Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection

Abstract

SummaryA complete understanding of host-parasite interactions must necessarily include the identification and characterization of gene products expressed by both parties during the infectious process. We have developed a new screen to identify bacterial genes that are transcriptionally induced during infection of a host animal. The method is based on pre-selection of strains carrying tnpR operon fusions (encoding resolvase, a site-specific DNA recombinase) which are not expressed in vitro, followed by screening for a subset of these strains that subsequently express resolvase within the host environment. The latter subset was recognized as recombinants that had deleted a resolvasespecific reporter construct. Thirteen transcription units of Vibrio cholerae were identified that were induced during infection in an infant mouse model of cholera. Five of these were predicted to encode polypeptides with diverse functions in metabolism, biosynthesis and motility; one encoded a secreted lipase; two appear to be antisense to genes involved in motility; and five are predicted to encode polypeptides of unknown function. Three of the transcripts were shown to be required for full virulence in infant mice, as assessed by competition experiments.

Environmentally regulated genes of Streptococcus suis: identification by the use of iron-restricted conditions in vitro and by experimental infection of piglets The GenBank accession numbers for the sequences determined in this work are AF302190–AF302207 (iri genes) and AF303226–303247 (ivs genes).

“…Clones ivs-6, 7, 13 and 14 showed similarity to a gene, iviVI, previously identified by IVET selection in V. cholerae (Camilli & Mekalanos, 1995). The function of iviVI is unknown.…”

“…suis, genes. Three of these sequences (iri-2, iri-1, 6 and 22, and iri-34) were similar to sequences of environmentally regulated genes previously selected by applying the IVET to Vibrio cholerae (Camilli & Mekalanos, 1995), Staphylococcus aureus (Lowe et al, 1998) and Pseudomonas aeruginosa (Wang et al, 1996), respectively. One, contained in iri-1, 6 and 22, was similar to the agrA gene of Staph.…”

“…suis gene putatively involved in virulence (Smith et al, 1993(Smith et al, , 1996 ; and ivs-31, which was similar to the fibronectin-binding protein of Streptococcus gordonii. Moreover, the sequences of two ivs genes (ivs-25 and ivs-6, 7, 13 and 14) were homologous to two environmentally regulated ivi genes, previously identified using IVET selection in other bacterial species (Camilli & Mekalanos, 1995 ;Lowe et al, 1998). Four ivs sequences (ivs-25 ; ivs-23 and 24 ; ivs-2, 4 and 28 ; and ivs-6, 7, 13 and 14) were also found when the library was selected using iron-restricted conditions.…”

“…Recently, several approaches have been reported that allow the identification of genes that are specifically expressed in the host. Examples are signature-tagged mutagenesis (STM) and in vivo expression technology (IVET ; Mahan et al, 1993Mahan et al, , 1995Camilli & Mekalanos, 1995 ;Hensel et al, 1995 ;Mei et al, 1997 ;Young & Miller, 1997 ;Chiang & Mekalanos, 1998 ;Coulter et al, 1998 ;Lowe et al, 1998 ;Polissi et al, 1998 ;Camacho et al, 1999 ;Darwin & Miller, 1999 ;Edelstein et al, 1999 ;Fuller et al, 1999 ;Zhao et al, 1999). In addition, important virulence proteins could also be identified by the selection of genes specifically expressed under conditions mimicking in vivo conditions, for example by growth under ironrestricted conditions (Litwin & Calderwood, 1993 ;Martinez et al, 1990).…”

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

“…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).…”