Hemolysin and the multifunctional autoprocessing RTX toxin are virulence factors during intestinal infection of mice with Vibrio cholerae El Tor O1 strains

Abstract

The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory toxins. In this study, we showed that 4- to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) toxin. Cholera toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory toxins were linked to virulence, a strain defective in the production of accessory toxins was still immunogenic since mice immunized with a multitoxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX toxin contribute to vaccine reactogenicity but that the genes for these toxins can be deleted from vaccine strains without affecting vaccine efficacy.

FIG. 1.

Rates of survival after infection with wild-type V. cholerae were dose dependent. C57BL/6 mice were inoculated with increasing doses of wild-type V. cholerae P27459, and survival was monitored over 7 days.

FIG. 2.

Survival after infection with V. cholerae. Mice were inoculated with various V. cholerae mutant strains expressing hemolysin (Y), MARTX_Vc (R), or HA/protease (P). Survival rates for mice inoculated with either wild-type P27459 (expressing CT, hemolysin, HA/protease, and MARTX_Vc; CYPR), CT mutant P4 (expressing hemolysin, HA/protease, and MARTX_Vc; YPR), or the multitoxin-deficient strain KFV101 (none) (A) or with strains expressing two accessory toxins (B) or only one accessory toxin as indicated (C) are shown. Quantitative data on survival and health are shown in Table 1.

FIG. 3.

Accumulations of sloughed cells (S) are observed in the lumina of mice inoculated with 10⁷ CFU of wild-type V. cholerae (A and B) but not in those of mice inoculated with the multitoxin-deficient mutant (C and D). No damage of the villus epithelia (V) in mice of any group is observed. Shown are representative H&E-stained areas of the distal small intestine 12 hpi. The scale bar represents 50 μm.

FIG. 4.

Hemolysin was not responsible for bacterial dissemination into the spleen and liver. Four mice were inoculated i.g. with 10⁸ CFU of CT mutant P4 (YPR) or KFV103 (P4 ΔhylA; PR). Small intestines (A), spleens (B), and livers (C) were collected 6 hpi, weighed, homogenized in 5 ml of PBS, and plated for CFU counting. Bacterial numbers per gram of organ for individual mice are shown, along with the medians for the groups.

FIG. 5.

Survival after infection with V. cholerae. (A) Mice were inoculated with 10⁸ CFU of either N16961 and Bah1 or the respective ΔhlyA mutants. (B) The entire hlyA gene was introduced into the lacZ loci of ΔhlyA strains KFV103 (PR) and KFV101 (none). Mice were inoculated with both ΔhlyA mutants and the complemented strains (PR::hlyA and none::hlyA). Rates of survival over 7 days are shown.

FIG. 6.

Prior vaccination with multitoxin-deficient mutant strain KFV101 protected mice from lethal challenge with wild-type P27459. Mice were either immunized with KFV101 (diamonds) or mock inoculated with PBS (triangles). On day 21, all mice were challenged with wild-type P27459, and survival was monitored.