The
 ompU
 Paralogue
 vca1008
 Is Required for Virulence of
 Vibrio cholerae

Osório, Carlos; Martinez-Wilson, Hector; Camilli, Andrew

doi:10.1128/jb.186.15.5167-5171.2004

Cited by 25 publications

(26 citation statements)

References 20 publications

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“…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.…”

Section: Discussionsupporting

confidence: 69%

“…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.…”

Section: Discussionmentioning

confidence: 94%

“…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).…”

Section: Improvements Of Rivetmentioning

confidence: 99%

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

et al. 2005

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We have constructed an improved recombination-based in vivo expression technology (RIVET) and used it as a screening method to identify Vibrio cholerae genes that are transcriptionally induced during infection of infant mice. The improvements include the introduction of modified substrate cassettes for resolvase that can be positively and negatively selected for, allowing selection of resolved strains from intestinal homogenates, and three different tnpR alleles that cover a range of translation initiation efficiencies, allowing identification of infection-induced genes that have low-to-moderate basal levels of transcription during growth in vitro. A transcriptional fusion library of 8,734 isolates of a V. cholerae El Tor strain that remain unresolved when the vibrios are grown in vitro was passed through infant mice, and 40 infection-induced genes were identified. Nine of these genes were inactivated by in-frame deletions, and their roles in growth in vitro and fitness during infection were measured by competition assays. Four mutant strains were attenuated >10-fold in vivo compared with the parental strain, demonstrating that infection-induced genes are enriched in genes essential for virulence.Much remains to be learned about genes that the facultative pathogen Vibrio cholerae induces during infection and how their protein products function during the complex and dynamic process in which this pathogen adapts to the human small intestine. Several new methods have been developed that are helping us to explore this process, including in vivo expression technology (IVET), signature-tagged mutagenesis, microarray technology, differential fluorescence induction, in vivo-induced antigen technology, and real-time reverse transcription-PCR, among others. A specific IVET method, recombination-based IVET (RIVET), has been used previously to identify V. cholerae genes that are induced during infection of infant mice (1, 4). RIVET is very sensitive to low or transient expression of in vivo-induced (ivi) genes during infection and is therefore capable of identifying members of this potentially interesting class of genes. However, this sensitivity is also a double-edged sword, as some ivi genes have low-to-moderate levels of expression in vitro and will therefore be lost during library construction, i.e., there will be premature excision (resolution) of the selectable cassette in such strains. In the present study, we have developed a modified RIVET that can overcome this main disadvantage and used it as a large-scale screening method to identify V. cholerae genes that are transcriptionally induced during infant mouse infection. Briefly, the new system incorporates two different resolvable cassettes that differ in the efficiency of excision, as well as three different alleles of the resolvase-encoding gene tnpR that have different efficiencies of translation initiation. These modifications extend the number of V. cholerae strains that are unresolved in vitro that can be generated in the final library. Additional modificatio...

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Section: Discussionsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 94%

Section: Improvements Of Rivetmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2005

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“…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).…”

Section: Genes Involved In Cell Envelope Structure and Modificationmentioning

confidence: 99%

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

Rediers

Rainey

Vanderleyden

et al. 2005

Microbiol Mol Biol Rev

140

120

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A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms

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“…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.…”

Section: Discussionmentioning

confidence: 80%

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

Hoeven

Forst

2009

J Bacteriol

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The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded ␤-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ⌬opnS strain. Coinjection of the wild-type and ⌬opnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ⌬opnS strain were colonized by the wild-type strain. In addition, the ⌬opnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ⌬opnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.

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The ompU Paralogue vca1008 Is Required for Virulence of Vibrio cholerae

Cited by 25 publications

References 20 publications

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

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

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

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

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