|dc.description.abstract||Bacteroides fragilis is a member of the normal microbiota that resides in the human
lower gastrointestinal tract. This bacterium is of clinical significance because it is the
most frequently isolated Gram-negative obligate anaerobe from peritoneal abscesses
and bloodstream infections. Human fibrinogen is a hexameric-glycoprotein that is
important for fibrin-mediated abscess formation and limiting the spread of infection.
B. fragilis can bind and degrade fibrinogen which may aid in its escape from
abscesses into the bloodstream, thereby promoting bacteraemia.
In addition to fibrinogen, binding of B. fragilis to fibronectin, a component of the
extracellular matrix, found in association with fibrinogen at wound sites, has also
been reported. An outer membrane protein, BF1705, expressed by B. fragilis was
found to share homology with BspA from Tannerella forsythia which is known to
bind fibrinogen. The gene encoding BF1705 was deleted from the B. fragilis NCTC
9343 genome in the present work using a markerless gene deletion technology.
Proteins derived from the outer membranes of wild-type B. fragilis were able to bind
fibronectin and fibrinogen in far-western blots. Similar protein extracts from the
ΔBF1705 strain did not bind fibrinogen and fibronectin, which confirms the role of
BF1705 in adhesive interactions with proteins of the host extracellular matrix.
The possible involvement of BF1705 in fibrinogen degradation was ruled out
because the ΔBF1705 strain still degraded fibrinogen. To identify the proteases
involved in degradation of fibrinogen, four genes encoding putative extracellular
metallo- and serine proteases in the size range 45-50 kDa were deleted from the
NCTC 9343 genome. All of the single and multiple mutants defective in these
selected proteases were still capable of degrading fibrinogen as determined by
zymography. Expression of eight B. fragilis proteases in E. coli did not lead to
detectable degradation of fibrinogen. These observations suggest that these proteases
alone cannot degrade fibrinogen and either that an unidentified protease is
responsible for degradation or that there is redundancy in the proteases involved.
Under conditions of nitrogen limitation bacteria resort to scavenging nitrogen from
the environment to replenish the depleting intracellular nitrogen content. By
examining the differential regulation of the B. fragilis transcriptome under nitrogen
replete and depleting conditions, a potential role for BF1705 and secreted proteases
in nutrient binding and assimilation were studied. Growth on conventional glucose
defined medium with ammonia as the nitrogen source was compared to growth in
defined medium with glutamine as nitrogen source. A reduced doubling time and
diauxic growth in the medium containing glutamine indicated nitrogen limitation.
Comparison of the transcriptome derived from cultures of B. fragilis grown on either
ammonia or glutamine by RNA-Seq did not reveal a significant upregulation of
BF1705 in response to nitrogen limitation. This observation in conjunction with its
inability to degrade fibrinogen suggests that the primary role of BF1705 might be as
an adhesin and does not act directly in nutrient binding and degradation.
Nevertheless, nitrogen limitation was found to induce the expression of four
protease-encoding genes by over a 2-fold (adjusted p value < 0.05). The molecular
weight of three of these proteases were identified to be within the size range of 45-55
kDa which corresponded to the lysis bands detected by fibrinogen zymography with
wild-type B. fragilis protein extracts. Therefore the possible involvement of these
three proteases in fibrinogen degradation could be assessed. A 155-fold upregulation
(adjusted p value < 0.05) in asnB, encoding a homologue of asparagine synthetase B,
under conditions of nitrogen limitation suggest a previously uncharacterised aspartate
metabolism pathway for ammonia generation via arginine catabolism in B. fragilis.
Ammonia thus formed might aid in sustaining B. fragilis growth under nitrogen
deprived conditions. In addition to nitrogen assimilation, significant upregulation
was observed in the expression of genes involved in regulation of oxidative stress
and metronidazole resistance. The observed changes in the transcriptome will add to
our understanding of the B. fragilis metabolism and potential assist with unravelling
the mechanisms of infection mediated by this important opportunistic pathogen.||en