S1 De voorjaarsbijeenkomst van de Nederlandse ... - NVMM

to modulate immune responses by suppressing IL-12
production by dendritic cells (DCs). Although this effect
could clearly be ascribed to the glucan, the underlying
mechanism remained unexplained. The goal of this project
was to unravel this mechanism by identifying the host
alpha-glucan receptors and investigating their role in
glucan-mediated immunomodulation. For this, we screened
the glucan-binding capacity of various lectins using
lectin-Fc constructs and found that alpha-glucan specifically
interacts with the C-type lectin DC-SIGN. This lectin was
previously shown to be important for the binding to and
modulation of DCs by mycobacteria. To see whether alphaglucan
could also bind to DC-SIGN on the cell surface, we
incubated fluorescently-labelled polystyrene beads coated
either with alpha-glucan or with human serum albumin
as a control together with Raji cells, Raji cells expressing
DC-SIGN, or DCs and analysed binding of the beads by
flow cytometry. The results showed that the glucan-coated
beads specifically bound to both the DCs and the Raji cells
expressing DC-SIGN, but not to the Raji cells alone, and
that this interaction could be abrogated by pre-incubating
with mannan or with antibodies that block DC-SIGN.
These results clearly demonstrate that mycobacterial alphaglucan
binds to DC-SIGN and that this interaction may be
important for its immunomodulatory activity.
Alpha-glucan is not unique to mycobacteria and similar
molecules can be found in a number of species including
some fungi. Interestingly, alpha-glucan from the fungus
Pseudallescheria boydii has been reported to induce the
secretion of pro-inflammatory cytokines in a mechanism
involving toll-like receptor 2 (TLR2). To test whether
mycobacterial alpha-glucan can also activate TLR2, we
incubated TLR2-transfected HEK293 cells with alphaglucan
and measured the TLR2-dependent production of
IL-8. Interestingly, our results showed that mycobacterial
alpha-glucan, like the fungal glucan, can activate TLR2.
Thus, besides binding to DC-SIGN, alpha-glucan can also
bind and activate TLR2. Exactly how this interaction occurs
and what influence it has on the host immune-response is
currently unknown. Our current experiments are aimed at
providing insight into these important questions.
O069
The Mycoplasma pneumoniae MPN229 gene encodes a
protein that selectively binds single-stranded dNA and
stimulates RecA-mediated dNA strand exchange
C. Vink, M. Sluijter, T. Hoogenboezem, N.G. Hartwig
Laboratory of Pediatrics, Dept. of Pediatric Infectious Diseases,
Erasmus University Medical Centre, Rotterdam
Mycoplasma pneumoniae has previously been characterised
as a micro-organism that is genetically highly
stable. In spite of this genetic stability, homologous DNA
Ned Tijdschr Med Microbiol 2008;16:Supplement
S30
recombination has been hypothesised to lie at the basis
of antigenic variation of the major surface protein, P1, of
M. pneumoniae. In order to identify the proteins that may
be involved in homologous DNA recombination in M.
pneumoniae, we set out to characterise the MPN229 open
reading frame (ORF), which bears sequence similarity
to the gene encoding the single-stranded DNA-binding
(SSB) protein of Escherichia coli. The MPN229 ORF has
the capacity to encode a 166-amino acid protein with a
calculated molecular mass of 18.4 kDa. The amino acid
sequence of this protein (Mpn SSB) is most closely related
to that of the protein predicted to be encoded by the MG091
gene from Mycoplasma genitalium (61% identity). The
MPN229 ORF was cloned, and Mpn SSB was expressed
in E. coli and purified. The Mpn SSB protein was found
to: i) exist primarily as dimer in solution, ii) selectively
bind single-stranded DNA in a divalent cation- and DNA
substrate sequence-independent manner, and iii) stimulate
E. coli RecA-promoted DNA strand exchange.
We conclude that Mpn SSB represents the M. pneumoniae
counterpart of the E. coli SSB protein. The results from this
study will pave the way for unraveling the DNA recombinatorial
pathways in M. pneumoniae.
O070
Comparative genomic profiling of dutch clinical Bordetella
pertussis isolates using dNA microarrays: identification of
genes absent from epidemic strains
A.J. King1 , T. van Gorkom1 , H. G. J. van der Heide1 , Q. He2 ,
J. Pennings1 , F.R. Mooi1 1Cib-Laboratory for Infectious Diseases, National Institute
for Public Health and the Environment (RIVM), Bilthoven,
2
KTL, Turku, Finland
Introduction: Bordetella pertussis causes whooping cough or
pertussis in humans and is particularly severe in infants.
Despite vaccination, whooping cough remains a public
health problem. Since the 1990s, a significant increase in
the B. pertussis incidence was observed in many countries.
Also in the Netherlands, an increase in B. pertussis has been
observed since 1996. Several causes for the re-emergence
have been suggested, there is evidence that pathogen
adaptation also plays a role. Numerous studies have demonstrated
that the B. pertussis population has changed since
the start of vaccination. In several countries, the B. pertussis
population was found to be variable since polymorphisms
in surface proteins were found. In the Netherlands, we
observed that strains with a particular allele of the ptx
promoter (ptxP) i.e. ptxP3 have expanded in the Dutch B.
pertussis population. The increase in frequency coincided
the increase in B. pertussis notifications in the Netherlands.
We aimed to use microarrays to identify additional changes
in strains associated with the Dutch epidemic.