Non-O1 Vibrio cholerae NRT36S produces a polysaccharide capsule that determines colony morphology, serum resistance, and virulence in mice

Abstract

Non-O1 Vibrio cholerae produced two distinct colony types, designated as opaque and translucent. NRT36S, a clinical isolate shown to be virulent in volunteers, produced predominantly opaque colonies, but translucent colonies appeared on subculture. Opaque variants were recovered exclusively following exposure to normal human serum or animal passage. A nonreverting translucent mutant of NRT36S, JVB52, was isolated following mutagenesis with the transposon Tn5 IS50L::phoA (TnphoA). Only translucent colonies were produced by a nonpathogenic environmental isolate, A5. Electron microscopic examination of the opaque form of NRT36S revealed thick, electron-dense, fibrous capsules surrounding polycationic ferritin-stained cells. The ferritin-stained material around translucent NRT36S or A5 was patchy or absent. JVB52 had a thin but contiguous capsular layer. The amount of ferritin-stained capsular material correlated with the amount of surface polysaccharide determined by phenol-sulfuric acid assay: opaque NRT36S had approximately three times as much polysaccharide as translucent NRT36S or A5 and four times as much as JVB52. The encapsulated, opaque variant of NRT36S was protected from serum bactericidal activity, while translucent non-O1 V. cholerae was readily killed. The encapsulated form also had increased virulence in mice. Our data provide the first indication that non-O1 V. cholerae strains can have a polysaccharide capsule. This capsule may be important in protecting the organism from host defenses and may contribute to the ability of some non-O1 V. cholerae strains to cause septicemia in susceptible hosts.