The ompU Paralogue vca1008 Is Required for Virulence of Vibrio cholerae

Abstract

We made single and combined mutations in ompU, ompT, and the two putative porin genes vca1008 and vc0972. The fitness of the strains was tested in vitro and in the infant mouse model of intestinal infection. We also studied the transcriptional induction of vca1008 in vitro and during mouse infection. We show that vca1008 is induced during infection and is necessary and sufficient (in the absence of ompU, ompT, and vc0972) for infection.Vibrio cholerae is a gram-negative bacterium and facultative pathogen that can cause an acute secretory diarrhea known as cholera. When V. cholerae enters a host it has to sense the new environment and induce an adaptive response that facilitates its survival and multiplication in the small intestine. ToxR is a transmembrane transcriptional activator that is part of a complex virulence gene regulon (the ToxR regulon) of more than 20 genes (9,12,17). The ToxR regulon is organized in two separate branches: the toxT-dependent and the toxT-independent branches. In the toxT-dependent branch, the transcriptional activator ToxT, controlled directly by ToxR, regulates the transcription of the cholera toxin and toxin-coregulated pilus and other factors essential for virulence (3). The toxTindependent branch includes two outer membrane porins called OmpU and OmpT (9). These two porins are directly and differentially regulated by ToxR in that ompU transcription is induced, whereas ompT transcription is repressed (2, 6, 9). There are some studies that suggest important functions for ompU during intestinal colonization, namely, increased resistance to bile and anionic detergents (13,14), an organic acid tolerance response (7), and adhesion to epithelial cells (18). However, one study reported that OmpU does not mediate adherence to rabbit intestinal epithelia (11), and another study reported that ⌬ompU and ⌬ompT strains exhibited no growth defect in vitro nor any detectable attenuation of virulence in infant mice (14).We recently used the recombination-based in vivo expression technology (RIVET) (1) to identify V. cholerae gene vca1008 as being transcriptionally induced during infection of the infant mouse small intestine (C. Osorio, J. Crawford, J. Michalsky, H. Martinez-Wilson, J. Kaper, and A. Camilli, unpublished data). This gene is one of three putative porin genes located on chromosome (Chr) II, and it encodes a protein that is closely related to the Chr I-encoded OmpU porin, having 33% identity and 55% similarity. The other Chr II-encoded putative porins, OmpS and OmpW, are orthologues of the Escherichia coli maltose-specific LamB and uncharacterized OmpW, respectively. The OmpU and VCA1008 paralogues are more closely related to the E. coli nonspecific porins OmpF, OmpC, and PhoE than are the other putative or known porins of V. cholerae (Fig. 1). In contrast, V. cholerae OmpT and VC0972 porins are only distantly related to these proteins (Fig. 1).The relatedness of OmpU and VCA1008 leads to the possibility of an overlap in their function, which might explain why mutations in ompU alone...

Second-Generation Recombination-Based In Vivo Expression Technology for Large-Scale Screening for Vibrio cholerae Genes Induced during Infection of the Mouse Small Intestine

“…This percentage (40%) is similar to the overall percentage (47%) of ivi genes that are required for virulence in animal models compiled from numerous IVET screening methods done on a number of different pathogens (1). Of the genes identified in this work, the most attenuated was VCA1008 (18). This result is interesting because VCA1008 was also the most highly induced ivi gene identified by our screening method.…”

“…In this and a related study (18), we found that 7 of the 10 ivi genes tested were important to achieve wild-type levels of intestinal colonization. Three of these mutants were only mildly attenuated, whereas four were attenuated Ͼ10-fold.…”

“…To determine if some of the identified ivi genes fall into the latter class, we made in-frame deletions of the entire coding sequence of nine ivi genes and tested each strain by competition assay during in vitro growth and infant mouse infection. The VCA1008 deletion strain was tested as part of a separate study of V. cholerae outer membrane porins (18); however, it should be noted that its level of attenuation in vivo was found in that study to be 40-fold but with no detectable growth defect in vitro. In this study, three deletion strains showed attenuation levels of more than 10-fold (Table 4).…”

Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

“…Likewise, a Vibrio cholerae vca1008 mutant shows no defective growth in vitro. However, during infection of mice, the vca1008 mutant strain is severely outcompeted by the wild-type strain, indicating that the Vca1008 porin is necessary and sufficient for virulence, while OmpU is dispensable (192).…”

OpnS, an Outer Membrane Porin ofXenorhabdus nematophila, Confers a Competitive Advantage for Growth in the Insect Host

“…It was recently shown that the constitutively expressed M35 porin of Moraxella catarrhalis was required for growth under nutrient-limiting conditions (10). In contrast, OpnS was not required for growth under nutrient- (7,31,33,34). We show that the ⌬opnS strain was not attenuated for virulence against M. sexta since it killed the insect host as well as the wild-type strain by both direct injection and by natural infection with nematodes.…”