Selection for in vivo regulators of bacterial virulence

Abstract

We devised a noninvasive genetic selection strategy to identify positive regulators of bacterial virulence genes during actual infection of an intact animal host. This strategy combines random mutagenesis with a switch-like reporter of transcription that confers antibiotic resistance in the off state and sensitivity in the on state. Application of this technology to the human intestinal pathogen Vibrio cholerae identified several regulators of cholera toxin and a central virulence gene regulator that are operative during infection. These regulators function in chemotaxis, signaling pathways, transport across the cell envelope, biosynthesis, and adherence. We show that phenotypes that appear genetically independent in cell culture become interrelated in the host milieu.

Figure 1

RIVET-based selection for positive regulators of virulence gene expression during infection. (A) A schematic diagram of RIVET-based selection strategy is shown. Transposon mutagenesis was performed on derivatives of the El Tor biotype V. cholerae strain, C6709–1, containing the res-tet-res cassette and either a ctxA∷tnpR or toxT∷tnpR transcriptional fusion. Three rounds of in vitro enrichment and animal passage were done as described in Materials and Methods. (B) After each animal passage, but before Tc-enrichment, a portion of the recovered bacteria was examined for previous induction of the toxT∷tnpR or ctxA∷tnpR fusion during infection by replica plating colonies onto LB agar containing Tc. An increase in the percentage of Tc^R cfu after each animal passage indicates an overall enrichment of mutant strains that fail to induce expression of the toxT∷tnpR or ctxA∷tnpR fusion during the period of infection.

Figure 2

Chemotaxis in semisolid agar and production of flagella. (A) Chemotactic ability of each mutant was determined by stabbing cells into swarm plates. Strain genetic backgrounds are indicated above each swarm location. The ΔvieSAB, ΔtoxR, ΔtoxT, and ΔtcpA strains were described (9, 10). (B–G) Production of the polar flagellum was determined by electron microscopy. The genetic background of each strain is indicated at bottom right corner.

Figure 3

Induction patterns of the toxT∷tnpR fusion in various strain backgrounds in vitro and during infection. (A) Induction profile of toxT∷tnpR fusion strains grown for 7 h either in LB broth (a normally noninducing condition), or in AKI broth (an inducing condition) was measured as percent Tc-sensitive cfu. The genetic backgrounds are denoted on the x axis. The data shown are the means from multiple independent experiments. (B and C) Induction kinetics of V. cholerae strains in infant mice. The data shown are the means from multiple independent experiments. Data at 5 h were tested for statistical significance by using the Student's two-tailed t test: Strains with reduced induction of the gene fusion compared with wild type (P < 0.1) are denoted by asterisks.

Figure 4

Colonization of the stomach and small intestine of infant mice by wild-type and nonchemotactic V. cholerae strains. Population dynamics of V. cholerae isogenic strains, AC-V66 (wild-type) and a nonchemotactic strain, cheY-3D52N, were determined during infection of infant mice. The stomach and small intestine were removed at 5 h after inoculation, and the latter was cut into six equal length segments as designated on the x axis. The number of bacteria recovered from each segment relative to the input inoculum is shown on the y axis. The values shown are the means from four mice per V. cholerae strain.

Figure 5

Induction patterns of the ctxA∷tnpR fusion in various strain backgrounds in vitro and during infection. (A) The induction of ctxA∷tnpR was determined as described in the Fig. 3 legend. †, Percentage of Tc-sensitive cfu for the ΔmotAB strain was determined by testing 16 independently constructed strains for Tc sensitivity. ND, induction with the ΔmotAB strain in AKI broth was not tested because of constitutive activity of the ctxA∷tnpR fusion in this strain background. Strains with reduced induction of the gene fusion compared with wild-type (P < 0.01) are denoted by asterisks. (B and C) The induction kinetics of ctxA∷tnpR during infection were determined and statistically analyzed as described in the Fig. 3 legend. Strains with reduced induction of the gene fusion compared with wild type (P < 0.02) are denoted by asterisks.