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

Abstract

A 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 resolvase-specific 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.

Fig. 1

Schematic diagram of the screening protocol used to identify V. cholerae transcripts that are induced in the host. See the Results for details.

Fig. 2

A. Similarities of deduced V. cholerae polypeptide partial sequences. Vertical lines indicate identical residues, highly conserved substitutions are represented by colons and conserved substitutions are represented by dots. Gaps were introduced into the sequences to maximize the similarities (see the Experimental procedures). For each V. cholerae polypeptide sequence shown, the C-terminal amino acid delineates the corresponding DNA site of fusion to tnpR–lacZY. B. Schematic diagram (not to scale) of predicted V. cholerae genes containing fusions to tnpR–lacZY (designated by triangles). Transcriptional orientations are shown by the arrows.

Fig. 3

Similarities of deduced V. cholerae polypeptide partial sequences (A) and predicted genes (B). Details are as in the caption to Fig. 2, except that only for IvilV does the C-terminal amino acid delineate the corresponding DNA site of fusion to tnpR–lacZY. The DNA site of fusion to tnpR–lacZY in AC-V233 and AC-V234 occurred 51 bp 3′ of the orf4 stop codon and within the codon for amino acid 45, respectively. Putative transcriptional terminators are designated by short stem–loop symbols. A putative σ²⁸-dependent promoter is shown immediately upstream of orf5. B. b. and P. a. refer to Bordetella pertussis and Pseudomonas aeruginosa, respectively.

Fig. 4

Similarities of deduced V choterae polypeptide partial sequences (A) and predicted genes (B). Details as in the legend to Fig. 2.

Fig. 5

Secreted lipase activity from wild-type V. cholerae C6709-1 and the hlyC mutant AC-V195. The plate contained LB agar plus emulsified tributyrin and was incubated at 37°C for 48h.

Fig. 6

Similarities of deduced V. cholerae polypeptide partial sequences (A) and predicted genes (B). Details are as in the caption to Fig. 2 except that the N-terminal amino acid for IviVI delineates the corresponding DNA site of fusion to tnpR–lacZY.