Genetic analysis of Vibrio parahaemolyticus intestinal colonization

Abstract

Vibrio parahaemolyticus is the most common cause of seafood-borne gastroenteritis worldwide and a blight on global aquaculture. This organism requires a horizontally acquired type III secretion system (T3SS2) to infect the small intestine, but knowledge of additional factors that underlie V. parahaemolyticus pathogenicity is limited. We used transposon-insertion sequencing to screen for genes that contribute to viability of V. parahaemolyticus in vitro and in the mammalian intestine. Our analysis enumerated and controlled for the host infection bottleneck, enabling robust assessment of genetic contributions to in vivo fitness. We identified genes that contribute to V. parahaemolyticus colonization of the intestine independent of known virulence mechanisms in addition to uncharacterized components of T3SS2. Our study revealed that toxR, an ancestral locus in Vibrio species, is required for V. parahaemolyticus fitness in vivo and for induction of T3SS2 gene expression. The regulatory mechanism by which V. parahaemolyticus ToxR activates expression of T3SS2 resembles Vibrio cholerae ToxR regulation of distinct virulence elements acquired via lateral gene transfer. Thus, disparate horizontally acquired virulence systems have been placed under the control of this ancestral transcription factor across independently evolved human pathogens.

Keywords: Vibrio parahaemolyticus; bacterial pathogenesis; pathogen evolution; transposon-insertion sequencing; type III secretion.

Fig. 1.

EL-ARTIST gene classifications. (A) Distribution of percentage disruption for genes with 10+ TA sites. Genes classified as underrepresented (UR), regional (R), and neutral (N) are represented within each bin and in aggregate. (B) Transposon-insertion profiles of representative underrepresented, regional, and neutral genes. The vertical axis indicates the number of reads mapped to a TA site along the horizontal axis.

Fig. S1.

TIS statistics. The number of reads mapped to TA sites in the V. parahaemolyticus genome, the number of unique TA sites disrupted, the percentage of TA sites disrupted, and the average number of mapped reads per insertion site are reported for each analysis of the V. parahaemolyticus transposon-insertion library.

Fig. 2.

Bioinformatic analysis of regional and underrepresented genes. (A) Abbreviated COG terms with statistically significant, differential representation in the regional and underrepresented datasets (exp.) relative to random sampling of the V. parahaemolyticus genome (sim.). (B) Status of E. coli and V. cholerae homologs of the V. parahaemolyticus genes classified as regional and underrepresented. E/DE, essential or domain-essential; NE, nonessential.

Fig. S2.

Additional analysis of datasets. COG terms associated with V. parahaemolyticus regional or underrepresented genes (yellow) compared with COG terms associated with V. cholerae domain-essential or essential genes (red) (14). *Adjusted-P < 0.05, a COG term with statistically significant, differential representation in either dataset (exp.) relative to random sampling of the organism’s genome (sim.).

Fig. 3.

Identification of conditionally depleted genes. (A) Distribution of percentage disruption for genes subjected to statistical analysis (Materials and Methods) in the bacterial inoculum (Inoculum) and a representative passaged library (Distal Small Intestine). Genes classified subsequently as conditionally depleted (blue) and all other genes queried (orange) are represented within each bin. (B) Transposon-insertion profiles of the neutral locus of vscN1 and of vscN2, a representative conditionally depleted gene. The vertical axis indicates the number of reads mapped to a TA site along the horizontal axis for the simulated inoculum and a representative passaged library (Distal SI).

Fig. 4.

Bioinformatic analysis of conditionally depleted genes. (A) Predicted structural schematic of T3SS2 indicating components that are conditionally depleted (blue), not queried in our analysis (black), or lacking known V. parahaemolyticus homologs (gray with dashed lines). (B) COG terms with statistically significant, differential representation in the conditionally depleted dataset (exp.) relative to random sampling of the 3,744 genes queried (sim.).

Fig. S3.

Conditionally depleted genes: expression and T3SS2 components. (A) Overlap between V. parahaemolyticus conditionally depleted genes and transcriptional profiling in vivo (8) for genes assessed in both studies (orange and blue), conditionally depleted genes (blue), and genes not queried in our analysis (black). (B) Conditionally depleted genes encoded in the T3SS2 gene cluster are listed along with functions inferred from previous reports or HHPred predictions.

Fig. 5.

Targeted validation of conditionally depleted genes. (A) Competitive indices of deletion mutants following coinfection of the distal small intestine (blue circles) or in vitro outgrowth (white squares). Deletion strains display in vivo competitive indices significantly lower (adjusted-P < 0.05) than the negative control (orange circles). Horizontal lines indicate the geometric mean of independent measurements. (B) T3SS2 activity of deletion strains (****P < 0.0001). vscN1 was deleted from all strains to eliminate T3SS1-mediated cytotoxicity.

Fig. 6.

toxR is required for induction of vtrB transcription. (A) Relative abundance of mRNA transcripts in the presence or absence of crude bile. ND, none detected. (B) VtrA-His₆ expression in the presence or absence of crude bile via immunoblotting (∼29 kDa). Due to elevated VtrA-His₆ levels, less lysate was run for samples containing pvtrA-his₆. IPTG, isopropyl β-d-1-thiogalactopyranoside; α-RNAP, α-RNA polymerase; wt, wild type.

Fig. 7.

Expression of vtrB bypasses the requirement for toxR. (A) Immunoblotting of the T3SS2 component VopD2 in ∆toxR strains grown in crude bile and exogenously expressing vtrB (pvtrB) or an unrelated gene (pcyA). (B) T3SS2 activity in ∆toxR strains grown in crude bile and exogenously expressing pvtrB or pcyA (****P < 0.0001). vscN1 was deleted from all strains to eliminate T3SS1-mediated cytotoxicity. ND, none detected.

Fig. S4.

toxR is required for T3SS2 expression in wild-type and Sm^R V. parahaemolyticus. T3SS2 expression was assessed for strains grown in the presence or absence of crude bile via immunoblotting against VopD2 (a component of T3SS2). (A) Wild-type (Wt) V. parahaemolyticus, the Sm^R parent strain, isogenic toxR mutants constructed in either background, and mutant strains exogenously expressing toxR (ptoxR) all grown in 1 μM isopropyl β-d-1-thiogalactopyranoside (IPTG). (B) ∆toxR strains exogenously expressing toxR (ptoxR), vtrA (pvtrA-his₆), or vtrB (pvtrB). α-RNAP, α-RNA polymerase.