SUMMARY
Escherichia coli bacteria are not only harmless intestinal inhabitants, but some
strains can also be highly adapted pathogens, capable of causing diseases. As a
pathogen, E. coli is best known for its ability to cause intestinal diseases. However, it
is also associated with extra-intestinal diseases. The most common extra-intestinal
pathogenic E. coli infections in humans are caused by uropathogenic E. coli (UPEC).
These cause urinary tract infections (UTIs), affecting millions of people each year.
Nearly half of all women will experience at least one UTI during their lifetime, and
25% of these women will experience frequent recurrent infections within months of
the initial infection. Subpopulations with an increased risk for UTIs are for example
the elderly, patients with diabetes or patients with catheters. With most pathogens
acquiring resistance to the available antibiotics today, the development of new
therapeutic approaches has taken centre stage. In order to develop new treatment
and prevention approaches for the control of UPEC infections, more has to be
known about the virulence mechanisms these strains use during the infectious
process.
The aims of this Ph.D. thesis concerned the contribution to the knowledge of the
virulence mechanisms of the human cystitis isolate E. coli UTI89, a frequently
studied model UPEC strain.
In the first part of the project, the focus lay on virulence factors with a potential role
in attachment and biofilm formation.
Attachment to host tissues is mediated by long hair-like extracellular appendages
called fimbriae. Type 1 and P fimbriae are proven and well-studied virulence factors
for E. coli in the urinary tract; therefore their role was not further investigated. The
potential role of the sfa, F17-like, yad, yqi, yeh and yfc fimbrial operons of E. coli
UTI89 was studied. Specific mutants of two important genes encoding the putative
usher and adhesin were constructed.
Not only is the attachment to host cells important for E. coli UTI89 pathogenesis, but
also the ability to form biofilms. Biofilms have been found on urinary catheters;
moreover UPEC strains can also form biofilm-like structures inside human bladder
epithelial cells. To gain more insight into the complex biofilm formation process, the
influence of genes which are known to contribute to biofilm formation in for
example E. coli K-12 (lrhA and csgD) or in E. coli ABU-strains (yqgA), were also
analyzed in UPEC. Newly identified genes, like for example yfaL, could also have a
putative but unknown role in biofilm formation.
Manipulation of the E. coli UTI89 genome, using the available methods, turned out to
be problematic. Therefore, construction of the specific mutants in E. coli UTI89 was
carried out using a modified Red-mediated gene targeting procedure with 500600 bp long homology regions that was developed for this aim (Derous et al., 2011).
After the successful construction of the specific mutants, their properties were
studied in different in vitro (i.e. biofilm formation, motility, adhesion to and invasion
in
human
bladder
epithelial
cell
assays)
and
in
vivo
models
(i.e. Caenorhabditis elegans colonization and mouse model).
Results do not provide evidence for a possible role of the putative usher and/or
adhesin of the yeh, yfc or yad operon of E. coli UTI89, in urinary tract infections.
However, the roles of other (putative) virulence factors like for example the F17-like
fimbriae, the yqi operon or the gene yfaL would be interesting to investigate further.
In the second part of this project, a library of transposon-insertion mutants was
constructed in which genes encoding exported proteins were targeted, in the effort
to look for new genes that could contribute to E. coli UTI89 pathogenesis.
Eleven in frame UTI89NalR::TnblaM mutans were identified. Genes which play a role
in different stages of the infection process of UPEC were targeted by this random
insertion method. For example, genes involved in iron acquisition (chuA, which is
responsible for heam uptake), genes which play a role in LPS synthesis (waaI,
waaL), in the flagellar capping protein FliD, genes belonging to the pap, yeh and F17like fimbrial operons, etc.
However, none of the targeted genes encoding exported proteins, of the random
insertion library, were interesting for further investigation in this project, as no
novel functions involved in UPEC pathogenesis were revealed. Known virulence
genes were targeted, which underlines that the transposon insertion strategy which
was used works.
The results from the UTI89NalR::TnblaM library constructed in this project suggest
that searching for unidentified genes in E. coli UTI89 by a random transposon
approach, may be rewarding.