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

Abstract

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...

An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers

“…8). There is a very good correlation between the genes identified in this human study and those identified in the screen of this same IVET library in the infant mouse model (3). Of the 40 genes that were confirmed to be in vivo induced in the mouse in that study, 83% were identified in this human screen by fusions to those genes or to the putative operons containing them.…”

“…The recombination-based IVET system used in this work and the construction of this exact library have been described in detail (3). The library consists of clones that contain a chromosomal transcriptional fusion to a promoterless resolvase gene (tnpR) and an unlinked neo-sacB cassette flanked by recognition sequences (res) for the resolvase [supporting information (SI) Fig.…”

“…Some are probably transcribed from a nearby convergent promoter; however, in some cases there is no candidate promoter. As discussed (3,27,28), these fusions may reflect cryptic protein-coding genes, or transcribed but nontranslated sequences such as antisense or small regulatory RNAs and may be worth further investigation given the increasing numbers of small RNAs with important regulatory functions (29).…”

“…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.…”

“…This reactogenicity (diarrhea and other symptoms) makes CVD110 unsuitable for vaccine purposes, but excellent for our goal of identifying genes induced in vivo in a human infection. This same IVET library was used previously in an infant mouse model (3), allowing comparison of the ivi gene sets. In this human IVET study we identified 217 putative human ivi genes of V. cholerae.…”