Characterization of the Role of the ToxR-Modulated Outer Membrane Porins OmpU and OmpT in Vibrio cholerae Virulence

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Abstract
ToxR, the transmembrane regulatory protein required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae , directly activates and represses the transcription of two outer membrane porins, OmpU and OmpT, respectively. In an attempt to dissect the role of the OmpU and OmpT porins in viability and virulence factor expression, in-frame chromosomal deletions were constructed in the coding sequences of ompU and ompT of V. cholerae . Two separate deletions were introduced into ompU ; the first (small) deletion, Δ ompU1 , removed the coding sequence for 84 internal amino acids (aa), while the second (large) deletion, Δ ompU2 , removed the coding sequence for the entire amino-terminal 274 aa. The Δ ompU1 strain had a growth defect that could not be complemented by episomal expression of full-length ompU . In contrast, a strain with Δ ompU2 displayed wild-type growth kinetics in rich media, suggesting that this is the true phenotype of a strain lacking OmpU and that the truncated OmpU protein, rather than the absence of OmpU, may be the cause for the Δ ompU1 phenotype. A large deletion removing the coding sequence for the entire N-terminal 273 aa of OmpT (Δ ompT ) was also constructed in wild-type as well as Δ toxR and Δ ompU2 strains, and these strains displayed wild-type growth kinetics in rich media. However, the Δ ompU2 strain was deficient for growth in deoxycholate compared to wild-type, Δ ompT , and Δ ompU2 Δ ompT strains, reinforcing a positive role for the OmpU porin and a negative role for the OmpT porin in V. cholerae resistance to anionic detergents. The Δ ompU2 , Δ ompT , and Δ ompU2 Δ ompT strains exhibited wild-type levels of in vitro virulence factor expression and resistance to polymyxin 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 (iii) OmpU and OmpT are not essential for virulence factor expression in vitro or intestinal colonization in vivo.