Characterization of Vibrio cholerae O1 El Tor galU and galE Mutants: Influence on Lipopolysaccharide Structure, Colonization, and Biofilm Formation

Abstract

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...

A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface

“…This is exemplified by P. aeruginosa, as the biofilm mode of growth confers antibiotic resistance within the lungs of patients with cystic fibrosis (Costerton et al, 1995). A frequently used assay for biofilm formation on abiotic surfaces is to grow the bacteria on polystyrene and then measure the absorbance after staining with crystal violet (Nesper et al, 2001;Solano et al, 2002); this assay is not appropriate for measurement of biofilm formation on a biotic surface such as C. elegans. We have shown the presence of an extracellular matrix containing bacteria on C. elegans infected with Y. pseudotuberculosis by EM (Fig.…”

“…Mutation of galU and galE in Vibrio cholerae causes production of an altered LPS core oligosaccharide; mutants are unable to synthesize exopolysaccharide (VPS) or to form a biofilm (Nesper et al, 2001). Clearly bacterial polysaccharides are intrinsic to the formation of the biofilm in the Y. pseudotuberculosis/C.…”

“…Wild-type and mutant strains of C. elegans were maintained in the laboratory on E. coli OP50. Biofilm formation on polystyrene, that is on an abiotic surface, was measured as described by Nesper et al (2001) with minor modifications. Briefly, bacteria were grown overnight in LB, diluted 1 : 10 in fresh LB, and 100 ml aliquots grown overnight in a round-bottom 96-well microtitre plate (Nunc) (in triplicate).…”

Ralstonia solanacearum RSc0411 (lptC) is a determinant for full virulence and has a strain-specific novel function in the T3SS activity

“…2d). Lessened tolerance to various antimicrobial compounds consistently reveals damage in the mutants' cell envelope integrity and may be due to the decreased LPS production (Martinez de Tejada et al, 1995;Nesper et al, 2001). However, the unaltered proliferation of the RSc0411 mutant in N. benthamiana leaf 24 h post infiltration (Fig.…”

Genome sequence of Yersinia pestis, the causative agent of plague

“…The O side chain mediates resistance to complement-mediated and phagocyte killing. It might also have a role in survival in the gut by protecting the bacterium from cationic peptides, such as those produced by Paneth cells in the human small intestine 25 . Yersinia pestis produces a rough LPS, lacking an O antigen as a consequence of these mutations within the biosynthesis cluster of the O antigen 3 , but the nature of the selective advantage this may confer to Y. pestis is unexplained.…”