Type III secretion is essential for the rapidly fatal diarrheal disease caused by non-O1, non-O139 Vibrio cholerae

Abstract

Cholera is a severe diarrheal disease typically caused by O1 serogroup strains of Vibrio cholerae. The pathogenicity of all pandemic V. cholerae O1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139 V. cholerae strains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139 V. cholerae has not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139 V. cholerae pathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected with V. cholerae O1. Unlike V. cholerae O1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain's virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139 V. cholerae represents a new type of diarrheagenic mechanism.

FIG 1

Gross and histologic features of infant rabbits infected with the wild-type or ΔvcsN2 V. cholerae AM-19226 strain. (A) Infant rabbits were orogastrically inoculated with either sodium bicarbonate buffer control or 10⁹ CFU of the wild-type (WT) or ΔvcsN2 AM-19226. All rabbits infected with wild-type AM-19226 developed severe watery diarrhea, which is evident as extensive wetness in the perianal region and hind legs. The arrow indicates the boundary between wet and dry fur. (B) Representative intestines from rabbits infected with the indicated strain or buffer control are shown (SI, small intestine). (C) Representative H&E-stained sections of the small intestines of infant rabbits are shown. Top images, ×10 magnification; bottom images, ×40 magnification.

FIG 2

Transcriptional levels of proinflammatory cytokines and chemokines are elevated in the small intestines of rabbits infected with V. cholerae AM-19226. RNA was isolated from homogenates of small intestines of infant rabbits inoculated with buffer (control) or the indicated strains. The levels of transcripts of TNF-α (Α), IL-8 (B), IL-1β (C), and IL-6 (D) were determined by quantitative real-time PCR, and all the values were normalized to the housekeeping gene HPRT. Each symbol represents the value for an individual rabbit, and the bar indicates the mean for the group. An asterisk indicates that the values for the WT samples were significantly different (P < 0.05) from those in the buffer control and ΔvcsN2 samples.

FIG 3

Intestinal colonization of WT and ΔvcsN2 AM-19226 strains. The numbers of bacterial CFU recovered from sections taken from the proximal, mid, and distal small intestines (SI), ceca, and midcolons of rabbits infected with WT AM-19226 or the ΔvcsN2 mutant are shown. Bars represent the mean values from 15 or 16 rabbits.

FIG 4

Representative confocal images of GFP-expressing WT and ΔvcsN2 AM-19226 strains in the rabbit small intestine. Distal small intestinal sections from rabbits infected with GFP-labeled WT or ΔvcsN2 AM-19226 were prepared at 12 hours postinfection. Tissues were stained with Alexa Fluor 568-labeled phalloidin (red) to visualize the F-actin and counterstained with wheat germ agglutinin (WGA) (blue) to visualize mucin.

FIG 5

Intestinal colonization of V. cholerae AM-19226 TTSS effector mutants. Infant rabbits were inoculated with the indicated AM-19226 derivatives containing deletions of genes encoding TTSS effectors. The numbers of bacterial CFU recovered from sections taken from the proximal (A), mid (B), and distal small intestines (SI) (C), ceca (D), and midcolons (E) of infant rabbits are shown. Bars represent the geometric mean values.

FIG 6

Importance of TCP and TTSS in V. cholerae V51 for intestinal colonization. Infant rabbits were inoculated with the indicated derivatives of the TCP⁺ CT⁺ TTSS⁺ strain V51. The numbers of bacterial CFU were determined in sections taken from the proximal (A), mid (B), and distal (C) small intestines (SI), ceca (D), and midcolons (E) of infant rabbits. Bars represent the geometric mean values.