Characterization of the role of the ToxR-modulated outer membrane porins OmpU and OmpT in Vibrio cholerae virulence

ToxR, the transmembrane regulatory protein required for expression of virul ence factors in the human diarrheal pathogen Vibrio cholerae, directly acti vates and represses the transcription of two outer membrane porins, OmpU an d OmpT, respectively. In an attempt to dissect the role of the OmpU and Omp T porins in viability and virulence factor expression, in-frame chramosomal deletions were constructed in the coding sequences of ompU and ompT of V. cholerae, Two separate deletions were introduced into ompU; the first (smal l) deletion, Delta ompU1, removed the coding sequence for 84 internal amino acids (aa), while the second (large) deletion, Delta ompU2, removed the co ding sequence for the entire amino-terminal 274 aa, The Delta ompU1 strain had a growth defect that could not be complemented by episomal expression o f full-length ompU, In contrast, a strain with Delta ompU2 displayed wild-t ype growth kinetics in rich media, suggesting that this is the true phenoty pe of a strain lacking OmpU and that the truncated OmpU protein, rather tha n the absence of OmpU, may be the cause for the Delta ompU1 phenotype, A la rge deletion removing the coding sequence for the entire N-terminal 273 aa of OmpT (Delta ompT was also constructed in wild-type as well as Delta toxR and Delta ompU2 strains, and these strains displayed wild-type growth kine tics in rich media, However, the Delta ompU2 strain was deficient for growt h in deoxycholate compared to wild-type, Delta ompT, and Delta ompU2 Delta ompT strains, reinforcing a positive role far the OmpU porin and a negative role for the OmpT porin in V. cholerae resistance to anionic detergents. T he Delta ompU2, Delta ompT, and Delta ompU2 Delta ompT strains exhibited wi ld-type levels of in vitro virulence factor expression and resistance to po lymyxin B and serum and in vivo colonization levels similar to a wild-type strain in the infant mouse intestine. Our results demonstrate that (i) OmpU and OmpT are not essential proteins, as was previously thought; (ii) these porins contribute to V. cholerae resistance to anionic detergents; and (ii i) OmpU and OmpT are not essential for virulence factor expression in vitro or intestinal colonization in vivo.