Francisella tularensis is able to survive and grow within macrophages, a trait that contributes to pathogenesis. Several genes have been identified that are important for intramacrophage survival, including mglA and iglC. F. tularensis is also able to survive within amoebae. It is shown here that F. tularensis mglA and iglC mutant strains are not only defective for survival and replication within the macrophage-like cell line J774, but also within Acanthamoebae castellanii. Moreover, these strains are highly attenuated for virulence in mice, suggesting that a common mechanism underlies intramacrophage and intraamoebae survival and virulence. A 2D gel analysis of cell extracts of wild-type and mglA mutant strains revealed that at least seven prominent proteins were at low levels in the mglA mutant, and one MglA-regulated protein was identified as the IglC protein. RT-PCR analysis demonstrated reduced transcription of iglC and several other known and suspected virulence genes in the mglA mutant. Thus, MglA regulates the transcription of virulence factors of F. tularensis that contribute to intramacrophage and intraamoebae survival.

SummaryThroughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a threedimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.

Recently we described the isolation of spontaneous bacteriophage K139-resistant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galU gene. We inactivated another gal gene, galE, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not the galE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that galU and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found that galU and galE are essential for the formation of a biofilm in a spontaneous phageresistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of galU in V. cholerae virulence, correlated with the observed change in LPS structure, and a role for galU and galE in environmental survival of V. cholerae.The causative agent of the intestinal disease cholera is Vibrio cholerae, a gram-negative motile bacterium. Of the more than 150 known serogroups, only the noncapsulated O1 and the encapsulated O139 serogroup have been found to be associated with epidemic cholera. Epidemic O139 strains are related to and were derived from O1 El Tor strains after genetic alterations of the O-antigen biosynthesis gene cluster (16). The ongoing seventh pandemic, which began in 1961, is caused by O1 El Tor strains (3). V. cholerae is a natural inhabitant of aquatic ecosystems and is known to attach to environmental surfaces such as plants, filamentous green algae, zooplankton, crustaceans, or insects (8). Recently, V. cholerae O1 El Tor was found to form a three-dimensional biofilm on abiotic surfaces (70). Biofilm formation may be important in the life cycle of pathogenic V. cholerae strains, because they reside within natural aquatic habitats during interepidemic periods. O1 El Tor strains are also able to switch to a rugose colony phenotype. This morphology correlates with the constitutively production of an exopolysaccharide allowing biofilm formation on abiotic surfaces (65...

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, ⌬ompU1, removed the coding sequence for 84 internal amino acids (aa), while the second (large) deletion, ⌬ompU2, removed the coding sequence for the entire amino-terminal 274 aa. The ⌬ompU1 strain had a growth defect that could not be complemented by episomal expression of full-length ompU. In contrast, a strain with ⌬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 ⌬ompU1 phenotype. A large deletion removing the coding sequence for the entire N-terminal 273 aa of OmpT (⌬ompT) was also constructed in wild-type as well as ⌬toxR and ⌬ompU2 strains, and these strains displayed wild-type growth kinetics in rich media. However, the ⌬ompU2 strain was deficient for growth in deoxycholate compared to wild-type, ⌬ompT, and ⌬ompU2 ⌬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 ⌬ompU2, ⌬ompT, and ⌬ompU2 ⌬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.