Research Report 2010 2011 - Helmholtz-Zentrum für ...

SCIENTIFIC REPORTS | Infection and Immunity | Pharmaceutical Research 113
05.4 Identification of Molecular Targets of Antiinfectives
PROJECT LEADER | Prof. Dr. Ursula Bilitewski | Research Group Biological Systems Analysis | ubi@helmholtz-hzi.de
PROJECT MEMBERS | Dr. Nina Klippel | Dr. Bianca Lüderitz | Anna Buschart | Shuna Cui | Mohammed El-Mowafy |
Daniela Evers | Katja Gremmer | Rabeay Hassan | Hani Kaba | Carolin Lewark | Dörthe Sokolis
New therapies of infections require new antimicrobial
compounds, which exploit new molecular targets to avoid
already existing antibiotic resistances. We focused on commensal
and persistent microorganisms, and chose the yeast
Candida albicans and the bacteria Mycobacterium bovis
(BCG) as substitute of the tuberculosis pathogen Mycobacterium
tuberculosis. These microorganisms are adapted to the
survival in the presence of host cells, and become infectious
in patients with an impaired immune system.
Histidine kinases as molecular targets of anti-infectives
Histidine kinases are mainly found in bacteria, but also
in fungi. Mostly they are sensor proteins and induce the
adaptation of the microorganisms to their environment. We
had previously shown that the histidine kinase CaNik1 from
Candida albicans is the molecular target of the myxobacterial
secondary metabolites ambruticin VS3 and jerangolid
A, as we transferred sensitivity for these fungicides to the
otherwise resistant yeast S. cerevisiae by heterologous
functional expression of the CaNik1 gene. We identified a
pairwise sequence of 9 so-called HAMP-domains, each of
which comprises approximately 50 amino acids. Deletion of
selected pairs of these domains from CaNik1 reduced the
sensitivity for fungicides, with the magnitude of the effect
being dependent on the compound. At present investigations
are performed to elucidate further details of the interaction
of the protein with fungicides. Gene expression analysis
showed that in the S. cerevisiae-transformants fungicides,
which target CaNik1, mainly activate the signal transduction
pathway of the osmotic stress defense network, which
includes effects on cell cycle and thus on cell proliferation.
Exploitation of new targets for anti-infectives Candida
albicans usually colonizes human skin and mucosa (mouth,
gastro-intestinal tract, vagina). Systemic infections
include penetration of the underlying cell layers, followed
by invasion of deeper tissues and spread to inner organs
via the bloodstream. An essential pathogenicity factor is
the capability to switch morphology between yeast and
hyphal forms. Among primary immune defense reactions
is phagocytosis and stimulation of the immune system by
activation of macrophages and neutrophils. Macrophages
are also involved in the immune defense against infections
of the lung by mycobacteria. However, these bacteria can
survive phagocytosis as persistent, dormant organisms and
are hardly eliminated by present antibiotics.
To empirically exploit new targets for anti-infectives, we
simulate environmental conditions, which the microorganisms
face in the human host. We test whether small molecules
can enhance the clearing efficiency of phagocytes,
The interaction between the yeast Candida albicans and cells
of the epithelium (cell line A431) in the presence of the peptide
LL37. LL37 belongs to the anti-microbial peptides and does not
inhibit the growth of C. albicans, but it supports the adhesion
of the yeast to human cells. After an incubation time of 6 hours
(conditions of cell cultures), C. albicans forms hyphen which
can be found in the cells of the epithelium as well, where they
also continue to grow. Photo: HZI, Rohde
block the morphology change of C. albicans and the survival
of dormant mycobacteria. Sources of those compounds are
libraries of chemical compounds, extracts from myxobacteria
and commercially available inhibitors. We could show
that all steps of the infectious process can be influenced
by chemical compounds, some of which are endogenous
products of the organisms. Human cells produce peptides,
which manipulate the adhesion of C. albicans to surfaces, in
particular to components of the extracellular matrix. The
formation of hyphae is suppressed by compounds which are
formed by C. albicans as quorum sensing compounds. Finally,
we discovered that genistein, a well-known constituent of
soybeans, stimulates phagocytosis of genistein-treated
C. albicans. Further studies aim at the elucidation of underlying
molecular mechanisms.
Wesolowski,J., Hassan,R.Y.A., Reinhardt,K., Hodde,S. & Bilitewski,U. (2010) Antifungal
compounds redirect metabolic pathways in yeasts: Metabolites as indicators of modes of
action, Journal of Applied Microbiology 108, 462 - 471
Klippel,N., Cui,S., Gröbe,L. & Bilitewski,U. (2010) Deletion of the Candida albicans histidine
kinase gene CHK1 improves recognition by phagocytes through an increased
exposure of cell wall ß-glucans, Microbiology 156, 3432 – 3431
Buschart,A., Gremmer,K., v.d.Heuvel,J. Müller,P.P. & Bilitewski,U. (2011) Repetitive
HAMP domains at the N-terminus of the CaNik1 histidine kinase of Candida albicans
mediate specific interactions with fungicides, Molecular Microbiology, submitted