Thymine auxotrophy as an attenuating marker in Vibrio cholerae

Vibrio cholerae CVD102 is a thymine-dependent auxotroph of CVD101, a cholera toxin A-B+ candidate live oral cholera vaccine. Previous clinical experience with these strains suggested that, by restricting intestinal growth, thymine auxotrophy is attenuating for V. cholerae. Studies in the infant mouse cholera model cast doubt upon this conclusion however. Stable thyA mutants selected from each of three pathogenic strains showed unimpaired gut colonization in mixed-infection competition experiments. Similar results were obtained using thyA mutants selected from two atoxigenic strains, including CVD101. Further studies with CVD102 showed that the reduced colonization potential of this strain could not be compensated by the provision of a functional thyA+ gene in trans. CVD102 shows reduced synthesis of toxin-coregulated pili (TCP) during in vitro growth, suggesting the presence of a second, undefined mutation in this strain. Given the critical role of TCP in intestinal colonization, it seems probable that this previously unrecognized mutation is responsible for the poor in vivo performance of CVD102.