Identification of an iron-regulated virulence determinant in Vibrio cholerae, using TnphoA mutagenesis

Several virulence determinants of bacteria are regulated by the concentration of iron in the medium, with increased expression occurring under low-iron conditions. Iron-regulated virulence factors have not been previously described in Vibrio cholerae. We used the transposon vector Tn5 IS50L::phoA (TnphoA) to obtain insertion mutations in iron-regulated genes of V. cholerae 0395. One strain, carrying an insertion mutation in iron-regulated gene irgA, had reduced virulence in an animal model and had lost the 77-kilodalton major iron-regulated outer membrane protein. The 50% lethal dose of this mutant strain (MBG40) in suckling mice was 3 x 10(5) bacteria compared with 4 x 10(3) bacteria for 0395 wild type. In an in vivo competition assay, the wild-type strain out competed the mutant almost 10-fold, suggesting a possible colonization defect in MBG40. The in vitro competitive index and in vitro growth curves in low- and high-iron media showed that the mutant strain had no discernable growth defect. These data suggest that the 77-kilodalton iron-regulated outer membrane protein may play a role in the pathogenesis of V. cholerae infection. It is not yet clear whether the reduction of virulence in MBG40, as assessed by 50% lethal dose analysis, is explained by a colonization defect or whether it relates to another defect associated with loss of the IrgA protein.