Functional analysis of the role of alternative sigma factors in Vibrio parahaemolyticus host pathogen interactions
Date
2015
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Vibrio parahaemolyticus, a ubiquitous Gram-negative marine bacterium, is the leading
cause of bacterial seafood borne gastroenteritis in humans. This organism must adapt
to both host and marine environmental stresses. One mechanism bacteria employ to
respond to rapid environmental changes is the switching on of specific gene
expression patterns through the use of alternative sigma factors. V. parahaemolyticus
has 11 sigma factors, compared to Escherichia coli which has 7. This work examined
the distribution and functionality of sigma factors in V. parahaemolyticus. Distribution
analysis of V. parahaemolyticus sigma factors among Vibrionacea revealed varying
levels of conservation of each sigma. All members of Vibrionaceae were found to
have single copies of sigmas RpoD and RpoH. A majority of Vibrio species possess
FliAP, the polar flagella sigma factor; conversely distribution of the lateral flagella
sigma factor, FliAL, is mostly clade specific and unique to V. parahaemolyticus,
compared to other notable pathogens Vibrio cholerae and Vibrio vulnificus. All
studied species possess a single copy of RpoS, however 22 species possess 1 to 3
additional copies of a divergent RpoS-like sigma factor. The greatest amount of
diversity is within the ECF subfamily; here we demonstrate there are 3 highly
conserved ECFs, which include RpoE, and several others which are less conserved
and show more variation. Upon finding that a number of V. parahaemolyticus sigma
factors are highly conserved, expression levels of these sigmas were compared in
minimal media (M9 glucose) to complex media (M9 mucus). RpoD, RpoH and RpoE
were found to be highly expressed under both conditions. Ecf3, which is highly
conserved, was found to be the only sigma factor to be highly induced in M9 mucus.
As alternative sigma factor, RpoE (VP2578), was highly expressed and is highly
conserved, its role in V. parahaemolyticus biology was investigated.
In this species, RpoE was shown to be important in in vivo fitness as well as
survival under polymyxin B, ethanol, and high temperature stresses. In contrast,
deletion of the regulator, RpoS, did not alter in vivo survival and is only limitedly
involved in stress response in this organism. Additionally, the role of the outer
membrane protein, OmpU, in RpoE signaling was investigated. OmpU is proposed to
be the sole activator of RpoE in Vibrio cholerae. We found that an ompU deletion
mutant and the rpoE mutant did not have overlapping phenotypes indicating OmpU is
not essential for RpoE function in V. parahaemolyticus under the conditions
examined. The function of the most divergent ECF (VP0055) found in V.
parahaemolyticus was also investigated.
VP0055 was found to be in close proximity to the gluconate catabolism gene
cluster. Previously gluconate catabolism genes were shown to be upregulated in an
rpoN mutant strain, as was VP0055. The rpoN mutant strain was a hypercolonizer of
an adult streptomycin treated mouse model compared to wild-type and carbon
catabolism differences may be involved in this phenotype. The role of VP0055 and
RpoN in the potential regulation of gluconate catabolism was investigated. Genes in
the gluconate catabolism gene cluster were found to be induced in the wild type in M9
gluconate relative to M9 glucose. The gluconate catabolism genes were found to be
unchanged in the vp0055 mutant, additionally, the vp0055 mutant did not demonstrate
a growth defect in M9 glucose, gluconate or mucus, suggesting it is not significantly
involved in gluconate catabolism. In contrast the rpoN mutant grows better than wildtype
in M9 gluconate and M9 mucus and genes involved in gluconate catabolism are
upregulated in this mutant, suggesting that RpoN is involved in regulation of
gluconate catabolism, acting as a repressor through an unknown indirect mechanism.
The significance of gluconate catabolism in V. parahaemolyticus was also investigated
through the construction of a deletion mutant of the canonical aldolase (VP0065) of
the Entner-Doudoroff pathway which is involved in gluconate catabolism.
Surprisingly, it was found that VP0065 (eda), is important but not essential for growth
in M9 gluconate or M9 mucus. In E.coli, EDA mutants are unable to utilize gluconate
as the sole carbon source. It was hypothesized that this unusual phenotype may be
attributed to either increased flux through the pentose phosphate pathway (PPP) or the
presence of non-canonical aldolases partially compensating for the loss of vp0065.
Bioinformatics analysis determined that the presence of two additional aldolases on
chromosome two (VPA0083 and VPA1708) in V. parahaemolyticus. Expression
analysis in M9 gluconate relative to M9 glucose demonstrated that VP1708, the first
gene in the pentose phosphate pathway, and all three putative aldolases are induced in
gluconate. These data indicate a potential role for multiple aldolases and PPP in
gluconate catabolism in this species. Both VPA1708 and VP1708 were expressed at
higher levels in the vp0065 mutant grown in gluconate compared to wild-type.
Together these data demonstrate that gluconate catabolism and regulation is complex
in V. parahaemolyticus.