Isolation and phenotypic characterization of virulence-deficient mutants of Vibrio cholerae

Abstract

Mutants of Vibrio cholerae was isolated on the basis of reduced ability to induce diarrhea in orally challenged infant mice. Nitrosoguanidine-treated clones were screened for low fluid accumulation ratios in individual mice, and presumptive mutants were confirmed in additional mouse tests. Mutants were examined for alterations in phage type, motility, toxin production, proteolytic activity, neuraminidase production, amylase production, morphology, growth requirements, carbohydrate fermentations, in vitro growth patterns, and cell surface alterations. The types of mutants found included several with previously recognized virulence-associated markers (rough, nonmotile, toxin deficient, protease deficient); several types with pleiotropic alterations (cell morphology, decreased extracellular products); and several with no previously recognized virulence-deficient phenotype (purine requiring, cell surface altered, rapid death in vitro, no defect found). Dose-response kinetics showed that most mutants could provoke diarrhea if given in 100-fold greater numbers than the dose used for screening. Recovery of viable organisms from the gut late in infection showed reduction of survival and/or multiplication capacity for the mutants, with variation in the degree of reduction for the different classes.

Identification of two previously unrecognized genes (guaA and argC) important for uropathogenesis

“…In fact, during the vital step of microbial proliferation in vivo, if certain nutrients are unavailable or withheld within the host, then the capacity of a pathogen to synthesize or acquire these nutrients becomes crucial. Although factors utilized to acquire iron are well accepted as being important for pathogenesis (Litwin and Calderwood, 1993), the importance of acquiring or synthesizing other nutritional factors is less appreciated but supported by a variety of both old (Bacon et al, 1950;Garber et al, 1952;Furness and Rowley, 1956;Levine and Maurer, 1958;Ivanovics et al, 1968;Hatch, 1975;Baselski et al, 1978) and more recent reports (Hosieth and Stocker, 1981;Straley and Harmon, 1984;Austin et al, 1987;Nnalue and Stocker, 1987;O'Callaghan et al, 1988;Bowe et al, 1989;Leung and Finlay, 1991;Mahan et al, 1993;Marquis et al, 1993;McAdam et al, 1995;Ullman and Carter, 1995). Furthermore, the frequent identification of biosynthetic genes as virulence traits in studies that have screened libraries of random gene fusions or randomly generated transposon mutants emphasizes the importance of metabolic genes in microbial pathogenesis (Fields et al, 1986;Leung and Finlay, 1991;Mahan et al, 1993).…”

Use of signature‐tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization

“…Although purine auxotrophs of several pathogens, including V. cholerae, have been shown previously to be attenuated (Ivanovics et al, 1968;Baselski et al, 1978;Straley and Harmon, 1984;McFarland and Stocker, 1987), biotin auxotrophy has never been reported to cause attenuation.…”

Identification and preliminary characterization of Vibrio cholerae outer membrane proteins

“…The influence of stage of growth on outer membrane proteins was examined by using outer membrane preparations from strain CA401 (grown in Syncase medium) at early log growth phase, middle log growth phase, and stationary 42,000-51,00.0b Red doublet 91.7 6 35,000…”

“…Rough cholera strains are avirulent (16), and recent mutant studies suggest that other cell surface changes may play a role in the virulence of V. cholerae (6). We studied the outer membrane proteins of V. cholerae strain CA401, a virulent wild-type strain.…”

“…MATERIALS AND MErHODS Bacterial strain. V. cholerae CA401 is a classical Inaba strain isolated in 1953 in Calcutta, India (22), which has been extensively characterized (3)(4)(5)(6)35). Stock cultures were kept lyophilized or frozen at -70°C in brain heart infusion broth plus 15% glycerol.…”