Functional analysis of the role of alternative sigma factors in Vibrio parahaemolyticus host pathogen interactions

Date
2015
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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.
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