology a partial C. albicans ORFeome that includes
531 ORFs and a corresponding collection of C. albicans over-expression strains,
each having a unique C. albicans gene controlled by a doxycycline-inducible
promotor and a unique molecular barcode. This collection has been used to
evaluate the fitness of each strain in competition experiments, taking advantage of
their molecular barcoding for individual microarray-based quantification. First, the
fitness of the strains in planktonic cultures with or without over-expression of the
genes has been studied, revealing 8 genes whose over-expression reduces fitness
(6 related to cell cycle, DNA damage and check-point; 2 involved in filamentation).
Second, the fitness of the strains in biofilms with or without over-expression of the
genes has been investigated. None of the genes whose over-expression altered
fitness in planktonic cultures were identified in this analysis. Strikingly, we observed
a strong enrichment for genes encoding cell wall proteins and genes involved in
budding regulation among those genes whose over-expression altered fitness in
biofilms. Current experiments are aimed at confirming the results of these pool
experiments through analysis of individual over-expression mutants and further
understanding the role of the identified genes in biofilm formation.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
56

Cell wall stress elicits a conserved response in Candida
albicans
Alice G. Sorgo, Clemens J. Heilmann, Sepehr Mohammady, Henk L.
Dekker, Stanley Brul, Chris de Koster, Leo J. de Koning and Frans M. Klis
Swammerdam Institute for Life Sciences, Universiteit van Amsterdam, Science Park 904,
1098 XH Amsterdam, The Netherlands
Both cell wall proteins and secreted proteins of C. albicans are critical for fitness,
virulence and adaptation to environmental challenges. We have recently shown that
fluconazole, an antifungal drug that affects ergosterol synthesis and increases
membrane fluidity, also causes cell wall stress as evidenced by decreased
resistance to wall-perturbing compounds and higher chitin levels in the wall (1). This
is accompanied by substantial changes in the wall proteome and secretome. We
found that the abundance of proteins mainly involved in wall biosynthesis and
integrity increased (Crh11, Pga4, Pir1, Phr1, Phr2, Sap9). However, the abundance
of hypha-associated proteins (Als3, Hwp1, Plb5) decreased in agreement with
reduced hyphal growth. Finally, fluconazole induced cell clustering. As membrane
fluidity also tends to increase at higher temperatures, we wanted to investigate
whether the response to thermal stress is similar to that of fluconazole and could
also result in cell wall stress. When C. albicans was cultured at 42°C, which resulted
in substantial growth inhibition, the cells showed indeed decreased resistance to
wall-perturbing agents, and chitin levels in the wall were elevated, both suggesting
cell wall stress. Likewise, the wall proteome response was similar to that of
fluconazole-treated cells, including increased incorporation levels of proteins
important for wall maintenance and integrity. Additionally, cell separation was
impaired, correlating with a severe reduction of the chitinases Cht2 and Cht3 and
the endoglucanase Eng1 in the medium. In summary, our data indicate that both
thermal stress and fluconazole-induced stress induce membrane stress, which
leads to cell wall stress and a conserved wall stress response.
References
(1) The effects of fluconazole on the secretome, the wall proteome and wall integrity of the clinical fungus
Candida albicans. Sorgo AG, Heilmann CJ, Dekker HL, Bekker M, Brul S, de Koster CG, de Koning LJ,
Klis FM. Eukaryot Cell. 2011. doi:10.1128/EC.05011-11.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
58

Candida albicans cell wall dynamics
Carol A. Munro, Louise A. Walker, Keunsook Lee, David Stead, Laura
Selway, Alistair J.P. Brown and Neil A.R. Gow
Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland
The outer surface of Candida albicans is composed of chitin and glucan
polysaccharides and an exposed fibrillar layer, rich in highly mannosylated proteins.
The co-ordinated assembly of the cell wall is regulated by a network of signalling
pathways that ensure cellular integrity is maintained in response to intracellular and
extracellular signals. Treatment with sub-inhibitory concentrations of the
echinocandin antifungal drugs, which inhibit beta-1,3-glucan synthesis, results in
significant changes in the cell wall architecture due to activation of compensatory
chitin synthesis and alterations in the cell wall proteome. A number of proteins that
are covalently attached to the cell wall are carbohydrate- active enzymes involved
in modulating and forming cross-links between chitin and glucan. These proteins,
such as Phr1 and Crh11 are positively regulated in response to applied cell wall
and membrane stresses and mutations in cell wall related genes. Changes in the
fungal cell wall can in turn alter susceptibility to antifungal drugs in vitro as well as
in vivo.
The cell surface is the interface between host and fungus and a number of virulence
attributes are cell wall localised. Alterations in cell wall composition and architecture
are likely to impinge on pathogenesis and recognition by host’s immune cells. Using
transcript profiling, biochemical assays, glycoproteomics and SILAC (Stable Isotope
Labelling with Amino Acids in Cell Culture) we have started to catalogue quantitative
and qualitative changes in the cell wall in response to pharmacological and genetic
blockade of cell wall biosynthesis. In addition we are studying the phenotypes of a
library of strains that are overexpressing cell wall related genes in order to better
understand the cell wall phenome.
60

Force-dependent activation of functional amyloid domains
increases cell adhesion in C. albicans
Peter N. Lipke 1 , David Alsteens 2 , Cho Tan 1 , Desmond N. Jackson 1 , Caleen
B. Ramsook 1 , Yves F. Dufrêne 2 , and Melissa C. Garcia 1
1 Department of Biology, Brooklyn College of the City University of New York; 2 Institute of
Condensed Matter, Université catholique de Louvain, Louvain-le-Neuve, Belgium
Yeast cell adhesion molecules encoded by diverse gene families have conserved
amyloid-forming sequences, and amyloid formation potentiates cell-cell interactions
(Ramsook et al., 2010 Euk. Cell 9:393-404 ; Alsteens et al., 2010 PNAS 107:20744-
9; Garcia et al., 2011 PLoS ONE 6: e17632. doi:10.1371/journal.pone.0017632 ).
Amyloid formation clusters the cell adhesion molecules, and increases cell-cell
binding by clustering of active sites in the same way that multivalence of antibodies
increases avidity relative to affinity of each site.
We show that amyloid formation is initiated by extension force, and results in
formation of nm-scale amyloid domains on the surface of the cell, with different
protein domains playing distinct roles to facilitate amyloid formation. Atomic force
microscopy of single molecules in situ unfolds successive domains in the Candida
albicans Als adhesins. This unfolding triggers local amyloid formation and
subsequent propagation of amyloid patches around the entire cell surface. Amyloid
formation is accompanied by activation of strong cell-cell and cell-substrate
adhesion. Both amyloid formation and activation of adhesion are inhibited by antiamyloid
dyes and peptides. In addition, a single site mutation in the amyloid core
sequence of Als5p abrogates amyloid formation and activation of adhesion,
although the mutant protein is well-folded and maintains in vitro binding activity.
Amyloid formation is also triggered by forces developed between cells during
assays or by prolonged vortex mixing of cells. The various protein domains in Als
proteins synergize to promote this force-induced amyloid formation. The
consequence is formation of nanoscale patches of amyloid on the cell surface to
potentiate cell interactions by formation of multiple intercellular bonds.
Supported by NIH SCORE grant SC1 GM083756
62

Iff2 is a cell surface protein involved in adhesion
Iaroslava Kos 1 , Grégory Da Costa 1 , Céline Monniot 1 , Anita Boisramé 1
Marie-Noëlle Bellon Fontaine 2 , Murielle Chauvel 3 , Christophe d’Enfert 3 and
Mathias L. Richard 1
1 Team “Virulence and Fungal Infection” Institut Micalis, UMR1319 INRA AgroParisTech,
78850 Thiverval-Grignon, FRANCE; 2 Team “Bioadhésion et Hygiène des Matériaux” Institut
Micalis, UMR1319 INRA AgroParisTech, 91744 Massy, FRANCE; 3 Team “Fungal Biology and
Pathogenicity”, Pasteur Institute, 75015 Paris, FRANCE.
In Candida albicans, the cell wall and especially cell wall proteins are known to play
a key role in the relationship between the fungal cell, the host and most indwelling
device, contributing to adhesion, immune response modulation and deep seated
infections. A specific class of cell wall proteins has been particularly under the
scope in the past decade: the glycosylphosphatidylinositol anchored proteins
(GpiPs). The largest family of GpiPs is a family of 12 proteins sharing a high similarity
with Hyr1. One of its members, Iff2 has been the subject of a large scale study in
our laboratory in order to decipher his putative function in C. albicans. The cell
localization of Iff2 was assessed using a V5-tagged protein and clearly showed that
Iff2 is a cell surface protein tethered to the cell wall proteins through alkali-Pir-like
links as well as di-sulfide bonds. We also demonstrated that Iff2 protrude from the
cell wall glucan network at the interface with the cellular environment. At the gene
level, we showed that IFF2 is a gene with an expression level stronger than most of
the member of Hyr/Iff family in laboratory conditions and that IFF2 is induced during
the stationary phase in the same media. An overexpressing strain with IFF2 under
the control of TEF1p was hyper adherent to different plastic but not to silicon,
resulting in the formation of stronger biofilm at the early stage of biofilm formation
compared to the reference strain. The future work is aimed to understand which
part of the protein is important for adhesion and if it is specific to Iff2 functional
domain. Additional experiments are also planned to monitor the consequences of
this overexpression in in vitro and in vivo models.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
64

Deciphering the role of CUG mistranslation on Candida
albicans adhesion and cell surface variation
Isabel Miranda 1,2 , Ana Silva-Dias 1,2 , Rita Rocha 1 , Teresa Gonçalves 3 , Cidália
Pina-Vaz 1,2,4 , Scott G. Filller 5,6 and Acácio G. Rodrigues 1,2,7
1 Department of Microbiology, Faculty of Medicine, University of Porto, 2 Cardiovascular
Research & Development Unit, Faculty of Medicine, University of Porto, Portugal, 3 Center
for Neurosciences and Cell Biology, Institute of Microbiology, Faculty of Medicine, University
of Coimbra, Portugal; 4 Department of Microbiology, Hospital S. João; 5 Department.of
Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center,
Torrance, California, USA; 6 David Geffen School of Medicine at UCLA, Los Angeles,
California, USA; 7 Burn Unit and Department of Plastic and Reconstructive Surgery, Hospital
S. João, Portugal.
Interactions with the surrounding environment represent a continuous stressing
challenge for microorganisms. Until now, the mechanisms regulating changes in
cell-surface in the most common human fungal pathogen still remain unclear. In
Candida albicans, the CUG codon is naturally mistranslated as two biochemically
distinct residues, serine and leucine. Interestingly, genes encoding adhesins such
as the ALS family are particularly enriched in CUG codons. In order to evaluate the
role of CUG mistranslation in C. albicans adhesion, we compared the behavior of
two strains: a wild-type, in which 3% of CUG codons are mistranslated as leucine
and a highly CUG mistranslating strain wherein 28% of CUG codons are
mistranslated as leucine. We found that the increase in leucine misincorporation
enhances cell wall hydrophobicity and the capacity of yeast cells to adhere to
biomaterials. This adhesion phenotype was further confirmed in Saccharomyces
cerevisiae, through expression of C. albicans ALS3 gene in its two isoforms: Als3p-
Leu and Als3p-Ser. Als3p-Leu yeasts prompted a strong adhesion phenotype,
resulting in flocculation in liquid media and two-to-fivefold more adhesion than
Als3p-Ser. The cell wall variability generated by CUG mistranslation may not be
confined to adhesion and could be relevant for immune system evasion. Indeed,
highly CUG mistranslating cells were less susceptible to phagocytosis by murine
macrophages than wild-type, probably due to the more efficient glucan masking
caused by CUG mistranslation. We propose that CUG mistranslation was
maintained during C. albicans evolution due to its potential to generate cell surface
variability.
A Silva-Dias is supported by a FCT (Fundação Ciência e Tecnologia) doctoral grant
SFRH/BD/44896/2008; I Miranda is supported by Ciência 2008 (FCT) and European Social Fund.
66

Interaction of a Candida glabrata transcription factor
knock-out library with the Drosophila melanogaster innate
immune system
Jane Usher 3 , Ilias Kounatidis 1 , Biao Ma 2 , Petros Ligoxygakis 1 &
Ken Haynes 3
University of Oxford 1 , Imperial College London 2 , University of Exeter 3
At least in part Candida species are thought to invade following initial
gastrointestinal colonisation. The mechanisms underpinning this translocation
depend on many factors including host intestinal flora, mucosal damage and
deficient host immunity. In addition fungal attributes eg the ability to acquire
nutrients and to adapt to host environmental insults are essential to disease
initiation and progression.
To analyse this interaction on a holistic level we have created a library of Candida
glabrata mutants and developed a Drosophila melanogaster model of
gastrointestinal candidosis. A library of ~200 bar coded mutant strains, each lacking
a specific DNA binding protein, was constructed in a derivative of C. glabrata ATCC
2001. Each of these was screened in a D. melanogaster larval GI infection model,
in which third instar larvae, containing a Drs-GFP allele, were fed with C. glabrata
infected food. Drosomycin (Drs) is activated in the larval fat body in response to
wild-type C. glabrata colonization. Of the 200 knock-out mutants 12 failed to
activate Drs-GFP indicating reduced activation of host innate immunity. We are
currently investigating the basis of this reduced activation.
68

Using zebrafish to study Candida albicans-mediated
systemic candidiasis in vivo
Thomas Evans, Simon Tazzyman, Timothy J.A. Chico, Martin H. Thornhill,
Craig Murdoch.
University of Sheffield, Sheffield, UK
Candida albicans is a common oral opportunistic pathogen that causes
mucocutaneous infections and rarely systemic candidiasis, a nosocomial infection
which involves the haematogenous spread of Candida to multiple organs and is
associated with high mortality. To cause tissue damage C.albicans must bind to the
endothelium of blood vessels, leave the circulation and invade tissues. However,
little is known about the mechanisms involved in these processes. This study aimed
to use transparent embryonic zebrafish (Danio rerio) as an in vivo infection model
in conjunction with an in vitro flow adhesion assay to study how C.albicans bind to
the endothelium, leave the circulation and invade tissues. Suspensions of viable or
treated C.ablicans were flowed over monolayers of endothelial cells and adherent
Candida counted. Zebrafish were injected with C.albicans and mortality determined
after 18h. Time-lapse confocal microscopy using fluorescent C.albicans and blood
vessels was performed to image dissemination. Viable C.albicans bound to
endothelium significantly (p

Thursday, October 13
73

The long pentraxin PTX3 as a paradigm of humoral pattern
recognition molecules in antimicrobial resistance
Alberto Mantovani
Laboratory of Immunology and Inflammation , Istituto Clinico Humanitas, Rozzano (Milan),
Italy; and Department of Translational Medicine, University of Milan, Italy
PTX3 is a multifunctional soluble pattern recognition receptor characterized by a
C-terminal domain highly homologous to C-reactive protein and serum amyloid P
component, associated to a N-terminal domain unrelated to other known proteins.
PTX3 is produced upon stimulation with proinflammatory cytokines and Toll-like
receptor engagement by different cell types, including endothelial cells,
monocytes/macrophages, dendritic cells, fibroblasts and epithelial cells. The
molecule binds with high affinity complement component, microbial moieties,
growth factors and apoptotic cells. PTX3 plays non-redundant functions including
innate immunity against selected microorganisms to regulation of inflammation.
PTX3 plays a nonredundant role in resistance against selected fungi, A. fumigatus
in particular. Moreover, levels of PTX3 and genetic polymorphisms are associated
with infections in humans. In addition PTX3 has a regulatory function. PTX3 serves
as a paradigm for the interplay between the cellular and the humoral arm of innate
immunity.
Bottazzi B, Doni A, Garlanda C, Mantovani A. An Integrated View of Humoral Innate Immunity: Pentraxins
as a Paradigm. Annu Rev Immunol. 2010, 28:157-83.
Deban L, Russo RC, Sironi M, Moalli F, Scanziani M, Zambelli V, Cuccovillo I, Bastone A, Gobbi M,
Valentino S, Doni A, Garlanda C, Danese S, Salvatori G, Sassano M, Evangelista V, Rossi B, Zenaro E,
Constantin G, Laudanna C, Bottazzi B, Mantovani A. Regulation of leukocyte recruitment by the long
pentraxin PTX3. Nat Immunol. 2010 Apr;11:328-34.
74

Pattern recognition and anti-fungal immunity: The role of
C-type lectins
Gordon D. Brown
Aberdeen Fungal Group, University of Aberdeen, UK
The innate ability to detect pathogens is essential for multicellular existence, and
has been achieved through the evolution of germ-line encoded receptors which
can recognise non-self structures, the so-called “pattern recognition receptors”
(PRR). One such receptor is Dectin-1, a type II transmembrane glycoprotein with a
single extracellular non-classical C-type carbohydrate recognition domain (CRD)
and a cytoplasmic tail possessing an immunoreceptor tyrosine-based activationlike
(ITAM) motif. Dectin-1 is predominantly expressed on myeloid cells and
recognises (1 3)-linked glucans. We and others have demonstrated that this
receptor mediates a variety of cellular responses to -glucans, including
phagocytosis, endocytosis and the oxidative burst and can induce the production
of arachidonic acid and numerous cytokines and chemokines. These responses
are triggered through the cytoplasmic ITAM-like motif of this receptor, utilising novel
signalling pathways involving a unique interaction with Syk kinase and collaborative
signalling with the TLRs. Dectin-1 is the first example of a signalling non-Toll-like
pattern recognition receptor being involved in the induction of protective immune
responses, and through these activities Dectin-1 plays a fundamental role in antifungal
immunity in mice and humans. Here, we will review new insights into the role
of Dectin-1 as well as other PRRs during fungal infections.
76

The Tyr238X dectin-1 polymorphism is a predisposing
factor for mucosal Candida albicans infections but not
candidemia
Diana C. Rosentul 1,2 , Theo S. Plantinga 1,2 , Marije Oosting 1,2 , William K.
Scott 3 , Digna R. Velez Edwards 4 , P. Brian Smith 5 , Barbara D. Alexander 5 ,
John C. Yang 6 , Gregory M. Laird 5 , Walter J. F. M. van der Velden 2,7 , Bart
Ferwerda 1,2 , Annemiek B. van Spriel 2,8 , Gosse Adema 2,8 , Ton Feuth 2,9 , J.
Peter Donnelly 1,2 , Gordon D. Brown 10 , Nicole M. A. Blijlevens 2,7 , Leo A.B.
Joosten 1,2 , Jos W. M. van der Meer 1,2 , John R. Perfect 5 , Bart-Jan
Kullberg 1,2 , Melissa D. Johnson 5,11 , Mihai G. Netea 1,2
1 Department of Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen,
The Netherlands; 2 Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud
University Nijmegen Medical Center, Nijmegen, The Netherlands; 3 Dr. John T. Macdonald
Foundation Department of Human Genetics and John P. Hussman Institute for Human
Genomics, University of Miami, Miller School of Medicine, Miami, FL, USA; 4 Department of
Obstetrics & Gynecology, Vanderbilt University Medical Center, Nashville, TN, USA; 5 Duke
University Medical Center, Durham, NC, USA; 6 National Jewish Health, Denver, CO, USA;
7 Department of Haematology, Radboud University Nijmegen Medical Center, Nijmegen, The
Netherlands; 8 Department of Tumor Immunology, Radboud University Nijmegen Medical
Center, Nijmegen, The Netherlands; 9 Department of Epidemiology, Biostatistics and Health
Technology Assessment, Radboud University Nijmegen Medical Center, Nijmegen, The
Netherlands; 10 Institute of Infectious Disease and Molecular Medicine, University of Cape
Town, Cape Town, South Africa; 11 Department of Clinical Research, Campbell University
School of Pharmacy, Buies Creek, NC, USA.
The genetic make up of innate immune genes is believed to play an important role
for susceptibility to fungal infections. We recently described the Tyr238X dectin-1
single nucleotide polymorphism (SNP) as an important factor influencing
susceptibility to vulvo-vaginal candidiasis and onychomycosis in a Dutch family
with recurrent fungal infections.
The aim of our studies was to assess the role of this polymorphism in the
susceptibility to mucosal and systemic Candida albicans infections. To achieve this
aim we have investigated the effect of this SNP in several cohorts of patients:
-142 Dutch patients undergoing hematopoietic stem cells transplantation (HSCT).
-331 Dutch and American candidemia patients and 351 non infected matched
controls.
Functional studies have shown that cells isolated from individuals heterozygous for
the 238X allele had a lower cytokine production upon stimulation with Candida. The
HSCT patients bearing the early stop variant of the dectin-1 gene presented a
higher C. albicans oral an gastrointestinal colonization compared to those who were
78

wild type for the polymorphism and required higher amounts of fluconazole in order
to avoid a disseminated invasion of the fungus.
In contrast, no effect if the Tyr238X dectin-1 SNP on the susceptibility to candidemia
and/or the clinical course of the infection was observed.
In conclusion, the dectin-1 signaling pathway is non-redundant in mucosal immunity
to C. albicans. The 238X allele results in a defective IL-6 and IL-17 response, but
normal IFN-gamma, upon stimulation with C. albicans.
A genetic deficiency of beta-glucan recognition has clear effect on the occurrence
of mucosal Candida infections, but a minor impact on susceptibility to candidemia.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
79

C-type lectin receptor signaling in innate anti-fungal
immunity
Jürgen Ruland
Institute for Molecular Immunology, Technical University Munich, Germany
Dectin-1 was the first C-type lectin receptor (CLRs) identified to be important in
innate anti-fungal immunity. This receptor couples to the tyrosine kinase Syk and
thereby triggers its own intra cellular signaling pathways for inflammatory responses
and host defence. Futher CLRs that can activate Syk and which mediate fungal
recognition are Dectin-2 and Mincle. Here we will discuss new insights into the
molecular mechanisms of CLR signaling and how they relate to anti-fungal defence.
80

Candida - epithelial interactions and innate immunity
David L Moyes 1 , Celia Murciano 1 , Oliver Bader 2 , Michael Weig 2 , Gary
Moran 3 , Bernhard Hube 4 and Julian R Naglik 1
1 King’s College London Dental Institute, King’s College London, London, UK; 2 University
Medical Centre Göttingen, Kreuzbergring 57, Germany; 3 Trinity College,Dublin 2, Ireland;
4 Hans Knoell Institute, Jena, Germany
Host mechanisms enabling discrimination between commensal and pathogenic
organisms are critical in mucosal immune defense and homeostasis. Of particular
interest are opportunistic pathogens such Candida albicans, which can act as both
commensal and pathogen. One of the key virulence factors contributing to C.
albicans pathogenicity at mucosal surfaces is hypha formation and recently we
reported a novel mechanism by which oral epithelial cells discriminate between the
yeast and hyphal forms of C. albicans. Discrimination is mediated via the MAPK
pathway and constitutes activation of the MAPK phosphatase 1 (MKP1) and c-Fos
transcription factor and is targeted against the hyphal form of C. albicans.
MAPK/MKP1/c-Fos activation correlates with proinflammatory responses and cell
damage in a dose-dependent manner, but is independent of TLR2, TLR4 and
Dectin-1 and is not triggered by cell wall polysaccharides (mannan, -glucan, chitin).
However, two fundamentally important issues remain unknown: the identity of the
hyphal moiety targeted by epithelial cells and the epithelial receptor that activates
the discriminatory pathway. To identify the hyphal moiety we screened a panel of
cell wall- and morphology-related C. albicans mutants and assayed for MKP1/c-
Fos activation, induction of cytokines and cell damage. This screen identified a
potential candidate hypha-specific protein that activates the epithelial response.
The respective null mutant remained in the hyphal phase but was unable to activate
MKP1/c-Fos, cytokines or cell damage. To verify this, the hyphal protein was
purified and was shown to directly activate c-Fos, cytokines and cell damage in a
dose-dependent manner. Our data indicate that epithelial cells target this hyphal
protein to initiate innate immunity against C. albicans and we propose that this
epithelial MAPK/c-Fos pathway represents a ‘danger response’ mechanism
informing the host of potentially dangerous levels of C. albicans hyphae, which may
be critical in identifying when this normally opportunistic fungus has become
pathogenic.
82

Candida albicans Secreted Aspartic Proteases Cause
Inflammatory Response Irrespective of Their Proteolytic
Activity
Neelam Pandey 1 , Donatella Pietrella 1 , Lydia Schild 2 , Francesco Bistoni 1 ,
Bernhard Hube 2 and Anna Vecchiarelli 1
1 Department of Experimental Medicine and Biochemical Science, University of Perugia,
Perugia, Italy; 2 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for
Natural Product Research and Infection Biology, Hans Knoell Institute, Jena, Germany
Candida albicans normally inhabits humans as a commensal, but causes fatal infections
when the immune system of patients is suppressed. It has a strong armory of virulence
attributes which facilitates bypassing the immune system and causes mucosal,
cutaneous and systemic infections. These attributes are the ability to colonize, invade
the host tissue and secrete aspartic proteases. We here report that recombinant secreted
aspartic proteases (rSaps) including rSap1, rSap2, rSap3 and rSap6 induce inflammatory
cytokine production by human monocytes to different degrees. rSap1, rSap2 and rSap6
induced the secretion of IL-1beta, TNF-alpha and IL-6 significantly, while rSap3
stimulated the secretion of IL-1beta and TNF-alpha. All these rSaps induced Ca 2+ influx
in monocytes. Pepstatin A, a potential inhibitor of aspartic proteases, has no effect on
the cytokine secretion induced by these rSaps. This suggests that the inflammatory
response due to rSaps is not because of their proteolytic activity, a hypothesis further
supported by the observation that the ability of rSaps to induce inflammatory cytokine
secretion was independent of protease-activated receptor (PAR) activation, and of the
optimal pH for individual rSap activity. Moreover, rSaps stimulated Akt activation and
thus induced IkB-alpha phosphorylation which mediated translocation of NFkB into the
nucleus in human monocytes. These findings give evidence that rSap1, rSap2 and rSap3
cause an inflammatory response irrespective of their proteolytic activity and mediate the
inflammatory response via activation of Akt/NF-kB. The inflammatory response
terminates with inflammasome activation. This occurs through a process requiring Sap
internalization via a clathrin dependent mechanism, and caspase-1 activation, and it is
mediated by K + efflux and ROS production. By demonstrating that the recognized
virulence factors of C. albicans such as Sap2 and Sap6 are potent inducers of
inflammasome activation, our results represent a step forward in the comprehension of
the close relationship between fungus virulence and host response mechanisms.
We are extending our study by using an in vivo experimental model of murine candidiasis
to analyze the role of Sap2 and Sap6 in the inflammatory response in the vaginal
candidiasis, and to investigate whether specific mAbs are indeed able to affect this
process.
This study was funded by the Eurpoean Commission through the FINSysB Marie Curie Initial Training
Network (PITN-GA-2008-214004).
84

A central role of complement for the Candida albicansinduced
cytokine production by human cells
Shih-Chin Cheng 1,2 , Tom Sprong 1,2 , Leo A.B. Joosten 1,2 , Jos W.M. van der
Meer 1,2 , Bart-Jan Kullberg 1,2 , Bernhard Hube 3,4 , Lone Schejbel 5 , Peter
Garred 5 Marcel van Deuren 1,2 , Mihai G. Netea 1,2
1 Departments of Medicine, Radboud University Nijmegen Medical Center, Nijmegen, the
Netherlands; 2 Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Nijmegen,
the Netherlands; 3 Department of Microbial Pathogenicity Mechanism, Leibniz Institute for
Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany;
4 Friedrich Schiller University, Jena, Germany; 5 Department of Clinical Immunology, Laboratory
of Molecular Medicine, Rigshospitalet, Copenhagen, Denmark.
In experimental studies, the role of complement for antifungal host defense has
been attributed to its ability of opsonization. In this study we report that in humans
an activated complement system mainly augments Candida albicans-induced host
pro-inflammatory cytokine production via C5a-C5a receptor signaling, while
Candida phagocytosis and intracellular killing is not influenced. By blocking the
C5a-C5aR signaling pathway either with anti-C5a antagonist antibodies or with the
C5aR antagonist W-54001, C. albicans-induced IL-6 and IL-1 levels were
significantly reduced. Recombinant C5a augmented cytokine production. In
addition, using serum from patients with various complement deficiencies, we
demonstrate a crucial role of C5, but not C6 or the membrane attack complex, for
C. albicans-induced IL-6 and IL-1 in monocytes. These findings reveal a central
role of anaphylatoxin C5a in augmenting host pro-inflammatory cytokine production
upon contact with C. albicans, and defines the role of the complement system for
anti-Candida host defense in humans.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
86

The role of pH-regulated antigen 1 of Candida albicans in
the interaction of the fungus with human neutrophils
Eliška Svobodová 1 , Josephine Losse 1 , Antje Heyken 2 , Bernhard Hube 2,4 ,
Peter F. Zipfel 3,4 and Mihály Józsi 1
1 Junior Research Group Cellular Immunobiology, 2 Department of Microbial Pathogenicity
Mechanisms and 3 Department of Infection Biology, Leibniz Institute for Natural Product
Research and Infection Biology - Hans Knöll Institute, Jena, Germany; 4 Friedrich Schiller
University, Jena, Germany
Candida albicans is an opportunistic human-pathogenic yeast and a leading cause
of life-threatening systemic fungal infections in immunocompromised individuals.
The virulence of the fungus is associated with its capability to filamentous growth.
The pH-regulated antigen 1 (Pra1) of C. albicans is a surface-associated and
secreted protein whose expression is upregulated in the hyphal form. Pra1 binds
to complement receptor 3 (CD11b/CD18) and can mediate adhesion to and
migration of human phagocytes. Here, we investigated the role of Pra1 in the
activation of human neutrophils. A C. albicans mutant strain lacking Pra1 (pra1 )
supported neutrophil migration to a lower extent than did the parental wild-type
strain. A Pra1-overexpressing C. albicans strain enhanced neutrophil migration and
adherence. While inactivated hyphae of the Pra1-overexpressing mutant enhanced
the production and release of reactive oxygen species, myeloperoxidase,
lactoferrin, and interleukin 8 by neutrophils, such responses were reduced when
stimulated with the pra1 strain. Pra1-overexpressing living hyphae also caused a
reduced neutrophil activation, indicating that fungal cells which express and release
more Pra1 can more efficiently inhibit the activation of these innate immune cells.
Fungal cells lacking Pra1 were more efficiently killed by neutrophils. The lack or the
overexpression of Pra1 did not significantly affect the generation of neutrophil
extracellular traps. In conclusion, surface-exposed Pra1 plays a role in the
recognition of C. albicans, especially hyphal cells, by human neutrophils and
enhances neutrophil antimicrobial responses. However, the fungus can counteract
some of these defense mechanisms likely by releasing Pra1.
88

Vaccines against opportunistic fungal infections
Antonio Cassone
Department of Infectious, Parasitic and Immuno-mediated Diseases, Istituto Superiore di
Sanità, Rome, Italy.
The rising threat represented by opportunistic fungal infections, the scarcity/high
cost of effective antifungal treatments in the setting of the immunocompromised
host, the advances in the knowledge of fungal pathogenicity and immune responses
are the main driving factors to recent studies aimed at generating active and passive
vaccination tools against human pathogenic fungi. Two vaccines, one specifically
designed to fight chronic recurrent mucosal candidiasis and another one for more
general anti-Candida prevention/therapy, are in clinical trials in Europe and US.
A third vaccine, made up by an algal -glucan (laminarin) conjugated with a protein
component (Lam-CRM vaccine) has been formulated to enter clinical investigation.
This vaccine has been shown to protect against experimental infections by the three
major opportunistic fungi such as Candida albicans, Aspergillus fumigatus and
Cryptococcus neoformans and has been therefore designated as pan-fungal or
“universal” vaccine. Protection appears to be largely if not exclusively mediated by
anti- -glucan antibodies with restricted specificity to 1-3 linked epitope and
capable to inhibit growth and virulence-associated fungal factors. Protective anti-
1-3 glucan monoclonal murine and human-chimeric antibodies have also been
generated thus opening perspectives for passive vaccination in
immunocompromized subjects.
Our research demonstrates the possibility of conveying into a single immunological
tool the potential to protect against multiple fungal infections. The above approach
could be theoretically extended to non-fungal infections by selecting the
appropriate molecular pattern shared by a given microbial group (e.g. peptidoglycan
for Gram-positive bacteria). Noteworthy, the molecular patterns are those highly
conserved pathogen-associated molecules which foster innate immunity through
their binding to the pattern-recognition structures and intracellular signalling, hence
shaping adaptive immune responses. Single-component, molecular pattern-based
vaccines would merge the broad target range typical of innate immunity with the
highly focussed specificity of the adaptive immunity.
90

Tackling the Candida spp infection spectrum with first-inclass
antimicrobial peptide technology
Dr Deborah A O’Neil
NovaBiotics Ltd, Cruickshank Building, Aberdeen, AB21 9TR, UK
Applying a biology-led, rational drug design approach, NovaBiotics has developed
patented antimicrobial peptide technology from which Novamycin ® , a potently
fungicidal API has been derived. Novamycin ® has a novel, membranolytic mode of
action against which acquired resistance cannot develop in target fungal pathogens
and is equally active against metabolically active and non-metabolising fungi.
Novamycin ® is active against all Candida spp, Cryptococcus spp and limited
Aspergillus spp. Novamycin ® demonstrates efficacy and is well tolerated in wellestablished,
clinically relevant in vivo models of Candida spp sepsis (neutropenic
and immunocompetant models), oral pharyngeal candidiasis and vaginal
candidiasis. Novamycin ® will enter the clinical phase of development in the very
near future for topical and mucosal indications whilst in parallel, formulation and
delivery for systemic application in sepsis is further optimised. Novamycin ® offers
a novel first-in-class API solution not only to the whole spectrum of Candida spp.
infections, but to those caused by other clinically relevant yeasts and moulds.
92

Wall proteins, absolute quantification and concatenated
vaccines: A new approach for Candida albicans therapy?
Clemens J. Heilmann, Alice G. Sorgo, Henk L. Dekker, Stanley Brul,
Chris G. de Koster, Leo J. de Koning and Frans M. Klis
Swammerdam Institute for Life Sciences, Universiteit van Amsterdam, Science Park 904,
1098XH Amsterdam
The cell wall and especially the proteins that are covalently anchored to it are the
first site of host-pathogen interaction. The wall proteins are promising targets for
the development of diagnostic biomarkers and vaccines. Previously, we described
the relative quantitative changes in up to 21 wall proteins during the yeast-to-hypha
transition (1) and upon exposure of cells to fluconazole (2). We identified hyphaassociated
wall proteins (Als3, Hwp2, Hyr1, Plb5, Sod5) conferring increased
adhesion and resistance to host defenses and yeast-associated proteins (Rhd3,
Sod4, Ywp1), which probably play a role in host dispersal and immune evasion.
Morphotype-independent proteins (Cht2, Crh11, Ecm33, Mp65) are mainly involved
in wall structure and remodeling.
However, relative quantification can only provide indications of how protein
abundance changes in response to an environmental change. To compare the
abundances of different proteins, absolute quantification is required. We are
developing a strategy for absolute quantification based on 15 N metabolic labeling
and concatenated proteins (Q-CON-CAT). By using the same 15 N reference culture
that was also used for relative quantification and determining the absolute amount
of a particular protein, we will be able to recalculate the relative quantification results
into absolute values.
These absolute values will be informative for the construction of peptide-based
vaccines. We propose that by concatenating peptides that are predicted to be
highly immunogenic and originate from abundant wall proteins an effective Q-CON-
CAT vaccine protein can be generated.
(1) Hyphal Induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein
profile. Heilmann CJ, Sorgo AG, Siliakus AR, Dekker HL, Brul S, de Koster CG, de Koning LJ, Klis FM.
Microbiology. 2011 May 20.
(2) The effects of fluconazole on the secretome, the wall proteome and wall integrity of the clinical fungus
Candida albicans. Sorgo AG, Heilmann CJ, Dekker HL, Bekker M, Brul S, de Koster CG, de Koning LJ,
Klis FM.Eukaryot Cell. 2011 May 27.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
94

scFv Phage Display Library against Complex
Immunomodulatory Glycans
Abhishek Saxena 1 , Soumya Palliyil 1,2 , Keith A. Charlton 3 , Alistair J.P.
Brown 1 & Andrew J.R. Porter 1,2,3
1 Department of Molecular and Cell Biology, Institute of Medical Sciences, Aberdeen Medical
School, University of Aberdeen, Aberdeen AB25 2ZD, UK; 2 Scottish Biologics Facility,
University of Aberdeen, Aberdeen AB25 2ZD, UK; 3 ImmunoSolv Ltd, Liberty Research centre,
University of Aberdeen, Aberdeen AB25 2ZP, UK
The concept of Antibody therapy against infectious diseases is still in its infancy.
Numerous studies in last two decades have focused on anti-glycan antibodies that
recognize fungal cell surface. The presence of these antibodies in blood has been
used to diagnose fungal infection. Candida albicans cell wall -glucan and -mannan
are very well established diagnostic and therapeutic targets because they represent
the most abundant cell surface epitopes responsible for generation of unique
antibodies against fungus. We have constructed a scFv M13 phage display library
against complex glycans of Candida albicans. To generate an antibody response
and construct a library, a Welch breed/Suffolk sheep was hyper-immunized (IgG
titres > 60,000) against a Candida albicans hyphal cell wall preparation. Binding
analysis of crude/purified polyclonal serum IgG indicates that cell wall glycans are
indeed primary targets because they dominate the immune response of the sheep.
We further demonstrated that polyclonal IgG has in vitro hyphae neutralization
potential in growth inhibition assay. A scFv library has been constructed by cloning
the immunoglobulin variable gene repertoire of peripheral blood lymphocytes
isolated from this hyper-immune animal. Variable heavy and variable light gene
segments of immunoglobulins were PCR amplified and linked through a cellulase
linker. Linked scFv segments were cloned into the phagemid vector pHEN2a. The
size of the VH-V and VH-V library in E.coli TG1 cells was 3.8 x 10 10 and 1.1 x 10 8
respectively. Repertoire diversity was confirmed by sequencing random clones
(approximately 45 clones) and CDRs of VH and V /V genes were found to be 100%
different. We have a panel of early monoclonal hits against both CA -glucan and
-mannan antigens. We are currently interested in pulling monoclonal phages from
library against variety of immunomodulatory glycans.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
96

Essential Genes as Broad-Spectrum Antifungal Targets
K S Dobb 1 , M Birch 1 , M J Bromley 2 , S Kaye 1 , L Stateva 3 , J D Oliver 1
1 F2G Ltd, Lankro Way, Manchester, UK. 2 Manchester Academic Health Centre,
The University of Manchester UK. 3 Faculty of Life Sciences, The University of
Manchester, UK
Aspergillus fumigatus and Candida albicans both cause serious, systemic disease
in vulnerable patients. Current antifungal drugs have many limitations and there is
a need for new classes of antifungal drugs. This study aims to investigate genes
essential for the growth of A. fumigatus and C. albicans as antifungal targets.
In silico analysis of a set of essential genes from A. fumigatus revealed those with
C. albicans homologs. To assess selectivity those with human homologs were also
identified. A subset of the A. fumigatus essential gene set was chosen for
essentiality studies in C. albicans.
One gene found to be essential in both A. fumigatus and C. albicans was
Phosphopantetheinyl transferase b (PPTb). This gene is involved in mitochondrial
fatty acid synthesis and catalyses the transfer of a phosphopantetheine group from
coenzyme A (CoA) to acyl carrier protein (ACP). To identify inhibitors of Pptb a
fluorescence polarisation (FP) assay was developed. The transfer of fluorophore
labelled phosphopantetheine group from CoA to ACP can be measured by a
change in FP value. As Pptb catalyses this transfer inhibition of this enzyme can
also be detected by a change in FP values. A statistically robust FP assay was
achieved using C. albicans ACP and proteins and fluorescently labelled CoA. Single
concentration and IC50 screening of a small molecule compound library has
identified inhibitors of Pptb. Other work on these “hits” has included antifungal
susceptibility testing (minimum inhibitory concentration, MIC) and mammalian cell
toxicology testing (MCTs). PPTb has so far shown good potential as a broadspectrum
antifungal target and future work will aim to further characterise these
small molecule inhibitors for their potential as drug leads.
98

Hyphal development in Candida albicans requires two
temporally linked changes in promoter chromatin for
initiation and maintenance
Yang Lu, Chang Su, Allen Wang and Haoping Liu
Department of Biological Chemistry, University of California, Irvine, CA 92697, USA.
Phenotypic plasticity is common in development. For Candida albicans, the most
common cause of invasive fungal infections in humans, morphological plasticity is
its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens
that exist primarily in either yeast or hyphal forms, C. albicans is able to switch
reversibly between yeast and hyphal growth forms in response to environmental
cues. Although many regulators have been found involved in hyphal development,
the mechanisms of regulating hyphal development and plasticity of dimorphism
remain unclear. Here we show that hyphal development involves two sequential
regulations of the promoter chromatin of hypha-specific genes. Initiation requires a
rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal
morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires
promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of
rapamycin) signalling. Hda1 deacetylates a subunit of the NuA4 histone
acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module
and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter
recruitment of Hda1 for hyphal maintenance happens only during the period when
Nrg1 is gone. The sequential regulation of hyphal development by the activation of
the cAMP-PKA pathway and reduced Tor1 signalling provides a molecular
mechanism for plasticity of dimorphism and how C. albicans adapts to the varied
host environments in pathogenesis. Such temporally linked regulation of promoter
chromatin by different signalling pathways provides a unique mechanism for
integrating multiple signals during development and cell fate specification.
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101

The transcription factor Sko1 represses the yeast-tohypha
transition and mediates the oxidative stress
response in Candida albicans.
Inês Correia, Elvira Román, David M. Arana, Daniel Prieto, Verónica
Urrialde, César Nombela, Rebeca Alonso-Monge and Jesús Pla. §
Departamento de Microbiología II. Facultad de Farmacia. Universidad Complutense de
Madrid. Plaza de Ramón y Cajal s/n. E-28040 MADRID. SPAIN
Cells adapt to external changes by triggering transcriptional responses. These
responses are complex and are mediated by different transcription factors. Mutants
lacking the transcription factor Sko1 were generated in Candida albicans and its
roles on the physiology of this opportunistic pathogen are reported. Genome-wide
transcriptional analysis revealed that Sko1 acts as a transcriptional repressor of
genes involved in pathogenesis and hyphal formation. The sko1 mutant displayed
an increased expression of the hyphal related genes ECE1 and HWP1 but showed
no drastic alterations in the mouse model of systemic infection. Deletion of SKO1
rendered mutants derepressed in the yeast-to-hypha transition. Moreover, Sko1
was shown to be involved in the response to oxidative stress and sko1 mutants
increased the sensitivity of hog1 mutants to the myelomonocytic cell line HL-60.
Alterations in the phosphorylation pattern of MAP kinase mutants in response to
oxidative challenge were also observed. Genome-wide transcriptional analysis after
hydrogen peroxide treatment revealed that sko1 mutants were able to generate an
adaptive response similar to wild type strains, although important differences were
detected in the magnitude of such transcriptional response. Collectively, these
results implicate Sko1 as a key mediator of the oxidative stress response and
morphological transition in C. albicans.
102

103

Calcineurin Signaling and Membrane Lipid Homeostasis
Regulates Iron Mediated MultiDrug Resistance Mechanisms
in Candida albicans
Saif Hameed, Sanjiveeni Dhamgaye, Ashutosh Singh, Shyamal K. Goswami
and Rajendra Prasad
School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
We previously demonstrated that iron deprivation enhances drug susceptibility of
Candida albicans by increasing membrane fluidity which correlated with the lower
expression of ERG11 transcript and ergosterol levels. The iron restriction dependent
membrane perturbations led to an increase in passive diffusion and drug
susceptibility. The mechanisms underlying iron homeostasis and multidrug
resistance (MDR), however, are not yet resolved. To evaluate the potential
mechanisms, we used whole genome transcriptome and electrospray ionization
tandem mass spectrometry (ESI-MS/MS) based lipidome analyses of iron deprived
Candida cells to examine the new cellular circuitry of the MDR of this pathogen.
Our transcriptome data revealed a link between calcineurin signaling and iron
homeostasis. Among the several categories of iron deprivation responsive genes,
the down regulation of calcineurin signaling genes including HSP90, CMP1 and
CRZ1 was noteworthy. Interestingly, iron deprived Candida cells as well as iron
acquisition defective mutants phenocopied molecular chaperone HSP90 and
calcineurin mutants and thus were sensitive to alkaline pH, salinity and membrane
perturbations. In contrast, sensitivity to above stresses did not change in iron
deprived DSY2146 strain with a hyperactive allele of calcineurin. Although, iron
deprivation phenocopied compromised HSP90 and calcineurin, it was independent
of protein kinase C signaling cascade. Notably, the phenotypes associated with
iron deprivation in genetically impaired calcineurin and HSP90 could be reversed
with iron supplementation. The observed down regulation of ergosterol (ERG1,
ERG2, ERG11 and ERG25) and sphingolipid biosynthesis (AUR1 and SCS7) genes
followed by lipidome analysis confirmed that iron deprivation not only disrupted
ergosterol biosynthesis, but it also affected sphingolipid homeostasis in Candida
cells. These lipid compositional changes suggested extensive remodeling of the
membranes in iron deprived Candida cells. Taken together, our data provide the
first novel insight into the intricate relationship between cellular iron, calcineurin
signaling, membrane lipid homeostasis and drug susceptibility of Candida cells.
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105

Functional analysis of H-loop residues of Cdr1p, an ABC
transporter of human fungal pathogen Candida albicans
Antresh Kumar 1 and Rajendra Prasad 1
1 School of Life Sciences, Jawaharlal Nehru University, New Delhi, INDIA
Nucleotide Binding Domains (NBDs) of multidrug transporter of Candida albicans,
Cdr1p possess unique divergent amino acids in their conserved motifs. For
example, NBD1 possesses divergent H-loop (IYQ) while the same motif is
conserved (IHQ) in NBD2.
We report here the contribution of these conserved and divergent H-loop motifs of
Cdr1p in ATP catalysis and drug transport. For this, we mutagenized two residues
of divergent (IYQ) and conserved H-loop region (IHQ) either by replacing them with
alanines or replacing residues with equiposition residues of another H-loop. These
mutations were introduced into GFP-tagged Cdr1p which was stably overexpressed
at the PDR5 locus in a heterologous host S. cerevisiae mutant strain, AD1-8u - which
lacks seven major ABC transporters.
All the Cdr1p mutant variants of H-loop region were properly expressed and
localized to the cell surface similar to wild-type protein. Alanine mutants of H-loop
region, Y361A in NBD1 has no effect on Cdr1p function and showed normal R6G
transport and ATPase activity whereas corresponding mutation (H1059A) in NBD2
abolished R6G transport without any significant loss of ATPase activity. Moreover,
cells expressing mutation like Q362A severely abrogated Cdr1p function while
Q1060A mutation in NBD2 showed no effect on Cdr1p function and measured
normal transport and ATPase activity similar to wild type. Though, Arginine
substitution of glutamine residue present in H-loop region of Cdr1p (Q362R and
Q1060R) displayed deferential effect on Cdr1 transport. These results suggest that
not only the conserved residue like H1059 but divergent Q362 is also crucial for
Cdr1p function.
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107

108

POSTER ABSTRACTS
109

110

The Pathogenic Armoury
111

Poster number: 01
Glucose promotes oxidative stress resistance in Candida
albicans via specific signalling pathways
Iryna Bohovych 1 , Pedro Miramón 2 , Aaron Mitchell 3 , Bernhard Hube 2 ,
Alistair JP Brown 1
1 Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, UK; 2 Microbial Pathogenicity, Hans Knoell Institute, Beutenbergstraße
11a, Jena 07745, Germany; 3 Department of Biological Sciences, Carnegie Mellon University,
Pittsburgh, Pennsylvania 15213, USA
Candida albicans, a successful human pathogen, displays the phenomenon of
glucose-enhanced oxidative stress resistance (Rodaki et al., 2009), which is not
observed in other yeast species tested. The molecular mechanisms that mediate
glucose-enhanced oxidative stress resistance in C. albicans are not clear. We
reasoned that glucose signalling might play a major role. Therefore we tested the
impact of specific C. albicans mutations to determine which of known signalling
pathways are required for glucose-enhanced oxidative stress resistance.
The Sugar Receptor-Repressor (Hgt4-Rgt1) pathway, homologous to the Rgt2/Snf3
pathway in S. cerevisiae, is not required for the phenotype. In contrast, a snf1
mutant, which lacks a key component of the Glucose Repression Pathway,
displayed high oxidative stress resistance on lactate, not altered by glucose. A kis1
mutant, lacking one of the subunits of Snf1 complex, displayed a related phenotype.
The data indicate that in the absence of glucose the Glucose Repression Pathway
represses glucose-enhanced oxidative stress resistance.
cAMP signalling also appears to play a role in glucose-enhanced oxidative stress
resistance. This phenotype was repressed by exogenous dibutyryl cAMP and by a
pde2 mutation that is thought to increase intracellular cAMP levels. Also the
phenotype was enhanced by inactivation of adenylyl cyclase (cyr1).
Having shown that both cAMP signalling and the Glucose Repression Pathway play
important roles in mediating glucose-enhanced stress resistance, the next step was
to identify targets of these pathways that might contribute to the phenotype.
Comparative analyses of transcriptomic profiles of C. albicans glucose- and lactategrown
cells in response to oxidative stress and glucose treatment correspondingly
revealed a small set of commonly up-regu lated genes (Enjalbert et al., 2006; Rodaki
et al., 2009). The impact of the cAMP and Glucose Repression pathways on these
genes is being determined.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
112

113

Poster number: 02
The Osmotic Stress Response of Candida glabrata
Emily Cook, Ken Haynes
Exeter University, Biosciences- Geoffrey Pope Building, Stocker Rd. Exeter, EX4 4QD
The osmotic stress response has been characterized in great detail in the model
yeast Saccharomyces cerevisiae however less is known in the opportunistic
pathogen Candida glabrata. We have approached our goal of better defining the
osmotic stress response in C. glabrata by using the current understanding in S.
cerevisiae as a framework.
Multiple differences between the described S. cerevisiae response and C. glabrata
were observed. The overall osmotic stress tolerance of C. glabrata was higher than
that of S. cerevisiae, as demonstrated by growth curves and plating assays. Quite
interestingly, we have observed that while C. glabrata did not grow under the highest
concentration of osmotic stress tested (2M NaCl) most cells remained viable when
transferred to normal growth media. We have measured change in cell size of C.
glabrata and S. cerevisiae and found that while S. cerevisiae decreased in size under
increasing concentrations of NaCl, C. glabrata increased in size by ≈10% under all
tested osmotic stress conditions. We are currently investigating biophysical
differences that could explain these results and their contribution to overall osmotic
stress tolerance. Glycerol production was found to be Hog1 independent as
comparable glycerol levels were measured by enzymatic assay in both wild-type
C. glabrata and a hog1 mutant. Differences in osmo-tolerance of single null mutants
in the Hog1 pathway suggest that a Hog1 independent response may be more
important in driving osmotic stress adaptation.
The osmotic stress response in C. glabrata is markedly different than S. cerevisiae,
highlighting the importance of further characterization of stress responses in
pathogenic species, even when closely related to model organisms. We are further
examining the novel mechanisms of osmotic stress adaptation in C. glabrata and
their potential contributions to pathogenicity.
114

115

Poster number: 03
The transcription factor Sko1 represses the yeast-tohypha
transition and mediates the oxidative stress
response in Candida albicans.
Inês Correia, Elvira Román, David M. Arana, Daniel Prieto, Verónica
Urrialde, César Nombela, Rebeca Alonso-Monge and Jesús Pla. §
Departamento de Microbiología II. Facultad de Farmacia. Universidad Complutense de
Madrid. Plaza de Ramón y Cajal s/n. E-28040 MADRID. SPAIN
Cells adapt to external changes by triggering transcriptional responses. These
responses are complex and are mediated by different transcription factors. Mutants
lacking the transcription factor Sko1 were generated in Candida albicans and its
roles on the physiology of this opportunistic pathogen are reported. Genome-wide
transcriptional analysis revealed that Sko1 acts as a transcriptional repressor of
genes involved in pathogenesis and hyphal formation. The sko1 mutant displayed
an increased expression of the hyphal related genes ECE1 and HWP1 but showed
no drastic alterations in the mouse model of systemic infection. Deletion of SKO1
rendered mutants derepressed in the yeast-to-hypha transition. Moreover, Sko1
was shown to be involved in the response to oxidative stress and sko1 mutants
increased the sensitivity of hog1 mutants to the myelomonocytic cell line HL-60.
Alterations in the phosphorylation pattern of MAP kinase mutants in response to
oxidative challenge were also observed. Genome-wide transcriptional analysis after
hydrogen peroxide treatment revealed that sko1 mutants were able to generate an
adaptive response similar to wild type strains, although important differences were
detected in the magnitude of such transcriptional response. Collectively, these
results implicate Sko1 as a key mediator of the oxidative stress response and
morphological transition in C. albicans.
116

117

Poster number: 04
Co-infection of an in vitro oral epithelium by Candida
glabrata and Candida albicans
Sónia Silva 1 , Mariana Henriques 1 , Rosário Oliveira 1 , David W Williams 2 ,
Joana Azeredo 1
1 Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar
4710-057, Braga, Portugal; 2 Tissue Engineering and Reparative Dentistry, School of Dentistry,
Cardiff University, Heath Park, Cardiff, CF14 4XY, UK
Candida albicans is regarded as the leading of cause of candidosis. However,
Candida glabrata has emerged as an important pathogen of oral mucosa, occurring
both singly or in mixed species infections, often with C. albicans. Compared with
C. albicans, little is known about the role of C. glabrata in oral infection. Thus, this
work aimed to examine single and mixed species infection of oral epithelium
involving C. glabrata, and to establish its ability to colonize, invade and damage an
oral epithelium.
A reconstituted human oral epithelium (RHOE) was infected with C. glabrata (n=7),
or with each C. glabrata plus one C. albicans oral isolate. The ability of both species
to colonize and invade an oral epithelium was examined by confocal laser scanning
electron microscopy (CLSM) using species-specific peptide nucleic acid (PNA)
probe for distinguishing C. glabrata of C. albicans cells. Qualitative oral epithelial
damage was assessed by determination of lactate dehydrogenase (LDH) released
by the oral epithelium cells.
This work revealed that all C. glabrata strains were able to colonise the RHOE,
however, in a strain dependent manner. Candida glabrata single infection after 12
h, revealed generally no invasion of the RHOE, which contrasted with extensive
tissue invasion demonstrated by C. albicans. Mixed infection showed that C.
albicans enhanced the invasiveness of C. glabrata, and led to increased LDH release
by the RHOE, which confirms the results observed by histological studies.
Summarizing, the results suggest that there is an enhanced invasion and increased
tissue damage caused by mixed C. glabrata and C. albicans infections, which has
important clinical significance and highlights the need to identify Candida species
involved in oral candidosis.
118

119

Poster number: 05
The N-terminal part of Als1 protein from Candida albicans
specifically binds fucose-containing glycans
Dagmara S. Donohue, Francesco S. Ielasi, Katty V. Y. Goossens and
Ronnie G. Willaert
Laboratory of Structural Biology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels,
Belgium.
The opportunistic pathogen Candida albicans expresses on its surface Als
(Agglutinin like sequence) proteins, which play an important role in the adhesion to
host cells and in the development of candidiasis. The binding specificity of these
proteins is broad, as they can bind to various mammalian proteins,such as
extracellular matrix proteins, and N- and E-cadherins. The N-terminal part of Als
proteins constitutes the substrate-specific binding domain and is responsible for
attachment to epithelial and endothelial cells. We have used glycan array screening
to identify possible glycan receptors for the binding domain of Als1p-N. Under those
conditions, Als1p-N binds specifically to fucose-containing glycans, which adds a
lectin function to the functional diversity of the Als1 protein. The binding between
Als1p-N and BSA-fucose glycoconjugate was quantitatively characterized using
surface plasmon resonance, which demonstrated a weak millimolar affinity between
Als1p-N and fucose. Furthermore, we have also quantified the affinity of Als1p-N
to the extracellular matrix proteins proteins fibronectin and laminin, which is situated
in the micromolar range. Surface plasmon resonance characterization of Als1p-N–
Als1p-N interaction was in the micromolar affinity range.
120

121

Poster number: 06
Impact of non-fermentative growth on the C. albicans cell
wall and dynamic responses to osmotic stress
Iuliana V. Ene, Alistair J. P. Brown
Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, UK
Candida albicans displays considerable metabolic flexibility and robust stress
responses in vivo. Both contribute to the virulence of this pathogen. C. albicans
assimilates both fermentative and non-fermentative carbon sources in vivo, often
occupying poor glucose niches in its mammalian host. We reasoned that changes
in carbon source might influence stress adaptation, but stress responses are
generally studied during growth on glucose.
We found that the C. albicans cell wall is extremely dynamic and susceptible to
variations in carbon source. Growth on lactate rather than glucose has a major
impact on cell wall structure and flexibility. Although the relative proportions of
chitin, -glucan and mannan are similar within the wall of lactate-grown cells, they
display increased porosity and decreased cell wall biomass. TEM confirmed major
differences in cell wall architecture, underlying the importance of the carbon source
in the cell biology of this pathogen. This in turn influences the responses of this
pathogen to stresses as these differences were reflected in the cell wall-related
phenotypes. Lactate-grown cells displayed higher resistance to Congo Red and
Calcofluor White and showed significantly increased resistance to various doses
of osmotic stress. This change was independent of Hog1 and Mkc1, but correlated
with large changes in cell volume following exposure to NaCl. Growth on lactate
also increased resistance to antifungals such as Caspofungin, Tunicamycin and
Amphotericin B, which is likely to have a major impact upon the behavior of C.
albicans during infection.
The impact of carbon source on stress and drug resistance was confirmed in other
pathogenic Candida species and for other carbon sources, resulting in variations
in resistance when compared to a glucose-grown control. Clearly carbon source
strongly influences cell wall architecture and the resistance of C. albicans to certain
stresses, which is likely to have a major impact on its in vivo behavior.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial
Training Network (PITN-GA-2008-214004).
122

123

Poster number: 07
Candida albicans apoptotic markers during the interaction
with macrophages
A. Gil-Bona 1 , L. Monteoliva 1 , V. Cabezón 2 , M. Ramsdale 2 , C. Gil 1 .
1 2 Facultad de Farmacia. Universidad Complutense de Madrid. School of Biosciences,
University of Exeter, UK
Candida albicans is a dimorphic fungus member of the human microbiota that
causes a range of opportunistic infections from superficial to systemic. The last one
remains as a high rate of morbidity-mortality in patients from intensive care units,
surgery or cancer treatment. This is related with the problems of the few available
drugs, as the appearance of antifungal-drug resistance, and with the diagnostic
limitations.
The host-pathogen interaction is being studied using an in vitro interaction model
between Candida albicans and murine macrophages to analyze the fungus answer.
Previous proteomics and genomics studies of C. albicans upon interaction with
macrophages allowed us to detect the fungus response to adapt to the new
environment. This response includes the expression of several genes and proteins
related to the regulation of apoptosis (Fernández-Arenas et al., 2007).
In order to verify the apoptotic death of C. albicans cells in the interaction with
macrophages, several apoptotic markers have been analyzed after host-pathogen
interaction; namely, caspase-like enzymatic activity, generation of intracellular
reactive oxygen species (ROS), phosphatidyl-serine externalization and cell viability.
Also, TUNEL (TdT-mediated X-Dutp Nick end labelling) assays and transmission
electron microscopy (TEM) studies have been made.
These analyses have been carried out at different incubation times: 3h, 6h, 8h, 12h,
14h, 16h, 18h, 20h and 24 hours. The results showed that after 6 hours of C.
albicans-macrophage interaction, ROS and active caspase are present in the cell,
positive TUNEL cells were detected after 8 hours of interaction and the viability of
the yeast at these times is not compromised. Furthermore, chromatin condensation
and nuclear fragmentation were observed by TEM after 12 hours of interaction.
Data obtained in this work confirm a percentage up to 30% of apoptotic death of
C. albicans upon interaction with macrophages in ratio 1:1.
124

125

Poster number: 08
Histidine kinase Sln1 interferes with mitochondrial
functions of Candida albicans
Rabeay Y.A. Hassan, Anna Buschart, and Ursula Bilitewski*
Helmholtz Centre for Infection Research, Biological Systems Analysis, Inhoffenstr. 7, 38124
Braunschweig, Germany. rabeay.hassan@helmholtz-hzi.de
In the pathogenic fungus Candida albicans, three histidine kinases (HK) have been
identified: Sln1p, Cos1p (also known as Nik1p) and Chk1p. They are involved in the
adaptation to stress, growth, morphogenesis and sensitivity to several antifungal
compounds. Deletion of SLN1 does not affect cell viability in rich media and only
slightly affects the tolerance to osmotic stress.
However, we show for the first time that deletion of the SLN1 perturbs mitochondrial
functions. The oxygen uptake rates of the three HKs deletion mutants, ∆sln1, ∆chk1
and ∆cos1 were measured and a faster rate of oxygen consumption was observed
in sln1 than in the wild-type. This can be explained by the up-regulated expression
of the alternative oxidase AOX2 in sln1, indicating that the alternative respiratory
pathway is activated in addition to the presence of the classical respiratory chain
(CRC). The alternative oxidative pathway is usually activated in response to
inhibitors of the CRC.
Rotenone-treatment of the wild type strain stimulated the production of reactive
oxygen species (ROS) and inhibited the reduction of dichlorophenolindophenol
(DCIP-complex I activity assay). However, rotenone had no or only a small effect
on the ROS production and the inhibition of the DCIP reaction in sln1. These
findings point to a low activity of complex I in sln1. This conclusion was supported
by whole-genome gene expression analysis, which showed a down-regulation of
genes encoding components of the mitochondrial NADH:ubiquinone
oxidoreductase complex (Complex I), namely NAD3, NAD4 and NAD5 in addition
to subunits of cytochrome c oxidase (complex IV) (COX2 and COX5) in sln1.
126

127

Poster number: 09
Phenotypic investigation of virulence factors in Candida
species isolated from different clinical specimens
Alina Maria Holban 1 , Crina Maria Saviuc 1,5 , Alexandru Mihai Grumezescu 2 ,
Coralia Bleotu 1,4 , Otilia Banu 3 , Dan Mihaiescu 2 , Paul Balaure 2 , Mariana
Carmen Chifiriuc 1 , Veronica Lazar 1
1 University of Bucharest, Faculty of Biology, Microbiology University Department; 2 University
Politechnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Organic
Chemistry Department; 3 Institute of Cardiovascular Diseases Prof. C.C. Iliescu, Bucharest;
4 S. Nicolau Institute of Virology, Bucharest; 5 S.C. Biotehnos S.A.
Introduction. The purpose of this study was to investigate the phenotypic expression
of the cell associated and soluble virulence factors in 30 fungal strains recently isolated
from different clinical specimens.
Materials and methods. Fungal strains belonging to Candida genus were isolated
from different clinical specimens (urinary tract isolates, plague secretions, upper airway
secretions) and identified using Vitek II automatic system. Susceptibility to currently
used antifungals was assessed by E-test. The phenotypic study of soluble virulence
factors was performed by specific enzymatic assays of haemolysins, lecithinase,
gelatinase, lipase, DNA-se, amylase and iron chelating agents production. Microbial
adherence capacity to the inert (plastic, catheter sections) and cellular substrata was
performed by microtiter method, Brun Buisson method and modified Cravioto method,
using HeLa cells. Fungal dimorphism was studied by filamentation assay. Statistics—
Data were compiled and analyzed using Microsoft Excel and GraphPad In Stat
software, using One-way Analysis of Variance ANOVA.
Results. All tested strains were susceptible to voriconazole, itraconazole, caspofungin
B, fluconazole, flucytosin and amphotericin B. The tested strains exhibited different
abilities to colonize the plastic/catheter sections as well as the cellular substrate, being
divided in non-adherent, moderately and highly adherent. Concerning the filamentation
assay, the strains were classified in the following descendant order: C. albicans, C.
tropicalis, C. glabrata, C. krusei, C. Famata. The profiles of the soluble virulence factors
have been statistically different, related to the species and clinical origin of the tested
strains. The tested strains abundantly secreted haemolysins, DNA-se, amylase and iron
chelating agents.
Conclusion. The phenotypic investigation of virulence factors production in Candida
strains revealed specific virulence profiles depending on the source of isolation and the
taxonomic affiliation of the tested strains, proving the usefulness of these assays in
predicting the clinical outcome of the fungal infections and in the adjustment of the
antifungal treatment.
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129

Poster number: 10
The Role of Hyphae and Hypha-specific genes in the
pathogenesis of Candida dubliniensis and Candida
albicans
Peter Hyde, Derek Sullivan, Gary Moran, David Coleman.
Oral Biosciences Division, Dublin Dental School and Hospital, Trinity College Dublin, Lincoln
Place, Dublin 2, Ireland
The ability of Candida albicans to cause invasive disease has been well
documented. Candida dubliniensis, the most closely related phylogenetic neighbour
of C. albicans, causes life-threatening infections at a much lower rate,
predominantly in immunocompromised individuals. Both species can grow as true
hyphae, pseudohyphae and yeast cells. Hyphae are typically associated with host
tissue adhesion and invasion. This study aims to characterise the contribution of
general hyphal morphology, in comparison to the expression of hypha-specific cell
wall proteins, to host tissue adhesion and invasion using molecular methods and
top-down “holistic” approaches. By expressing C. albicans-specific hyphaassociated
cell wall proteins in a C. dubliniensis background under the control of a
tetracycline-inducible promoter, the ability of proteins CaALS3p, CaHWP1p, and
CaHWP1p, to cause tissue adhesion and invasion is being measured, independent
of cell morphology. In addition, by priming the C. dubliniensis mutant strains to
produce hyphae at different rates the combined effect of hypha-associated cell wall
proteins and hyphal morphology is being investigated. The ability of a panel of C.
dubliniensis strains, including systemic isolates, to produce hyphae has also been
examined and has shown high levels of inter-species variation. Interestingly, the
highest rates of true hyphae formation have been associated with strains isolated
from systemic infections, suggesting a significant contribution from the filamentous
morphology in the virulence of C. dubliniensis. This study is exploring the basis
behind the relative lack of pathogenicity of C. dubliniensis and further elaborating
on the role of hyphae and hypha-specific proteins as virulence factors in the
pathogenesis of C. albicans.
130

131

Poster number: 11
Mini-chromosomes in Candida glabrata clinical isolates
originate through two different mechanisms
Olena P. Ishchuk 1 , Khadija Mohamed Ahmad 1 , Linda Hellborg 1 , Gloria
Jørgensen 2 , Miha Škvar 1 , Jørgen Stenderup 3 , Dorte Jørck-Ramberg 2 ,
Silvia Polakova 1 and Jure Piškur 1
1 Department of Biology, Lund University, Sölvegatan 35, Lund SE-223 62, Sweden,
Olena.Ishchuk@biol.lu.se; 2 BioCentrum-DTU, Technical University of Denmark, DK-2800
Lyngby, Denmark; 3 Department of Clinical Microbiology, Regionshospitalet Herning,
DK-7400 Herning, Denmark
We analyzed around two hundred strains of the pathogenic yeast Candida glabrata,
collected from immuno-compromised patients in Danish hospitals during 1985 -
1999. In this report we showed that these strains were closely related but exhibited
large karyotype polymorphism. Nine strains contained mini-chromosomes, which
were smaller than 0.4 Mb. These strains had an independent origin, regarding the
year, patient and hospital, and the analyzed mini-chromosomes were structurally
not related to each other (i.e., they contained different sets of genes). We inferred
two mechanisms involved in their origin: (i) through a segmental duplication which
covered the centromeric region, and (ii) by a translocation event moving a larger
chromosome arm to another chromosome thereby leaving the centromere part with
the shorter arm. The first type of mini-chromosomes carrying duplicated genes
exhibited mitotic instability, while the second type, which contained the
corresponding genes in only one copy in the genome, was mitotically stable.
Apparently, in patients C. glabrata chromosomes are frequently reshuffled resulting
in various genetic configurations, including appearance of mini-chromosomes,
which could increase the fitness in certain “environments”.
132

133

Poster number: 12
Functional analysis of C. albicans infection-associated
genes with unknown function during interaction with host
cells and in complex infection models
Ilse D. Jacobsen 1 , Duncan Wilson 1 , Francois Mayer 1 , Bernhard Hube 1,2
1 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product
Research and Infection Biology - Hans-Knöll-Institute, Jena, Germany; 2 Friedrich-Schiller
University, Jena
Candida albicans is able to cause a variety of clinical infections. We hypothesized
that C. albicans unknown function genes, which were transcriptionally upregulated
during oral infections, liver invasion or incubation in human blood (“infection
associated genes”) might contribute to the infection process. We selected a subset
of infection-associated genes with unknown function for further analysis.
Twentyfour isogenic deletion mutants were constructed and subsequently analyzed
both in vitro and in infection models of increasing complexity. Sixteen mutants were
impaired in their ability to damage monolayers of human endothelial and/or
epithelial cells. Within this subset, seven mutants additionally displayed decreased
stress resistance in vitro. Only two mutants showed filamentation defects. To
determine whether the deleted genes influenced virulence in more complex
infections models, all 24 mutants were tested in ovo for their ability to kill chicken
embryos infected on the chorio-allantoic membrane. We recently showed that
mortality in this model depends on the fungal ability to invade the membrane and
that the pro-inflammatory host response likely contributes to pathogenesis.
Surprisingly, only seven mutants were attenuated in this model, of which five were
also attenuated in damaging epithelial cells in vitro. Three of the mutants, which
were attenuated in ovo, and one mutant, which was attenuated in damaging
endothelial and epithelial cells but fully virulent in chicken embryos, were
subsequently analyzed in a systemic mouse model. Virulence in mice mirrored the
results obtained in ovo.
Our results demonstrate that genes essential for causing full damage of epithelial
or endothelial cells in vitro are not necessarily crucial for full virulence in complex
infection models. We conclude that either such damage potential is not critical for
full virulence in the complex host situation or that the greater complexity of the
environment in animal models stimulates additional fungal regulatory networks
leading to compensation of the defect.
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135

Revisiting the Hog Pathway of C. glabrata
136
Poster number: 13
Zeljkica Jandric, Wolfgang Reiter, Gustav Ammerer, Christoph Schüller
Max F. Perutz Laboratories, Department of Biochemistry University of Vienna, Dr.Bohr-
Gasse 9/5, A-1030 Vienna, Austria
Signal transduction networks mediated by mitogen – activated protein kinases
(MAPK) play essential roles in eukaryotic cells. Yeast cells have developed
osmoadaptation pathways to cope with osmotic changes in the microenvironment.
In Saccharomyces cerevisiae five MAPK pathways are well characterized. One of
these, especially associated with stress response, is the High Osmolarity Glycerol
(HOG) pathway . Stress induced phosphorylation triggers the activation of the key
MAPK, Hog1. After being phosphorylated by the upstream MAPKK Pbs2, Hog1
enters the cell nucleus and modulates the transcription of its target genes.
Orthologues of the all HOG pathway compounds found in Saccharomyces
cerevisiae have been identified in fungi and animals.
The human fungal pathogen Candida glabrata is closely related to Saccharomyces
cerevisiae. It is a common commensal but in immunocompromised cancer and
transplant patients or elder persons it can turn into an opportunistic pathogen.
Depending on the host environment, Candida glabrata has to adapt to
environmental changes during invasion and adaptation steps. We investigated the
role of Hog1 in Candida glabrata. Several stress sources were used to identify HOGspecific
phenotypes and to analyze the role of Hog1 in Candida glabrata. As
expected, the deletion of Hog1 caused osmostress hypersensitivity. Physiological
stress conditions, such as nutrients starvation, were simulated by infection of
macrophages with Candida glabrata and determination of the number of surviving
Candida cells. Cghog1∆ cells showed a much higher sensitivity to macrophages
compared to wild type cells. Further investigations focus on stress induced gene
transcription in Cghog1∆ cells and comparison of the transcription pattern to cells
without Hog1 deletion.

137

Poster number: 14
Functional analysis of H-loop residues of Cdr1p, an ABC
transporter of human fungal pathogen Candida albicans.
Antresh Kumar 1 and Rajendra Prasad 1
1 School of Life Sciences, Jawaharlal Nehru University, New Delhi, INDIA
Nucleotide Binding Domains (NBDs) of multidrug transporter of Candida albicans,
Cdr1p possess unique divergent amino acids in their conserved motifs. For
example, NBD1 possesses divergent H-loop (IYQ) while the same motif is
conserved (IHQ) in NBD2.
We report here the contribution of these conserved and divergent H-loop motifs of
Cdr1p in ATP catalysis and drug transport. For this, we mutagenized two residues
of divergent (IYQ) and conserved H-loop region (IHQ) either by replacing them with
alanines or replacing residues with equiposition residues of another H-loop. These
mutations were introduced into GFP-tagged Cdr1p which was stably overexpressed
at the PDR5 locus in a heterologous host S. cerevisiae mutant strain, AD1-8u - which
lacks seven major ABC transporters.
All the Cdr1p mutant variants of H-loop region were properly expressed and
localized to the cell surface similar to wild-type protein. Alanine mutants of H-loop
region, Y361A in NBD1 has no effect on Cdr1p function and showed normal R6G
transport and ATPase activity whereas corresponding mutation (H1059A) in NBD2
abolished R6G transport without any significant loss of ATPase activity. Moreover,
cells expressing mutation like Q362A severely abrogated Cdr1p function while
Q1060A mutation in NBD2 showed no effect on Cdr1p function and measured
normal transport and ATPase activity similar to wild type. Though, Arginine
substitution of glutamine residue present in H-loop region of Cdr1p (Q362R and
Q1060R) displayed deferential effect on Cdr1 transport. These results suggest that
not only the conserved residue like H1059 but divergent Q362 is also crucial for
Cdr1p function.
138

139

Poster number: 15
Target specificity of the Efg1 regulator in Candida albicans
Lassak, T., Lagadec, Q., Kurtz, D., Bussmann, M. and Ernst, J. F.
Heinrich-Heine-Universität Düsseldorf, Institut für Mikrobiologie, Molekulare Mykologie
Efg1 is a general transcription factor, which regulates numerous morphogenetic
and metabolic processes in C. albicans. We previously found that overexpression
of EFG1 and activation of Efg1 during hypha formation leads to downregulation of
EFG1 promoter activity and that EFG1 downregulation is necessary to allow the
formation of true hyphae and prevent pseudohyphal growth.
To explore the Efg1 binding specificity its binding to the EFG1 promoter was
analyzed by EMSA and footprint analyses. These experiments revealed binding of
Efg1 to a promoter sequence close to the transcript start site, which contained a
repeated 8-mer motif (referred to as APSES response element, ARE). Surprisingly,
promoter fusions to the RLUC reporter lacking the ARE motif were still able to
downregulate the EFG1 promoter during hypha formation in an Efg1-dependent
manner. Furthermore, extensive deletions of the 10 kb-large 5 untranslated region
of EFG1 still did not prevent regulation suggesting multiple binding sites for Efg1
within the EFG1 promoter. We subsequently analyzed genome-wide occupation of
chromosomal sequences by Efg1 using ChIP chip analysis. These results revealed
the entire 10 kb EFG1 upstream region as a major site of Efg1 occupancy.
Unexpectedly, Efg1 binding to this region was rapidly lost during hypha induction
suggesting a regulatory model, in which Efg1 acts as transcriptional activator
specifically during yeast growth. In further experiments we attempted to reconstitute
the EFG1 autoregulatory circuit in the yeast S. cerevisiae. In this system the
presence of Efg1 was shown to downregulate EFG1 promoter activity, although
not to the same extent as was observed in C. albicans. This finding hints at
co-regulators that together with Efg1 mediate autoregulation in C. albicans.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
140

141

Poster number: 16
The contributions of polyubiquitin to stress resistance,
metabolic adaptation and virulence in Candida albicans
Michelle D. Leach, Doblin Sandai, David Stead, Evelyn Argo, Donna
MacCallum and Alistair J.P. Brown
Aberdeen Fungal Group, School of Medical Sciences, Aberdeen University, Institute of
Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
Microorganisms exist in complex and dynamic environments. To survive these
challenges they must continuously monitor environmental change and adapt
appropriately. The major fungal pathogen of humans, Candida albicans can be
subjected to diverse stresses during commensalism, colonisation of mucosa and
systemic infection. These include chemical and enzymic insults that damage
external and internal structures. Not surprisingly, robust stress adaptation is vital
for the virulence of C. albicans. In eukaryotic cells post-translational modifications
such as ubiquitination are known to play crucial regulatory roles in many cellular
processes including stress adaptation. Indeed, the highly conserved 76 amino acid
protein, ubiquitin, is induced by stresses, mediating protein turnover via the
proteasome in stressed cells. We have combined molecular, cellular and proteomic
approaches to examine the roles of ubiquitination in C. albicans, as little is known
about ubiquitination in this major fungal pathogen of humans. Independent null
(ubi4/ubi4) and conditional (MET3p-UBI4/ubi4) mutations were constructed at the
C. albicans polyubiquitin-encoding locus. These mutants displayed morphological
and cell cycle defects, as well as sensitivity to thermal, oxidative and cell wall
stresses. Furthermore ubi4/ubi4 cells rapidly lost viability under starvation
conditions. Consistent with these phenotypes, proteins with roles in stress
responses (Gnd1, Pst2, Ssb1), metabolism (Acs2, Eno1, Fba1, Gpd2, Pdx3, Pgk1,
Tkl1) and ubiquitination (Ubi4, Ubi3, Pre1, Pre3, Rpt5) were amongst the
ubiquitination targets we identified, further indicating that ubiquitination plays key
roles in growth, stress responses and metabolic adaptation in C. albicans. Clearly
ubiquitination plays key roles in the regulation of fundamental cellular processes
that underpin the pathogenicity of this medically important fungus. This was
confirmed by the observation that the virulence of C. albicans ubi4/ubi4 cells is
significantly attenuated.
142

143

Poster number: 17
Dissecting the response of Candida albicans to the attack
by neutrophils
Pedro Miramón 1 , Iryna Bohovych 4 , Alistair J. P. Brown 4 , Oliver Kurzai 2,3 ,
Bernhard Hube 1,3
1 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product
Research and Infection Biology - Hans Knoell Institute (HKI), Jena, Germany; 2 Department of
Fungal Septomics, Leibniz Institute for Natural Product Research and Infection Biology - Hans
Knoell Institute (HKI), Jena, Germany; 3 Friedrich-Schiller Universität, Jena, Germany; 4 Aberdeen
Fungal Group, Institute of Medical Sciences, University of Aberdeen, UK
Candida albicans successfully colonises diverse niches in the human host without causing any
harm thanks to a balance with the microflora and possibly due to a constant surveillance of
patrolling immune cells. However, under certain conditions, this fungus is able to cause lifethreatening
deep-seated infections. Although it is know that neutrophils are key players in
controlling and eliminating C. albicans, it is unclear how C. albicans is killed by neutrophils and
how the fungus counteracts these phagocytes.
By means of single-cell expression profiling using GFP reporter strains, we characterised
different responses of C. albicans to three main neutrophil-imposed stresses: nutrient starvation,
oxidative stress and nitrosative stress.
We observed that phagocytosed fungal cells responded to carbohydrate starvation by
upregulating the glyoxylate cycle, but not the glycolytic pathway. Moreover, a mutant lacking a
key gene of the glyoxylate cycle (icl1 ) exhibited decreased resistance in the presence of
neutrophils.
Next, we investigated the contribution of oxidative stress to killing by neutrophil. The catalase
gene CTA1 was induced upon phagocytosis. However, the surface-associated superoxide
dismutase gene SOD5 was induced in yeast cells even before phagocytosis took place,
suggesting that C. albicans can sense and encounter an extracellular oxidative attack. In a
similar fashion, the thioredoxin gene TRX1 was also upregulated upon extracellular contact with
neutrophils. Strikingly, neither cta1 nor trx1 mutants exhibited decreased resistance to
neutrophils, suggesting that they are not essential for normal resistance of C. albicans. However,
sod5 was more sensitive to neutrophils, indicating that Sod5 is an important detoxifying enzyme
to cope with neutrophil-generated oxidative stress. C. albicans also responded to nitrosative
stress generated in the intracellular milieu and this response was essential for full resistance as
a yhb1 mutant, defective in nitric oxide dioxygenase, exhibited decreased survival.
In summary, our study provides evidence that C. albicans displays specific responses to
overcome either extracellular or intracellular attack by neutrophils.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network (PITN-GA-
2008-214004).
144

145

Poster number: 18
A C-terminal hyphal activation motif of the transcription
factor Tec1p is essential for biofilm formation and
nematode infection in Candida albicans
Schröppel K., Singh A., Dietsche T., Eickhoff H., Wiesmüller K.-H., Fischer
K., and Abu Rayyan W.
Institute of Medical Microbiology and Hygiene, University Hospital and Clinics, University of
Tübingen, Tübingen, Germany
Hyphal formation of Candida albicans is a prerequisite for penetration of host
tissues. The formation of biofilms allows this pathogen to persist and to increase
resistance against anti-fungal compounds. In order to define a structure function
relationship for the important transcription factor Tec1p, we integrated modified
open reading frames (ORFs) of the wild type TEC1 under the control of the C.
albicans adapted reverse Tet-dependent transactivator (rtTA) into the tec1/tec1
mutant. We screened a library of such C. albicans mutants for their ability of in vitro
biofilm formation and morphogenetic development, and examined their virulence
in a nematode host using C. elegans as an infection model. Additionally, we
characterized the antifungal activity of EMC120B12, a novel (S)-2-aminoalkyl
benzimidazole derivative, against the library of C. albicans TEC1 mutants in vitro
and in vivo. In comparison to a full length TEC1 ORF, the ORF constructs either with
a deletion of the C-terminus from position 637 aa to the stop codon at 744 aa or a
deletion of the TEA domain from 216 to 244 aa showed abrogation in restoring
hyphal growth and biofilm formation in the tec1/tec1 mutant. Interestingly, the
tec1/tec1 phenotype was rescued in a mutant containing an ORF with an additional
72 aa that spanned the region from 1 to 709 aa. The deletion of the proposed Cterminal
hyphal activation motif CHAM of Tec1p did not affect DNA binding to a
TEA consensus sequence TCS probe.
In the C. elegans infection model we found that the C. albicans mutant lacking
CHAM showed less virulence in terms of hyphal invasion and killing of the nematode
host. During in vivo infection studies, we could demonstrate that EMC120B12 can
inhibit filamentation of C. albicans and prolong survival of the C. albicans infected
nematodes.
146

147

Poster number: 19
Resistance of Candida albicans biofilms formed on
catheters and probes and the discovery of a
chlamydospore in the biofilm
S.M.L. Seddiki a, , Z. Boucherit-Atmani a , K. Boucherit a ,
L. Sari-Belkharoubi a , D. Kunkel b
a Laboratoire antibiotique antifongique, physico- chimique synthèse et activité biologique.
Département de biologie, Faculté des sciences de la nature et de la vie et des sciences de
la terre et de l’univers, Université Abou bekr belkaid. BP 119, Imama 13000 Tlemcen.
ALGERIE; b Dennis Kunkel Microscopy, Inc. P.O. Box 2008 Kailua, HI 96734, USA
In Algeria, many studies on bacterial biofilms were clarified but those of fungal
origin, particularly caused by the yeast Candida albicans remained unidentified.
This work was performed at the hospital Chabane Hamdoune of Maghnia (Algeria),
where 51 strains of C. albicans representing 16.94% of all samples taken were
isolated. They were collected from catheters and probes from different hospital
services with variable rates, and the most concerned service was the ICU (40.74%)
followed by the gynecology department (17.39 %), while that of general surgery
came in third (15.79 %). Testing the antifungal amphotericin B (AmB) showed clearly
that the sessile cells of C. albicans were much more resistant than their planktonic
counterparts (suspended cells), especially at the resistance increases during the
different phases of biofilm formation until it reaches its threshold at the ripening
stage (48 hrs). Furthermore, scanning electron microscopy of the isolated strains
in the laboratory proved the formation of biofilms on catheters by Candida albicans.
Surprisingly, observations revealed a new structure in these biofilms: The
chlamydospore.
keywords: Candida albicans; nosocomials infections; catheters; biofilms;
chlamydospore.
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149

Poster number: 20
Investigation of combinatorial stress responses in C.
albicans
Anna Theresa Tillmann 1 , Elahe Radmaneshfar 2 , Tao You 2 , Despoina
Kaloriti 1 , Mette Jacobsen 1 , Zhikang Yin 1 , Marco Thiel 2 , Celso Grebogi 2 ,
Neil A.R. Gow 1 , Mike Gustin 3 , Alistair J.P.Brown 1
1 School of Medical Sciences, University of Aberdeen, UK; 2 Institute of Complex Systems
and Mathematical Biology, University of Aberdeen, UK; 3 Department of Cell Biochemistry
and Cell Biology, Rice University, USA.
Candida albicans is a major human fungal pathogen that causes mucosal and
systemic infections. Immune cells combat Candida infections with reactive nitrogen
and reactive oxygen species. Meanwhile Candida has evolved strategies to
minimise the damage caused by these environmental stresses, and the success of
this pathogen depends partly on its robust adaptation to these stresses. We
examined three infection-associated stresses: nitrosative, oxidative and osmotic
stress. First we examined responses to each individual stress. Then we analysed
responses to stress combinations because C. albicans cells are exposed to such
combinatorial stresses in vivo. The Hog1 SAPK, the cell wall integrity and the Cta4-
Yhb1 pathways contribute to these combinatorial stress responses. The
involvement of these pathways was confirmed by sensitivity testing of null mutants
to NO, H 2 O 2 and NaCl, and by testing the activation of the corresponding MAP
kinases by Western blotting. Furthermore transcription factor localisation and the
expression of downstream target genes provided further evidence for this activation.
The relative contributions of these nitrosative, oxidative and/or osmotic stress
resistance pathways during C. albicans infections are discussed.
150

151

Poster number: 21
Agent based modelling and host pathogen interactions
Katarzyna M. Tyc 1 , Clemens Kühn 1 , Edda Klipp 1
1 Theoretische Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany
Mathematical models of pathogen and host interactions provide a tool for medical
applications and potential for pharmaceutical companies for testing novel drug
treatments and consequences of their application. Host pathogen interactions are
highly complex and experimental techniques even when combined with different
computational methods allow only for partial investigation of their mutual
dependences.
Based on the available literature we reconstruct a computational model of a human
oral epithelium invasion by the pathogenic fungus Candida albicans. We investigate
the system’s dynamics using the agent based modelling approach (ABM) where
cells (Candida cells, neutrophils (PMNs)) are represented as individual agents. These
agents interact in different ways: fungal cells form hyphal colonies on the epithelium
and then epithelial cells secrete cytokines upon contact with the hyphae form of
fungi, indicating inflammation. Cytokine secretion is one of the signals stimulating
PMNs recruitment to the site of infection. Mix of different gradients such as lactate
dehydrogenase (LDH) release from damaged epithelium, cytokines, and secreted
hyphal particles influences PMN activity. In our model, we investigate the essential
parameters that establish the system’s dynamics. We also look at the
consequences of an antifungal drug treatment on the Candida albicans survival rate
and its dose dependence. We test the optimal intervals and doses for new
application.
With agent based models, we can investigate the effect of individual dynamics on
global patterns, enabling an intuitive view on host-pathogen interactions that can
provide relevant predictions for further experiments.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
152

153

Poster number: 22
Candida albicans unique genes: specific roles in host cell
damage
Duncan Wilson 1 , Francois Mayer 1 , Ilse Jacobsen 1 , Bernhard Hube 1,2
1 Hans Knoell Institute, Jena, Germany; 2 Friedrich Schiller University, Jena, Germany
Candida albicans is amongst the most pathogenic species of fungi and causes both
superficial (high prevalence) and systemic (high mortality) infections. Because the
major human pathogenic fungal species are distantly related, we hypothesized that
they may possess unique genes which account for their high pathogenic potential.
Using C. albicans as a model pathogen, we performed in silico genome subtraction
and transcriptional analysis to identify [C. albicans] species-unique genes
expressed during infection. Gene deletion combined with a series of infection
models (including biochemical analysis of cellular damage and fluorescence
microscopy-based morphological, adhesion and invasion assays for in vitro
epithelial, endothelial and macrophage infection models, as well as an in vivo mouse
virulence model) were used to determine the role of C. albicans unique genes during
infection.
A total of 65 C. albicans unique genes, expressed during infection, were identified
and six were deleted. Five of the resultant homozygous mutants exhibited
significantly altered phenotypes in at least one infection model. One gene, PGA16,
was found to be required both for epithelial destruction and virulence in a mouse
model of hematogenously disseminated candidiasis. Furthermore, this gene was
required for hyphal ramification and optimal dissemination within epithelia.
This study therefore provides molecular evidence that filament branching of a fungal
pathogen contributes to virulence.
154

155

Poster number: 23
Fitness Profiling in Candida albicans by Systematic Gene
Overexpression
Sadri Znaidi 1,2 , Vitor Cabral 1,2 , Melanie Legrand 1,2 , Arnaud Firon 1 , Murielle
Chauvel 1 , Carol. A. Munro 3 and Christophe d’Enfert 1,2 .
1 Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Département Génomes et
Génétique, F-75015 Paris, France; 2 INRA, USC2019, F-75015 Paris, France; 3 School of
Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK
Background: Gene overexpression is a powerful strategy for mapping pathways
and phenotypes. It mimics gain-of-function mutations, complements loss-offunction
phenotypes and allows to study the function of both essential and non
essential genes. Because lack of a complete sexual cycle and aneuploidy hamper
the use of classical genetics in C. albicans, we hypothesized that combining gene
overexpression and strain barcoding would help in understanding gene function in
C. albicans.
Methods: Pools of 531 barcoded C. albicans strains, each overexpressing a unique
ORF under the control of a doxycycline (dox)-inducible promoter, were grown i) for
18 generations in rich medium in the absence or presence of dox or ii) in doxsupplemented
medium in the presence or absence of caspofungin (~IC 90% ). The
relative abundances of the strains were determined using barcode arrays. To
validate the microarray data, growth rates of the strains were measured individually,
using spot or liquid growth assays.
Results: We found 8 genes whose overexpression decreased C. albicans fitness in
rich medium by at least 1.5-fold (p

157

158

The Key Battlefields
159

Poster number: 24
Characterization of the Candida albicans PKH1 and YPK1
genes for their roles in biofilm formation and cell wall
integrity
Vitor Cabral 1,2 , Arnaud Firon 1,2 , Tobias Schwarzmüller 3 , Karl Kuchler 3 ,
Christophe d’Enfert 1,2 and Sophie Bachellier-Bassi 1,2
1 Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Département Génomes et
Génétique, F-75015 Paris, France; 2 INRA, USC2019, F-75015 Paris, France; 3 Medical
University Vienna, Max F. Perutz Laboratories, Christian Doppler Laboratory for Infection
Biology, Department of Medical Biochemistry, A-1030 Vienna, Austria
In Saccharomyces cerevisiae, the Pkh1/Pkh2 and Ypk1/Ypk2 protein kinases are
part of a signaling cascade important for maintenance of cell wall integrity. Our
analysis of a large collection of Candida glabrata knock-out mutants for their ability
to form biofilms has revealed that the C. glabrata Pkh2 protein kinase
(CAGL0I07513g) is necessary for growth on solid surfaces and efficient biofilm
formation while Pkh1/CAGL0G04609g is not (Schwarzmüller et al., in preparation).
Candida albicans has only one gene for the Pkh protein kinase and one gene for
the Ypk protein kinase that we have named PKH1 and YPK1, respectively. In order
to test whether the Pkh1 and Ypk1 protein kinases also play a role in biofilm
formation in C. albicans, heterozygous and homozygous knockout mutants for the
YPK1 and PKH1 genes have been constructed. Preliminary data show that the
ypk1∆/ypk1∆ knock-out mutant has altered sensitivity to the cell wall perturbing
agents Congo Red and Calcofluor White suggesting that Ypk1 might perform a
function similar to that of Ypk1 and Ypk2 in S. cerevisiae. Additional studies of these
mutants and complemented strains are ongoing and will be presented.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
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161

Poster number: 25
Insights into Candida tropicalis virulence factors
Melyssa Negri 1 , Sónia Silva 1 , Mariana Henriques 1 , Terezinha Svidzinski 2 ,
Joana Azeredo 1 , Rosário Oliveira 1
1 Institute for Biotechnology and Bioengineering, Centre of Biological Engineering,
Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal; 2 Teaching and
Research in Clinical Analysis Laboratory, Division of Medical Mycology, Universidade
Estadual de Maringá, Av. Colombo, 5790, postal code 87020-900, Maringá, Paraná, Brazil.
Candida tropicalis is a common nosocomial species related to candidemia and
candiduria. Several virulence factors seem to be responsible for C. tropicalis
infections, which lead to high mortality. Adhesion to surfaces (medical devices and
host cells) and biofilm formation are considered important factors that contribute
to the development of candidosis. Therefore, adhesion to urinary catheters and
biofilm formation were assessed in an optimized in vitro flow model, using silicone
and latex urinary catheters and artificial urine (AU). Moreover, biofilm matrices were
also evaluated in terms of proteins and carbohydrates. Regarding adhesion to biotic
surfaces, the interaction of C. tropicalis with host cells was determined using three
different human epithelial cell lines: TCC-SUP (urinary bladder); HeLa (cervical
carcinoma) and Caco-2 (colorectal adenocarcinoma). Specifically, the degree of
human cells damage and activity reduction induced by C. tropicalis adhesion and
the role of Candida tropicalis aspartyl proteinases (SAPT) genes expression were
assessed. Additionally, the influence of C. tropicalis biofilm cells with different ages
(24 - 120 h) on TCC-SUP cells integrity was also studied. Another important
Candida factor is its resistance to antifungal agents, which was also assessed and
related with the expression of enzymes and hyphae formation.
In summary, C. tropicalis strains were able to form biofilms in AU, in static or
dynamic mode, although, with differences among strains. It is important to
emphasize that human cells response to C. tropicalis adhesion, as well as SAPs
production, is strain and cell line dependent. Additionally, it should be highlighted
that C. tropicalis cells detached from biofilms are able to colonize human cells and
cause injury and reduction of metabolic activity. In addition SAPT3 was highly
expressed compared to other SAPT genes. Therefore, it should be pointed out that
C. tropicalis presented a set of different virulence factors that might be responsible
for its high degree of infection.
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163

Poster number: 26
Iff2 is a cell surface protein involved in adhesion
Iaroslava Kos 1 , Grégory Da Costa 1 , Céline Monniot 1 , Anita Boisramé 1
Marie-Noëlle Bellon Fontaine 2 , Murielle Chauvel 3 , Christophe d’Enfert 3 and
Mathias L. Richard 1
1 Team “Virulence and Fungal Infection” Institut Micalis, UMR1319 INRA AgroParisTech,
78850 Thiverval-Grignon, FRANCE. 2 Team “Bioadhésion et Hygiène des Matériaux” Institut
Micalis, UMR1319 INRA AgroParisTech, 91744 Massy, FRANCE. 3 Team “Fungal Biology and
Pathogenicity”, Pasteur Institute, 75015 Paris, FRANCE.
In Candida albicans, the cell wall and especially cell wall proteins are known to play
a key role in the relationship between the fungal cell, the host and most indwelling
device, contributing to adhesion, immune response modulation and deep seated
infections. A specific class of cell wall proteins has been particularly under the
scope in the past decade: the glycosylphosphatidylinositol anchored proteins
(GpiPs). The largest family of GpiPs is a family of 12 proteins sharing a high similarity
with Hyr1. One of its members, Iff2 has been the subject of a large scale study in
our laboratory in order to decipher his putative function in C. albicans. The cell
localization of Iff2 was assessed using a V5-tagged protein and clearly showed that
Iff2 is a cell surface protein tethered to the cell wall proteins through alkali-Pir-like
links as well as di-sulfide bonds. We also demonstrated that Iff2 protrude from the
cell wall glucan network at the interface with the cellular environment. At the gene
level, we showed that IFF2 is a gene with an expression level stronger than most of
the member of Hyr/Iff family in laboratory conditions and that IFF2 is induced during
the stationary phase in the same media. An overexpressing strain with IFF2 under
the control of TEF1p was hyper adherent to different plastic but not to silicon,
resulting in the formation of stronger biofilm at the early stage of biofilm formation
compared to the reference strain. The future work is aimed to understand which
part of the protein is important for adhesion and if it is specific to Iff2 functional
domain. Additional experiments are also planned to monitor the consequences of
this overexpression in in vitro and in vivo models.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial
Training Network (PITN-GA-2008-214004).
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165

Poster number: 27
C. glabrata cell wall attached and intracellular proteinases
Pirjo Pärnänen 1,2 , Pirjo Nikula-Ijäs 1
1 Department of Biosciences, Division of Biochemistry, University of Helsinki, Finland;
2 Institute of Dentistry, University of Helsinki, Finland
Background: C. glabrata is the most common non-albicans Candida species
causing oral mucosal infections and the third most common microbe that causes
invasive mortal hospital infections. C. glabrata has an an innate immunity to the
most common antifungal group azoles. Proteinases cause tissue protein
degradation and are involved in Candida virulence and invasion. Compared to the
most studied C. albicans, C. glabrata proteinases are mostly unfound or
uncharacterized. Against this background we studied clinical C. glabrata species
to locate and characterize these proteinases.
Materials and methods: Eight C. glabrata clinical strains, both mucosal and blood
isolates from hospitalized patients,were included. Gelatin zymography was used
for preliminary scanning of potential active proteinases and their inhibition by
proteinase inhibitors. The most gelatinolytic blood isolate (T-1639) was used for
further studies. Cell wall bound proteinases were enzymatically released and yeast
cells were sonicated to isolate plasma membrane and intracellular fractions in a
native state. Further activity testing was performed by fluorometric detection of
degraded synthetic L-Arg-AMC proteinase substrate.
Results: All the strains showed variable gelatinolytic activity by zymography. The
strain T-1639 showed enzymatic activity of approximate 25 kDa size released from
the 10x concentrated cell wall fraction, and more pronounced intracellular activity
of approximately 75 kDa size, which were both verified with fluorometry. The cell
wall bound proteinase was inhibited by a serine proteinase inhibitor (PMSF),and
the intracellular proteinase by a metalloproteinase inhibitor (EDTA).
Conclusions: The found proteinases are potentially new to our knowledge and
merrit further studies. The found proteinases belong to two different proteinase
classes and most probably have distinct roles in protein degradation and
pathogenic properties of C. glabrata.
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167

Poster number: 28
Dolichol-dependent glycosylation; a prerequiste of
Candida albicans morphogenesis
Mateusz Juchimiuk 1 , Jacek Orłowski 1 , Joachim Ernst 2 Katarzyna
Gawarecka 1 , Ewa Kula Świeżewska 1 and Grażyna Palamarczyk 1
1 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi skiego 5a,
02 106 Warsaw, Poland; 2 Department of Biology, Molecular Mycology, Heinrich-Heine-
Universitaet, Duesseldorf, Germany
An interesting feature of C. albicans biology is its ability to grow as unicellular
budding yeast, pseudohyphae, true hyphae and chlamydospore. The yeast to
hyphae transition is considered as a prerequisite of C. albicans virulence. On the
other hand, recent studies on the role of mannoproteins in C. albicans virulence
have demonstrated the importance of protein glycosylation for the fungal
pathogenesis. In the present work we characterized Candida albicans RER2 gene,
[cis-prenyltransferase (Rer2p) encoding]. Rer2p is the first enzyme of the
mevalonate pathway committed to the biosynthesis of dolichol, the isoprenoid lipid,
involved in protein glycosylation. A conditional mutant, expressing the C. albicans
ortholog of RER2 from the regulatable MET3 promoter, contained only 4 % of cisprenyltransferase
activity and markedly diminished amount of dolichols, when
cultivated in the repressive condition. Furthermore, at 28 o C the growth of the strain
was retarded and the strain was unable to grow at the restrictive temperature of 37
°C. Particularly, decreased dolichol level prevented morphological differentiation of
the strain leading to hyphae formation. Biochemical analysis confirmed
glycosylation disorder and compromised cell wall integrity. Our results suggest that
biosynthesis and level of dolichol and its ability to enter glycosylation pathway upon
phosphorylation, are essential not only for protein glycosylation and the cell wall
integrity but also for the growth and morphological differentiation of C. albicans
leading to virulence.
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169

Poster number: 29
Localization of the Phr1p, a glucan remodeling enzyme
crucial for morphogenesis and virulence in Candida
albicans
G. Degani and L.Popolo
Università degli Studi di Milano, Dipartimento di Scienze Biomolecolari e Biotecnologie, Via
Celoria 26, Milano, Italy
PHR1 is a pH-regulated gene belonging to a family of five genes of C. albicans.
PHR1 expression is triggered at pH values of the growth medium > 5.5. Loss of
PHR1 induces morphological defects at neutral-alkaline ambient pH, inability to
support hyphal growth, to adhere and invade human epithelia. PHR1 mutants are
avirulent in mouse models of systemic infection. At the restrictive pH, cells lacking
Phr1p have an irregular surface and higher and delocalized content of chitin in the
abnormal germ tubes which the mutant produces. Phr1p is a glycoprotein endowed
with beta(1,3)-glucan elongase activity (GH72) similarly to Gas1p of S. cerevisiae
and Gel proteins of A. fumigatus. We obtained a GFP fusion to determine its cellular
localization. During growth in yeast form Phr1p-GFP was predominantly detected
at the bud periphery, bud-neck and septum. During hyphal growth Phr1p-GFP
concentrated at the tip of the germ tubes and gradually distributed along the lateral
side of the hyphae. Phr1p also localized to the septa of the hyphae. By exploiting
the conditional nature of PHR1 and the intrinsic instability of the fluorescent hybrid,
we performed pH shift-down experiments. Differently from the cell wall-bound form
of ScGas1p which is stabilized by cross-links to the chitin ring and remains in the
bud scars after generations, Phr1p-GFP did not persist. Further studies were
performed using mutants lacking CHS3 or members of the CRH family whose
homologues are known to be required for Gas1p immobilization to the bud scar.
Our results suggest that the activity of Phr1p is required at the sites of wall formation
where the incorporation of newly synthesized beta(1,3)-glucan is strictly coupled
to morphogenic events. Moreover, our studies revealed that the mechanisms active
in tethering Gas1p to the bud scars in S. cerevisiae are not operative in C. albicans.
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171

Poster number: 30
A Novel Flow Cytometric Protocol for Assessment of Yeast
Cell Adhesion
A. Silva-Dias 1,2 , I.M. Miranda 1,2 , R. Rocha 1 , M. Monteiro-Soares 3 ,
A. G. Rodrigues 1,2,4 , C. Pina-Vaz 1,2,5
1 2 Department of Microbiology, Faculty of Medicine, University of Porto, Cardiovascular
Research & Development Unit, Faculty of Medicine, University of Porto; 3CINTESIS, Department of Biostatistics and Medical Informatics, Faculty of Medicine, University of Porto,
4 5
Burn Unit and Department of Plastic and Reconstructive Surgery, Hospital S. João,
Department of Microbiology, Hospital S. João, Porto, Portugal.
The capacity of microorganisms to adhere to each other, to biomaterials, to biotic
substrates and tissues is an important trait at environmental, industrial or medical
level. Pathogenic yeasts belonging to genus Candida are frequently involved in
systemic infections, mainly due to its ability to adhere and colonize tissues and
medical indwelling devices. In order to quantify this attribute, several methods have
been used, with advantages and drawbacks. Some of the major limitations found
within these methodologies concern the incubation time, the small number of cells
analyzed and the operator`s subjectivity. In order to overcome these aspects we
have developed a quantitative method to measure yeast cells` adhesion through
flow cytometry. The adhesion assay is based upon a simple principle: yeast cells
became fluorescent when attached to highly green fluorescent microspheres.
Therefore by flow cytometry a quantitative distinction between non-adherent yeast
cells (non fluorescent) and adherent cells (fluorescent) is achieved. By measuring
the intensity of fluorescence emitted by each cell it is possible to evaluate the
number of beads that are attached to each cell. Additionally, fluorescent beads
could be coated with different substrates to which yeast adhesion ability can be
tested.
A suspension of yeast cells (1x10 6 cell.ml -1 ) is mixed with green fluorescent
polystyrene microspheres, uncoated or coated with host proteins, and incubated
for 30 minutes. Samples (50.000 cells) are analyzed in the FL3 fluorescence channel
(FACSCalibur BD Biosciences). Within two hours an adhesion profile is obtained
based on three parameters: percentage of adherent cells, mean of fluorescence
intensity (MFI) and cells-microsphere population`s distribution pattern. Comparing
to the classical assays, flow cytometry protocol represents a useful tool to quantify
yeast adhesion to different substrata in a large scale, providing manifold data in a
speedy and informative manner.
A Silva-Dias is supported by a FCT (Fundação Ciência e Tecnologia) doctoral grant
SFRH/BD/44896/2008; I Miranda is supported by Ciência 2008 (FCT) and European Social Fund.
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173

Poster number: 31
Cell wall stress elicits a conserved response in Candida
albicans
Alice G. Sorgo, Clemens J. Heilmann, Sepehr Mohammady, Henk L.
Dekker, Stanley Brul, Chris de Koster, Leo J. de Koning and Frans M. Klis
Swammerdam Institute for Life Sciences, Universiteit van Amsterdam, Science Park 904,
1098 XH Amsterdam, The Netherlands
Both cell wall proteins and secreted proteins of C. albicans are critical for fitness,
virulence and adaptation to environmental challenges. We have recently shown that
fluconazole, an antifungal drug that affects ergosterol synthesis and increases
membrane fluidity, also causes cell wall stress as evidenced by decreased
resistance to wall-perturbing compounds and higher chitin levels in the wall (1). This
is accompanied by substantial changes in the wall proteome and secretome. We
found that the abundance of proteins mainly involved in wall biosynthesis and
integrity increased (Crh11, Pga4, Pir1, Phr1, Phr2, Sap9). However, the abundance
of hypha-associated proteins (Als3, Hwp1, Plb5) decreased in agreement with
reduced hyphal growth. Finally, fluconazole induced cell clustering. As membrane
fluidity also tends to increase at higher temperatures, we wanted to investigate
whether the response to thermal stress is similar to that of fluconazole and could
also result in cell wall stress. When C. albicans was cultured at 42°C, which resulted
in substantial growth inhibition, the cells showed indeed decreased resistance to
wall-perturbing agents, and chitin levels in the wall were elevated, both suggesting
cell wall stress. Likewise, the wall proteome response was similar to that of
fluconazole-treated cells, including increased incorporation levels of proteins
important for wall maintenance and integrity. Additionally, cell separation was
impaired, correlating with a severe reduction of the chitinases Cht2 and Cht3 and
the endoglucanase Eng1 in the medium. In summary, our data indicate that both
thermal stress and fluconazole-induced stress induce membrane stress, which
leads to cell wall stress and a conserved wall stress response.
References
(1) The effects of fluconazole on the secretome, the wall proteome and wall integrity of the clinical fungus
Candida albicans. Sorgo AG, Heilmann CJ, Dekker HL, Bekker M, Brul S, de Koster CG, de Koning LJ,
Klis FM. Eukaryot Cell. 2011. doi:10.1128/EC.05011-11.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
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175

176

The Defensive Shields
177

Poster number: 32
Human cells response to Non- Candida albicans Candida
infection
Cláudia Botelho 1 , Melyssa Negri 1 , Catarina Seabra 1 , Mariana Henriques 1 ,
Joana Azeredo 1 , Rosário Oliveira 1
1 Institute for Biotechnology and Bioengineering, Centre of Biological Engineering,
Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
This study evaluated the influence of adhered Candida spp (C.tropicalis ATCC 750,
C. parapsilosis ATCC 2201, C. glabrata ATCC 2001 and C. albicans SC5413) on the
biological behavior of human cells (HeLa and TCC-SUP). A yeast suspension was
added to a confluent layer of human cells and after 2 and 24h of incubation yeast
cells were detached for CFU counts. All Candida spp assayed adhered to epithelial
cells, although C. glabrata was the species adhering in higher extent to both
epithelial cell lines. From 2 to 24h there is a significant increase in the number of C.
glabrata adhered to TCC-SUP cells, while no increase in the number of C. glabrata
colonizing HeLa cells was observed. An opposite result was observed for C.
tropicalis; there was no increase in the number of adherent cells to TCC-SUP cells,
but there was an increase for HeLa cells. The metabolic activity of HeLa cells
decreased after a period of 24h of Candida infection, especially in the presence C.
tropicalis. The biological response of TCC-SUP cells was different from the one
observed with HeLa cells, the presence of C. albicans and C. parapsilosis induced
an increase in the metabolic activity of TCC-SUP cells, while C. tropicalis and C.
glabrata promoted a decrease.. After 24h of C. albicans adhesion, the highest
human cells damage was detected in HeLa cells (measured by LDH release), while
C.tropicalis seems to induce higher LDH release after 24h in contact with TCC-SUP
cells. The biological response of human cells showed to be dependent on their
origin and on the colonizing yeast species/strain, and no direct relation between
the number of adherent cells and the biological response was observed.
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179

Poster number: 33
Using zebrafish to study Candida albicans-mediated
systemic candidiasis in vivo
Thomas Evans, Simon Tazzyman, Timothy J.A. Chico, Martin H. Thornhill,
Craig Murdoch.
University of Sheffield, Sheffield, UK
Candida albicans is a common oral opportunistic pathogen that causes
mucocutaneous infections and rarely systemic candidiasis, a nosocomial infection
which involves the haematogenous spread of Candida to multiple organs and is
associated with high mortality. To cause tissue damage C.albicans must bind to the
endothelium of blood vessels, leave the circulation and invade tissues. However,
little is known about the mechanisms involved in these processes. This study aimed
to use transparent embryonic zebrafish (Danio rerio) as an in vivo infection model
in conjunction with an in vitro flow adhesion assay to study how C.albicans bind to
the endothelium, leave the circulation and invade tissues. Suspensions of viable or
treated C.ablicans were flowed over monolayers of endothelial cells and adherent
Candida counted. Zebrafish were injected with C.albicans and mortality determined
after 18h. Time-lapse confocal microscopy using fluorescent C.albicans and blood
vessels was performed to image dissemination. Viable C.albicans bound to
endothelium significantly (p

181

Poster number: 34
Candida albicans Secreted Aspartic Proteases Cause
Inflammatory Response Irrespective of Their Proteolytic
Activity
Neelam Pandey 1 , Donatella Pietrella 1 , Lydia Schild 2 , Francesco Bistoni 1 ,
Bernhard Hube 2 and Anna Vecchiarelli 1
1 Department of Experimental Medicine and Biochemical Science, University of Perugia,
Perugia, Italy; 2 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for
Natural Product Research and Infection Biology, Hans Knoell Institute, Jena, Germany2;
Candida albicans normally inhabits humans as a commensal, but causes fatal infections
when the immune system of patients is suppressed. It has a strong armory of virulence
attributes which facilitates bypassing the immune system and causes mucosal, cutaneous
and systemic infections. These attributes are the ability to colonize, invade the host tissue
and secrete aspartic proteases. We here report that recombinant secreted aspartic
proteases (rSaps) including rSap1, rSap2, rSap3 and rSap6 induce inflammatory cytokine
production by human monocytes to different degrees. rSap1, rSap2 and rSap6 induced
the secretion of IL-1beta, TNF-alpha and IL-6 significantly, while rSap3 stimulated the
secretion of IL-1beta and TNF-alpha. All these rSaps induced Ca 2+ influx in monocytes.
Pepstatin A, a potential inhibitor of aspartic proteases, has no effect on the cytokine
secretion induced by these rSaps. This suggests that the inflammatory response due to
rSaps is not because of their proteolytic activity, a hypothesis further supported by the
observation that the ability of rSaps to induce inflammatory cytokine secretion was
independent of protease-activated receptor (PAR) activation, and of the optimal pH for
individual rSap activity. Moreover, rSaps stimulated Akt activation and thus induced IkBalpha
phosphorylation which mediated translocation of NFkB into the nucleus in human
monocytes. These findings give evidence that rSap1, rSap2 and rSap3 cause an
inflammatory response irrespective of their proteolytic activity and mediate the inflammatory
response via activation of Akt/NF-kB. The inflammatory response terminates with
inflammasome activation. This occurs through a process requiring Sap internalization via a
clathrin dependent mechanism, and caspase-1 activation, and it is mediated by K + efflux
and ROS production. By demonstrating that the recognized virulence factors of C. albicans
such as Sap2 and Sap6 are potent inducers of inflammasome activation, our results
represent a step forward in the comprehension of the close relationship between fungus
virulence and host response mechanisms.
We are extending our study by using an in vivo experimental model of murine candidiasis
to analyze the role of Sap2 and Sap6 in the inflammatory response in the vaginal candidiasis,
and to investigate whether specific mAbs are indeed able to affect this process.
This study was funded by the European Commission (FINSysB Marie Curie Initial Training Network grant PITN-
GA-2008-214004).
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183

Poster number: 35
The Tyr238X dectin-1 polymorphism is a predisposing
factor for mucosal Candida albicans infections but not
candidemia
Diana C. Rosentul 1,2 , Theo S. Plantinga 1,2 , Marije Oosting 1,2 , William K.
Scott 3 , Digna R. Velez Edwards 4 , P. Brian Smith 5 , Barbara D. Alexander 5 ,
John C. Yang 6 , Gregory M. Laird 5 , Walter J. F. M. van der Velden 2,7 , Bart
Ferwerda 1,2 , Annemiek B. van Spriel 2,8 , Gosse Adema 2,8 , Ton Feuth 2,9 , J.
Peter Donnelly 1,2 , Gordon D. Brown 10 , Nicole M. A. Blijlevens 2,7 , Leo A.B.
Joosten 1,2 , Jos W. M. van der Meer 1,2 , John R. Perfect 5 , Bart-Jan Kullberg 1,2 ,
Melissa D. Johnson 5,11 , Mihai G. Netea 1,2
1 Department of Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen,
The Netherlands; 2 Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud
University Nijmegen Medical Center, Nijmegen, The Netherlands; 3 Dr. John T. Macdonald
Foundation Department of Human Genetics and John P. Hussman Institute for Human
Genomics, University of Miami, Miller School of Medicine, Miami, FL, USA; 4 Department of
Obstetrics & Gynecology, Vanderbilt University Medical Center, Nashville, TN, USA; 5 Duke
University Medical Center, Durham, NC, USA; 6 National Jewish Health, Denver, CO, USA;
7 Department of Haematology, Radboud University Nijmegen Medical Center, Nijmegen, The
Netherlands; 8 Department of Tumor Immunology, Radboud University Nijmegen Medical
Center, Nijmegen, The Netherlands; 9 Department of Epidemiology, Biostatistics and Health
Technology Assessment, Radboud University Nijmegen Medical Center, Nijmegen, The
Netherlands; 10 Institute of Infectious Disease and Molecular Medicine, University of Cape
Town, Cape Town, South Africa; 11 Department of Clinical Research, Campbell University
School of Pharmacy, Buies Creek, NC, USA.
The genetic make up of innate immune genes is believed to play an important role
for susceptibility to fungal infections. We recently described the Tyr238X dectin-1
single nucleotide polymorphism (SNP) as an important factor influencing
susceptibility to vulvo-vaginal candidiasis and onychomycosis in a Dutch family
with recurrent fungal infections.
The aim of our studies was to assess the role of this polymorphism in the
susceptibility to mucosal and systemic Candida albicans infections. To achieve this
aim we have investigated the effect of this SNP in several cohorts of patients:
-142 Dutch patients undergoing hematopoietic stem cells transplantation (HSCT).
-331 Dutch and American candidemia patients and 351 non infected matched
controls.
Functional studies have shown that cells isolated from individuals heterozygous for
the 238X allele had a lower cytokine production upon stimulation with Candida. The
HSCT patients bearing the early stop variant of the dectin-1 gene presented a
184

higher C. albicans oral an gastrointestinal colonization compared to those who were
wild type for the polymorphism and required higher amounts of fluconazole in order
to avoid a disseminated invasion of the fungus.
In contrast, no effect if the Tyr238X dectin-1 SNP on the susceptibility to candidemia
and/or the clinical course of the infection was observed.
In conclusion, the dectin-1 signaling pathway is non-redundant in mucosal immunity
to C. albicans. The 238X allele results in a defective IL-6 and IL-17 response, but
normal IFN-gamma, upon stimulation with C. albicans.
A genetic deficiency of beta-glucan recognition has clear effect on the occurrence
of mucosal Candida infections, but a minor impact on susceptibility to candidemia.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
185

Poster number: 36
Survival of Candida glabrata within phagocytes
Lydia Schild, Katja Seider, Pedro Miramón, Sascha Brunke and Bernhard
Hube
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product
Research and Infection Biology – Hans Knoell Institute (HKI), Jena, Germany
Candida glabrata is both a human commensal and a pathogenic fungus. Immune
evasion strategies likely play a key role during infection since C. glabrata elicits little
inflammation in mice. To investigate immune evasion as a pathogenic strategy, we
characterize the interaction of C. glabrata with human phagocytes such as
macrophages and neutrophils.
C. glabrata is able to survive and even replicate within human macrophages in vitro.
Low pro-inflammatory response to fungal infection and inhibition of macrophage
ROS production by C. glabrata support the view that this fungus can persist in
macrophages as an immune evasion strategy. Our data suggest that C. glabrata
modifies phagosome maturation, residing in a non-acidic phagosome. While the
inhibition of ROS production is affected by viability of fungal cells, inhibition of
phagosome maturation does not require metabolic activity of the fungus. Instead,
an UV-indestructible fungal attribute seems to be involved this process.
To elucidate which C. glabrata factors are responsible for modification of
phagosome maturation and persistence in phagocytes, we analyzed a set of
deletion mutants for survival in macrophages and identified 24 genes that are crucial
for intracellular survival of C. glabrata within macrophages. Phenotypic analyses of
these mutants point to an important role of cell wall integrity and stress resistance
for macrophage survival of C. glabrata. Furthermore, nutrient acquisition, in
particular iron uptake has been shown to play a role during intra-phagosomal
persistence.
Similar attributes seem to be important for resistance to neutrophils, as mutants
lacking genes with suggested functions in cell wall integrity and nutritional sensing
are attenuated in survival in both, neutrophils and macrophages.
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187

Poster number: 37
The role of pH-regulated antigen 1 of Candida albicans in
the interaction of the fungus with human neutrophils
Eliška Svobodová 1 , Josephine Losse 1 , Antje Heyken 2 , Bernhard Hube 2,4 ,
Peter F. Zipfel 3,4 and Mihály Józsi 1
1 Junior Research Group Cellular Immunobiology, 2 Department of Microbial Pathogenicity
Mechanisms and 3 Department of Infection Biology, Leibniz Institute for Natural Product
Research and Infection Biology - Hans Knöll Institute, Jena, Germany; 4 Friedrich Schiller
University, Jena, Germany
Candida albicans is an opportunistic human-pathogenic yeast and a leading cause
of life-threatening systemic fungal infections in immunocompromised individuals.
The virulence of the fungus is associated with its capability to filamentous growth.
The pH-regulated antigen 1 (Pra1) of C. albicans is a surface-associated and
secreted protein whose expression is upregulated in the hyphal form. Pra1 binds
to complement receptor 3 (CD11b/CD18) and can mediate adhesion to and
migration of human phagocytes. Here, we investigated the role of Pra1 in the
activation of human neutrophils. A C. albicans mutant strain lacking Pra1 (pra1 )
supported neutrophil migration to a lower extent than did the parental wild-type
strain. A Pra1-overexpressing C. albicans strain enhanced neutrophil migration and
adherence. While inactivated hyphae of the Pra1-overexpressing mutant enhanced
the production and release of reactive oxygen species, myeloperoxidase,
lactoferrin, and interleukin 8 by neutrophils, such responses were reduced when
stimulated with the pra1 strain. Pra1-overexpressing living hyphae also caused a
reduced neutrophil activation, indicating that fungal cells which express and release
more Pra1 can more efficiently inhibit the activation of these innate immune cells.
Fungal cells lacking Pra1 were more efficiently killed by neutrophils. The lack or the
overexpression of Pra1 did not significantly affect the generation of neutrophil
extracellular traps. In conclusion, surface-exposed Pra1 plays a role in the
recognition of C. albicans, especially hyphal cells, by human neutrophils and
enhances neutrophil antimicrobial responses. However, the fungus can counteract
some of these defense mechanisms likely by releasing Pra1.
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189

190

Defeating The Enemy
191

Poster number: 38
Alkali-metal-cation homeostasis of Candida species is
affected by subinhibitory concentrations of azoles
Hana Elicharová, Hana Sychrová
Department of Membrane Transport, Institute of Physiology AS CR, v. v. i., Videnska 1083,
14220 Prague, Czech Republic, elicharova@biomed.cas.cz
Homeostasis of alkali-metal cations is essential for all living cells including the
pathogenic Candida species. Candida cells sustain high internal concentration of
potassium cations that are involved in osmotic and pH regulations, enzymatic
function and other processes. In contrast, Candida cells keep the internal
concentration of sodium low due to its intracellular toxicity. Plasma membrane and
transporters play a key role in maintenance of cation homeostasis. Azoles form the
most important group of drugs with antifungal activity. They destabilize plasma
membrane of Candida cells. The combination of subinhibitory concentrations of
fluconazole and NaCl has been shown to inhibit the growth of both fluconazolesensitive
and fluconazole-resistant C. albicans strains (1).
We have tested the influence of subinhibitory concentrations of azoles on the alkalimetal-cation
homeostasis of four Candida species (C. albicans, C. dubliniensis,
C. parapsilosis and C. glabrata).We have found that all tested Candida species can
generally tolerate high concentrations of azoles or salts separately, but the
combination of both compounds inhibits their growth effectively. The level of
inhibition of single Candida species differs strongly. Presence of subinhibitory
concentration of fluconazole increases the intracellular content of toxic Na + in all
four Candida species. Estimation of relative membrane potential together with the
use of Candida mutants lacking plasma-membrane transporters of alkali metal
cations should explain how alkali-metal-cation homeostasis is affected by presence
of fluconazole.
(1) Kolecka et al., Can J Microbiol 2009 55(5):605-10
Supported by MSMT LC531
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193

Poster number: 39
Calcineurin Signaling and Membrane Lipid Homeostasis
Regulates Iron Mediated MultiDrug Resistance
Mechanisms in Candida albicans
Saif Hameed, Sanjiveeni Dhamgaye, Ashutosh Singh, Shyamal K. Goswami
and Rajendra Prasad
School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
We previously demonstrated that iron deprivation enhances drug susceptibility of
Candida albicans by increasing membrane fluidity which correlated with the lower
expression of ERG11 transcript and ergosterol levels. The iron restriction dependent
membrane perturbations led to an increase in passive diffusion and drug
susceptibility. The mechanisms underlying iron homeostasis and multidrug
resistance (MDR), however, are not yet resolved. To evaluate the potential
mechanisms, we used whole genome transcriptome and electrospray ionization
tandem mass spectrometry (ESI-MS/MS) based lipidome analyses of iron deprived
Candida cells to examine the new cellular circuitry of the MDR of this pathogen.
Our transcriptome data revealed a link between calcineurin signaling and iron
homeostasis. Among the several categories of iron deprivation responsive genes,
the down regulation of calcineurin signaling genes including HSP90, CMP1 and
CRZ1 was noteworthy. Interestingly, iron deprived Candida cells as well as iron
acquisition defective mutants phenocopied molecular chaperone HSP90 and
calcineurin mutants and thus were sensitive to alkaline pH, salinity and membrane
perturbations. In contrast, sensitivity to above stresses did not change in iron
deprived DSY2146 strain with a hyperactive allele of calcineurin. Although, iron
deprivation phenocopied compromised HSP90 and calcineurin, it was independent
of protein kinase C signaling cascade. Notably, the phenotypes associated with
iron deprivation in genetically impaired calcineurin and HSP90 could be reversed
with iron supplementation. The observed down regulation of ergosterol (ERG1,
ERG2, ERG11 and ERG25) and sphingolipid biosynthesis (AUR1 and SCS7) genes
followed by lipidome analysis confirmed that iron deprivation not only disrupted
ergosterol biosynthesis, but it also affected sphingolipid homeostasis in Candida
cells. These lipid compositional changes suggested extensive remodeling of the
membranes in iron deprived Candida cells. Taken together, our data provide the
first novel insight into the intricate relationship between cellular iron, calcineurin
signaling, membrane lipid homeostasis and drug susceptibility of Candida cells.
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195

Poster number: 40
Wall proteins, absolute quantification and concatenated
vaccines: A new approach for Candida albicans therapy?
Clemens J. Heilmann, Alice G. Sorgo, Henk L. Dekker, Stanley Brul, Chris
G. de Koster, Leo J. de Koning and Frans M. Klis
Swammerdam Institute for Life Sciences, Universiteit van Amsterdam, Science Park 904,
1098XH Amsterdam
The cell wall and especially the proteins that are covalently anchored to it are the
first site of host-pathogen interaction. The wall proteins are promising targets for
the development of diagnostic biomarkers and vaccines. Previously, we described
the relative quantitative changes in up to 21 wall proteins during the yeast-to-hypha
transition (1) and upon exposure of cells to fluconazole (2). We identified hyphaassociated
wall proteins (Als3, Hwp2, Hyr1, Plb5, Sod5) conferring increased
adhesion and resistance to host defenses and yeast-associated proteins (Rhd3,
Sod4, Ywp1), which probably play a role in host dispersal and immune evasion.
Morphotype-independent proteins (Cht2, Crh11, Ecm33, Mp65) are mainly involved
in wall structure and remodeling.
However, relative quantification can only provide indications of how protein
abundance changes in response to an environmental change. To compare the
abundances of different proteins, absolute quantification is required. We are
developing a strategy for absolute quantification based on 15 N metabolic labeling
and concatenated proteins (Q-CON-CAT). By using the same 15 N reference culture
that was also used for relative quantification and determining the absolute amount
of a particular protein, we will be able to recalculate the relative quantification results
into absolute values.
These absolute values will be informative for the construction of peptide-based
vaccines. We propose that by concatenating peptides that are predicted to be
highly immunogenic and originate from abundant wall proteins an effective Q-CON-
CAT vaccine protein can be generated.
(1) Hyphal Induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein
profile. Heilmann CJ, Sorgo AG, Siliakus AR, Dekker HL, Brul S, de Koster CG, de Koning LJ, Klis FM.
Microbiology. 2011 May 20.
(2) The effects of fluconazole on the secretome, the wall proteome and wall integrity of the clinical fungus
Candida albicans. Sorgo AG, Heilmann CJ, Dekker HL, Bekker M, Brul S, de Koster CG, de Koning LJ,
Klis FM.Eukaryot Cell. 2011 May 27.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
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197

Poster number: 41
Dynamic assessment of the antifungal effect of different
plant essential oils by flow cytometry
Crina Maria Saviuc 1,5 , Alexandru Mihai Grumezescu 2 , Coralia Bleotu 1,4 ,
Alina Maria Holban 1 , Otilia Banu 3 , Dan Mihaiescu 2 , Paul Balaure 2 , Mariana
Carmen Chifiriuc 1 , Veronica Lazar 1
1 University of Bucharest, Faculty of Biology, Microbiology University Department; 2 University
Politechnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Organic
Chemistry Department; 3 Institute of Cardiovascular Diseases Prof. C.C. Iliescu, Bucharest;
4 S. Nicolau Institute of Virology, Bucharest; 5 S.C. Biotehnos S.A.
Introduction: The in vitro and in vivo models already demonstrated the microbicidal
as well as the influence of the essential oils on the coordinated expression of the
virulence factors in fungal strains. In the context of incomplete standardization of
susceptibility testing assays, our aim was to evaluate a rapid tool for the
assessment of the antifungal activity of the essential oils using the flow citometry.
Materials and methods: Four essential oils were extracted by microwave assisted
hydrodistilation from different vegetative and reproduction organs of Eugenia
caryophyllata, Anethum graveolens, Mentha piperita and Rosmarinus officinalis.
Chemical composition of the essential oils was established by GC-MS analysis.
The bioassays were performed on five Candida spp. strains recently isolated from
clinical specimens. The qualitative screening of the antimicrobial effect and its
evolution on time was assessed after 1’, 3’, 5’, 15’ and 30’ of contact by viable cell
counts. Minimum inhibitory concentrations (MIC) was assessed by the classical
twofold micro-dilution technique in 96 multiwell plates and also by flow cytometry
after 15’ and 24h of contact.
Results. The essential oils proved to be reach in terpenes and terpenoids in a
balanced composition wich explained the antimicrobial activity. The essential oil
exhibited an early microbicidal effect, with MIC ranging from 0.75 to 7.5µL/mL,
values also confirmed by flow cytometry.
Conclusion. Our results are proving that the tested essential oils exhibited a strong
antifungal effect, probably due to their good diffusion rates and easy contact with
the target structures. Essential oils complex composition and their volatility raise
specific problems in susceptibility testing, due to their poor solubility, unknown
diffusion pattern in solid media and vapor phase effect. In this context flow
cytometry technique could be considered a reliable tool for evaluating the antifungal
activity of this type of complex mixtures.
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Poster number: 42
The role of fungal cell wall composition and remodelling in
echinocandin drug tolerance
Louise Walker, Kerstin Nather, Laura Selway, David Stead, Keunsook Lee,
Alistair Brown, Carol Munro
Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Aberdeen, UK
Echinocandins are antifungal agents, which affect fungal cell wall integrity by
inhibiting (1,3)-glucan synthesis. Assembly of the fungal cell wall is a dynamic
process. Echinocandin treatment below the MIC activates compensatory
mechanisms that upregulate chitin biosynthesis and result in altered cross-linking
and changes in the cell wall proteome, in an attempt to reinforce the cell wall.
Proteomic analysis and transcript profiling have identified cell wall proteins (CWP)
and genes that are more abundant and have elevated expression in response to
treatment with sub-MIC caspofungin. These include transglycosidases and crosslinking
proteins involved in modifying cell wall polysaccharides (Utr2, Crh11, Phr1,
Cht2), as well as novel proteins (Pga31). Changes in cell wall architecture and
composition can alter a cell’s sensitivity to the echinocandins. Using a tetracycline
inducible promoter, genes of CWPs which were upregulated in response to
caspofungin treatment, were overexpressed to determine whether this altered
susceptibility to caspofungin. Overexpression of CHT2 lead to reduced
susceptibility whereas overexpression of UTR2 and PHR1 resulted in increased
sensitivity to caspofungin. To determine whether altered susceptibility to
caspofungin was observed in vivo, larvae of Galleria mellonella were used as a
preliminary screening model. Overexpression of UTR2 lead to reduced susceptibility
to caspofungin in G. mellonella. Treatment with caspofungin increased the survival
time of larvae infected with the UTR2 overexpression strain compared to larvae
infected with the wild-type. Fungal burdens were significantly reduced in infected
larvae treated with caspofungin and this reduction was greater in larvae infected
with the UTR2 overexpression strain. The number of immune cells also determines
the fitness of G. mellonella. Larvae infected with wild-type and UTR2 overexpression
strain had more immune cells after treatment with caspofungin, indicating an
increase in fitness. Therefore, overexpression of fungal cell surface proteins can
affect the susceptibility of C. albicans to caspofungin in vitro and in vivo.
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Poster number: 43
Genetic code ambiguity accelerates evolution of drug
resistance in Candida albicans
Tobias Weil, Ana R. Bezerra, João Simões and Manuel A. S. Santos
Department of Biology – CESAM, University of Aveiro, 3810-193 Aveiro, Portugal.
The opportunistic fungal pathogen Candida albicans has evolved to survive within
human hosts where it can cause serious diseases in immunocompromised patients.
The emergence of drug-resistant C. albicans strains has become a severe clinical
problem and the mechanisms of fungal drug resistance are still poorly understood.
In the present study we are testing the hypothesis that mRNA mistranslation is
relevant for the evolution of drug resistance. A characteristic feature of C. albicans
biology is the reassignment of the leucine CUG codon to serine. Remarkably, the
serine tRNA that decodes the CUG codons is aminoacylated in vivo with both serine
(3%) and leucine (97%). Such tRNA ambiguity is incorporated into proteins and
creates a statistical proteome. It is not yet clear whether proteome variation is
relevant for C. albicans parthogenesis, however, the potential of CUG ambiguity to
alter cell wall proteins and remodel surface antigens may help C. albicans evading
the immune system and gain advantages in terms of drug resistance. To determine
whether increasing mistranslation levels have important implications for C. albicans
drug resistance we have constructed a series of C. albicans strains that mistranslate
leucine CUG codons as serine at a rate up to 65% and exposed them to azoles,
which are commonly used to treat patients with fungal infections. Interestingly,
these strains developed drug resistance quite rapidly confirming our hypothesis
that mistranslation is indeed relevant for drug resistance. We are now applying in
vivo approaches combined with systems biology techniques based on gene
expression profiling with microarrays and deep genome sequencing in order to
identify novel putative drug target genes and pathways.
Acknowledgements: TW is supported by the European FP7 framework programme
SYBARIS.
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204

Barkay, Naama
Weizmann Institute
Meyer Building 404
Rehovot 76100
Israel
naama.barkai@weizmann.ac.il
Bohovych, Iryna
University of Aberdeen
Institute of Medical Science
Aberdeen, AB25 2ZD, UK
i.bohovych@abdn.ac.uk
Botelho, Claudia
University of Minho
Centre of Biological Engineering
4710-057 Braga
Portugal
claudiabotelho@deb.uminho.pt
Brown, Alistair
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
al.brown@abdn.ac.uk
Brown, Gordon
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
gordon.brown@abdn.ac.uk
Cabral, Vitor
Pasteur Institute
25 rue du Docteur Roux
75015, Paris
France
vitor.cabral@pasteur.fr
Cassone, Antonio
Istituto Superiore di Sanità
Viale Regina Elena 299
00161 Rome
Italy
antonio.cassone@iss.it
List of Participants:
205
Cavalieri, Duccio
Università di Firenze
Viale Pieraccini 6
50139, Firenze
Italy
duccio.cavalieri@unifi.it
Chen, Shin-Chin
Radboud University Nijmegen Medical
Center
Geert Grooteplein-Zuid 10
6525 GA Nijmegen
The Netherlands
S.Cheng@AIG.umcn.nl
Citulio, Francesco
Hans Knoell Institute (HKI)
Beutenbergstrasse 11a
07745 Jena
Germany
francesco.citiulo@hki-jena.de
Cook, Emily
University of Exeter
Geoffrey Pope Building
Exeter, EX4 4QD, UK
E.Cook@exeter.ac.uk
Correia, Inês
University of Madrid
Plaza de ramón y Cajal s/n
28040 Madrid
Spain
inescorreia351@gmail.com
D’Enfert, Christophe
Pasteur Institute
25 rue du Docteur Roux
75015, Paris
France
denfert@pasteur.fr
Dobb, Kate
F2G Ltd
PO Box 1, Lankro Way
Eccles
Manchester, M30 0BH, UK
KDobb@f2g.com

Donohue, Dagmara
Vrije Universiteit Brussel
Pleinlaan 2
1050 Brussels
Belgium
dszestak@vub.ac.be
Elicharová, Hana
Academy of Sciences CR
Videnska 1083
14220 Prague 4
Czech Republic
elicharova@biomed.cas.cz
Ene, Iuliana
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
iuliana.ene@abdn.ac.uk
Ernst, Joachim
University of Düsseldorf
Universitaetsstr. 1, Geg 26.12
40225 Düsseldorf
Germany
joachim.ernst@uni-duesseldorf.de
Filler, Scott
Harbor-UCLA Medl Cntr-Inf Dis
1124 West Carson Street
Torrance, CA 90509
USA
sfiller@ucla.edu
Gil de Bona, Ana
University of Madrid
Plaza de ramón y Cajal s/n
28040 Madrid
Spain
anagildebona@farm.ucm.es
Gustin, Mike
Rice University
W100 George R. Brown Hall
P.O. Box 1892, MS-140,
Houston, Texas 77251-1892
USA
gustin@rice.edu
206
Hameed, Saif
School of Life Sciences,J.N.U
Membrane Biology Lab #101
New Delhi-110067
India
saifhameed@yahoo.co.in
Hassan, Rabeay
Helmholtz Centre for Infection Research
Inhoffenstr. 7
38124 Braunschweig
Germany
Rabeay.Hassan@helmholtz-hzi.de
Heilmann, Clemens
University of Amsterdam
Nieuwe Achtergracht 166
1018 WV Amsterdam
The Netherlands
C.J.Heilmann@uva.nl
Henriques, Mariana
University of Minho
Centre of Biological Engineering
4710-057 Braga
Portugal
mcrh@deb.uminho.pt
Holban, Alina
University of Bucharest
Faculty of Biology,
Portocalelor Street, nr 1-3
Bucharest
Romania
alina_m_h@yahoo.com
Hube, Bernhard
Hans Knoell Institute (HKI)
Beutenbergstrasse 11a
07745 Jena
Germany
Bernhard.Hube@hki-jena.de
Hyde, Peter
Trinity College Dublin
Lincoln Place, Dublin 2,
Ireland
Peter.Hyde@dental.tcd.ie

Ishchuk, Olena
Lund University
Sölvegatan 35
Lund SE-223 62
Sweden
olenkaishchuk@gmail.com
Jacobsen, Ilse
Hans Knoell Institute (HKI)
Beutenbergstrasse 11a
07745 Jena
Germany
Ilse.Jacobsen@hki-jena.de
Jandric, Zeljkica
University of Vienna
Dr.Bohr-Gasse 9
A-1030 Vienna
Austria
zeljkica.jandric@univie.ac.at
Juchimiuk, Mateusz
Polish Academy of Sciences
Pawinskiego 5a
02-106 Warszawa
Poland
mjuchimiuk@ibb.waw.pl
Klipp, Edda
Humboldt University
Theoretical Biophysics
Invalidenstraße 42
D-10115 Berlin, Germany
edda.klipp@rz.hu-berlin.de
Klis, Frans
University of Amsterdam
Nieuwe Achtergracht 166
1018 WV Amsterdam
The Netherlands
F.M.Klis@uva.nl
Kos, Iaroslava
INRA/AGROPARITECH
UMR Micalis 1319
CBAI
78850 Thiverval Grignon, France
ikos@grignon.inra.fr
207
Kumamoto, Carol
Tufts University
136 Harrison Ave.,
Boston, MA,
USA
carol.kumamoto@tufts.edu
Kumar, Antresh
School of Life Sciences, J.N.U
Membrane Biology Lab #101
New Delhi-110067
India
antreshkumar@gmail.com
Lagadec, Quentin
University of Düsseldorf
Universitaetsstr. 1, Geg 26.12
40225 Düsseldorf
Germany
quentin.lagadec@uni-duesseldorf.de
Lavie-Richard, Mathias
INRA/AGROPARITECH
UMR Micalis 1319
CBAI
78850 Thiverval Grignon, France
mathias.lavie-richard@grignon.inra.fr
Leach, Michelle
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
michelle.leach@abdn.ac.uk
Lipke, Peter
Brooklyn College
City University New York
2900 Bedford Avenue, Brooklyn,
NY 11210, USA
plipke@brooklyn.cuny.edu
Liu, Haoping
University of California, Irvine
D250, D288 MED SCI I
Irvine, CA 92697
USA
h4liu@uci.edu

Lorenz, Michael
University of Texas
6431 Fannin, Houston
TX 77030
USA
Michael.Lorenz@uth.tmc.edu
Mantovani, Alberto
University of Milan
MANZONI 56,
20089 ROZZANO
ITALY
Alberto.Mantovani@humanitasresearch.it
McLean, Malcolm
Weizmann Institute
Meyer Building 404
Rehovot 76100
Israel
malcolm.mclean@weizmann.ac.il
Miramón, Pedro
Hans Knoell Institute (HKI)
Beutenbergstrasse 11a
07745 Jena
Germany
pedro.miramon@hki-jena.de
Miranda, Isabel
University of Porto
Alameda Prof. Hernani Monteiro
4200-319 Porto
PORTUGAL
imiranda@med.up.pt
Mitchell, Aaron
Carnegie Mellon University
4400 Fifth Avenue,
Pittsburgh, PA,
USA, 15213
apm1@cmu.edu
Morschhäuser, Joachim
Universität Würzburg
Josef-Schneider-Str. 2, Bau D15
97080 Würzburg
Germany
joachim.morschhaeuser@uni-wuerzburg.de
208
Munro, Carol
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
c.a.munro@abdn.ac.uk
Murdoch, Craig
University of Sheffield
Claremont Crescent
Sheffield
S10 2TA, UK
c.murdoch@sheffield.ac.uk
Naglik, Julian
King's College London
St Thomas Street
London
SE1 9RT, UK
julian.naglik@kcl.ac.uk
Netea, Mihai
Radboud University Nijmegen Medical
Center
Geert Grooteplein-Zuid 10
6525 GA Nijmegen
The Netherlands
M.Netea@aig.umcn.nl
Oliver, Jason
F2G Ltd
PO Box 1, Lankro Way
Eccles
Manchester, M30 0BH, UK
O’Neil, Deborah
NovaBiotics Ltd
Cruickshank Building, Craibstone
Aberdeen
AB21 9TR, UK
deborah@novabiotics.co.uk
Pandey, Neelam
University of Perugia
Via del Giochetto
06122 Perugia
ITALY
neelambiotech@yahoo.co.in

Pärnänen, Pirjo
University of Helsinki
P.O.Box 56 (Viikinkaari 5D)
FIN-00014
Finland
pirjo.parnanen@helsinki.fi
Popolo, Laura
Università degli Studi di Milano
Via Celoria 26
Milano
Italy
laura.popolo@unimi.it
Porter, Andy
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
a.porter@abdn.ac.uk
Posas, Francesc
Universitat Pompeu Fabra
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88 ; E-08003 Barcelona
Spain
francesc.posas@upf.edu
Quinn, Janet
Newcastle University
I Framlington Place
Newcastle upon Tyne
NE2 4HH
janet.quinn@ncl.ac.uk
Rosentul, Diana
Radboud University Nijmegen Medical
Center
Geert Grooteplein-Zuid 10
6525 GA Nijmegen
The Netherlands
D.RosentulAmram@aig.umcn.nl
Ruland, Jürgen
Technical University Munich
Ismaningerstr. 22,
81675 München,
Germany
jruland@lrz.tum.de
209
Saviuc, Crina
University of Bucharest
Faculty of Biology,
Portocalelor Street, nr 1-3
Bucharest
Romania
crina.saviuc@yahoo.com
Saxena, Abishek
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
abhisheksaxena@abdn.ac.uk
Schild, Lydia
Hans Knoell Institute (HKI)
Beutenbergstrasse 11a
07745 Jena
Germany
Lydia.Schild@hki-jena.de
Schröppel, Klaus
University of Tübingen,
Elfriede-Aulhorn-Straße 6
Tübingen
Germany
Klaus.Schroeppel@med.uni-tuebingen.de
Christoph Schüller
University of Vienna
Dr.Bohr-Gasse 9
A-1030 Vienna
Austria
Christoph.Schueller@univie.ac.at
Sedikki, Sidi Mohammed Lahbib
Université Abou bekr belkaid,
BP 119, Imama
13000 Tlemcen
Algeria
sdadek@hotmail.com
Silva-Dias, Ana
University of Porto
Alameda Prof. Hernani Monteiro
4200-319 Porto
PORTUGAL
asilvadias@med.up.pt

Silva, Sónia
University of Minho
Centre of Biological Engineering
4710-057 Braga
Portugal
soniasilva@deb.uminho.pt
Sorgo, Alice
University of Amsterdam
Nieuwe Achtergracht 166
1018 WV Amsterdam
The Netherlands
a.g.sorgo@uva.nl
Sullivan, Derek
Trinity College Dublin
Lincoln Place
Dublin 2
Ireland
derek.sullivan@dental.tcd.ie
Svobodová, Eliška
Hans Knoell Institute (HKI)
Beutenbergstrasse 11a
07745 Jena
Germany
Eliska.Svobodova@hki-jena.de
Tillmann, Anna
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
anna.tillmann@abdn.ac.uk
Tyc, Katarzyna
Humboldt-University
Theoretical Biophysics
Invalidenstraße 42
D-10115 Berlin, Germany
katarzyna.tyc@staff.hu-berlin.de
Usher, Jane
University of Exeter
Geoffrey Pope Building
Exeter
EX4 4QD, UK
J.Usher@exeter.ac.uk
210
Vecchiarelli, Anna
University of Perugia
Via del Giochetto
06122 Perugia
ITALY
vecchiar@unipg.it
Walker, Louise
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
louise.walker@abdn.ac.uk
Weil, Tobias
University of Aveiro
Campus Universitário de Santiago
3810-193 Aveiro
Portugal
tobias@ua.pt
Whiteway, Malcolm
NRC/BRI
6100 Royalmount Ave,
Montreal, Quebec
Canada H4P 2R2
malcolm.whiteway@nrc-cnrc.gc.ca
Wilson, Duncan
Hans Knoell Institute (HKI)
Beutenbergstrasse 11a
07745 Jena
Germany
Duncan.Wilson@hki-jena.de
Zabke, Claudia
University of Aberdeen
Institute of Medical Science
Aberdeen
AB25 2ZD, UK
c.zabke@abdn.ac.uk
Znaidi, Sadri
Pasteur Institute
25 rue du Docteur Roux
75015, Paris
France
sadri_znaidi@hotmail.com

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