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...

In vivo expression technology (IVET) has been widely used to study gene expression of human bacterial pathogens in animal models, but has heretofore not been used in humans to our knowledge. As part of ongoing efforts to understand Vibrio cholerae pathogenesis and develop improved V. cholerae vaccines, we have performed an IVET screen in humans for genes that are preferentially expressed by V. cholerae during infection. A library of 8,734 nontoxigenic V. cholerae strains carrying transcriptional fusions of genomic DNA to a resolvase gene was ingested by five healthy adult volunteers. Transcription of the fusion leads to resolvasedependent excision of a sacB-containing cassette and thus the selectable phenotype of sucrose resistance (Suc R ). A total of Ϸ20,000 Suc R isolates, those carrying putative in vivo-induced fusions, were recovered from volunteer stool samples. Analysis of the fusion junctions from >7,000 Suc R isolates from multiple samples from multiple volunteers identified 217 candidate genes for preferential expression during human infection. Of genes or operons induced in three or more volunteers, the majority of those tested (65%) were induced in an infant mouse model. VC0201 (fhuC), which encodes the ATPase of a ferrichrome ABC transporter, is one of the identified in vivo-induced genes and is required for virulence in the mouse model. gene expression ͉ genetics ͉ vaccinology ͉ virulence O ur understanding of the complex interactions between bacterial pathogens and humans relies heavily on the use of animal and tissue culture model systems that serve as surrogates of human infection. One useful genetic tool for discovering genes and pathways involved in virulence is in vivo expression technology (IVET), which was designed to identify genes of pathogens that are preferentially expressed during infection and has been extensively used in model systems (reviewed by refs. 1 and 2). IVET is a promoter-trapping strategy in which cells carrying a library of transcriptional fusions of genomic DNA to a reporter gene are used to infect a model host and those carrying fusions that are expressed in vivo can be identified by either a genetic screen or selection. This technique allows the identification of genes that may be expressed only under in vivo conditions [in vivo-induced genes (ivi genes)]. Such genes may be very difficult to identify during growth under laboratory conditions, but are likely to be important in the host for survival and virulence. One form of IVET, used in this work, is recombination-based IVET (3, 4) in which the reporter gene encodes a resolvase that effects a permanent genetic change, allowing a direct selection for cells that expressed the resolvase even transiently during infection.Here, we describe the use of recombination-based IVET to identify genes of Vibrio cholerae, the causative agent of the diarrheal disease cholera, that are expressed during human infection. This approach has the potential to identify important virulence genes that may not be expressed in vitro or during in...

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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...