Identifying Unusual Bacterial- Eukaryotic Homologs

1
www.pathogenomics.bc.ca
Introduction Genomics and bioinformatics provide
powerful new tools for the study of microbial
pathogenicity, hence the development of a new
field, Pathogenomics. Our Pathogenomics project
utilizes a combination of informatics,
evolutionary biology, microbiology and eukaryotic
genetics to identify pathogen genes which are
more similar to host genes than expected, and
likely to interact with, or mimic, their hosts
gene functions. Currently, our project has been
divided into two complementary fields of
phylogenetic and functional analysis. Within the
phylogenetic analysis, we have developed software
which aids identification of horizontally
acquired sequences in hope that this approach
will enabled us to not only identify new
potential virulence factors, but also gain
insight into the frequency of horizontal gene
transfer within the bacteria, and between the
three domains of life of Bacteria, Eukarya, and
Archaea. Candidate virulence factors identified
by our informatics approach are being targeted
for further functional study using a
Caenorhabditis model for infection. The
utilization of Caenorhabditis as a model organism
offers numerous advantages for functional genetic
analysis including its small size, ease of
maintenance, transparent morphology, rapid
generation time, completely sequenced genome, and
the large availability of well-developed genetic
and molecular tools. In addition, recent
published literature has demonstrated that C.
elegans can be successfully infected with
Pseudomonas aeruginosa, Bacillus megaterium, and
Salmonella typhimurium, thus demonstrating
C.elegans as a suitable host model for functional
analysis of virulent genes during the infection
process.
Caenorhabditis as a model for infection Rationale
Previous literature has demonstrated successful
infection in C. elegans using pathogens such as
Pseudomonas aeruginosa, Bacillus megaterium, and
Salmonella typhimurium, therefore we rationalized
that it would be feasible to establish similar
infection models using C. elegans and enteric
bacteria such as Yersinia enterocolitica and
Listeria monocytogenes. Infection Assay
Attempts Initial infection assays between C.
elegans and Y. enterocolitica failed to
establish a successful infection model and we
investigated several possible factors that would
contribute to lack of bacterial infection within
the C. elegans host model. These factors
included choice of liquid and solid media, pH,
salt content, and incubation temperature.
Utilization of Thermotolerant Caenorhabditis
species Temperature Incubation Dilemma The
optimal growth temperature for C. elegans is 20oC
and the maximum temperature that C. elegans
remains viable and fertile is 25oC. Temperatures
exceeding 25oC result in worm infertility and
death. However, virulent gene expression in
enteric pathogens is regulated by temperature,
with the optimal temperature being 37oC. For
example, in Yersinia species, the virulent genes
yadA and psaA, as well as the yop operons, are
up-regulated at 37oC and down-regulated at
temperatures below 26oC. We theorized that a
possible explanation of unsuccessful infection of
C. elegans with a pathogenic enteric bacterium
could be due to the lack of virulent gene
expression during room temperature incubation.
To circumvent this problem, a compromise between
the optimal bacterial temperature and the optimal
nematode temperature was suggested and we
proposed the utilization of a thermotolerant
worm. C. briggsae as a Thermotolerant Host The
search for Caenorhabditis mutants that are
capable of remaining viable and fertile at higher
temperatures than 25oC, resulted in the
acquisition of two Caenorhabditis species namely,
a C. elegans daf-2 mutant and the wildtype C.
briggsae, var. Gujarati, G16. We performed
thermotolerant testing on both candidate
Caenorhabditis species to evaluate the maximum
temperature each organism could survive and
remain fertile. From our analysis, we determined
that C. briggsae exhibited the highest
thermotolerance, remaining viable and fertile at
30oC after 72 h. Genetic Analysis of the
Pathogenecity Process Currently we are utilizing
C. briggsae G16 as the host model
of choice in our infection assays with
Y. enterocolitica and L. monocytogenes at
30oC with the goal of successfully establishing
an infection model. Once established, functional
analysis of the putative gene products that are
conserved within C. elegans and bacteria will be
performed to elucidate possible virulent factors
that are involve during the infection process.
Identifying Unusual Bacterial- Eukaryotic
Homologs Rationale Pathogen proteins have been
identified that manipulate host cells by
interacting with, or mimicking, host proteins.
We wondered whether we could identify selected
novel virulence factors by identifying bacterial
pathogen genes more similar to host genes than
you would expect based on phylogeny. A web-based
tool we developed investigates this, producing a
database of such proteins. It is also useful for
identifying cross-domain lateral gene transfer
events between the three domains of life of
Bacteria, Archaea and Eukarya, hence we named
this database BAE-watch. This tool was used to
aid identification of any possible cases of
cross-domain horizontal gene transfer between all
complete bacterial and eukaryotic genomes,
including the C. elegans genome. Description of
the BAE-watch database Proteins in a given
pathogen genome that are more similar to
eukaryote proteins than other proteins (and vice
versa) are identified through BLAST analysis,
followed by use of a StepRatio scoring system
we developed (to screen out of the analysis most
proteins that are highly conserved in all
organisms, that BLAST may list as most similar to
a protein from another Domain by chance).
Various taxonomic levels of organisms are
filtered from the BLAST results to aid
identification of putative lateral transfers that
occurred before or after species, genus, family
etc divergence. This database includes an
analysis of the C. elegans genome (see next
poster section). Analysis of complete bacterial
genomes A comprehensive analysis of all complete
bacterial genomes for eukaryotic homologs, using
BAE-watch and subsequent phylogenetic analysis,
suggests that recent horizontal gene transfer
between bacteria and eukaryotes has been rare.
However, some unusual cases of bacterial-eukaryoti
c homology have been identified and are being
targeted for further functional study, with the
aim of using C. elegans as an infection
model.
Acknowledgements This project is funded by the
Peter Wall Institute for Advanced Studies

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