SummaryWzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzz ST that results in long (L) modal length LPS with 16-35 Oag repeat units (RUs), we now report that wzz fepE , a homologue of Escherichia coli fepE , is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain > 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzz ST / wzz fepE double mutant, with relatively short, random-length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella . The presence of wzz fepE homologues in the genomic sequences of strains of Escherichia coli , Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.

A panel of isogenic Salmonella enterica serovar Typhimurium strains that vary only in the length of the O antigen was constructed through complementation of a wzz double mutant (displaying unregulated O-antigen length) with one of two homologous (wzz ST and wzz fepE ) or three heterologous (wzz O139 of Vibrio cholerae and wzz SF and wzz pHS-2 of Shigella flexneri) wzz genes. Each gene was functional in the S. enterica serovar Typhimurium host and specified production of O-antigen polymers with lengths typical of those synthesized by the donor bacteria (ranging from 2 to >100 O-antigen repeat units). By use of this panel of strains, it was found that O-antigen length influences invasion/uptake by macrophage cells; this is the first time this has been shown with Salmonella. O-antigen length was confirmed to be related to complement resistance, with a minimum protective length of >4 and <15 repeat units. O antigen of 16 to 35 repeat units was found to activate complement more efficiently than other lengths, but this was unrelated to complement resistance. No evidence was found to suggest that modifying the length of the O-antigen polymer affected expression of the O1, O4, or O5 antigenic factors.The lipopolysaccharide (LPS) of smooth gram-negative bacteria comprises three components-lipid A, the core oligosaccharide, and O antigen (Oag). Oag is a polymer of a sugar repeat unit (RU); the numbers of RUs attached to lipid A-core are clustered around a modal value determined by Wzz proteins (3, 26). Each wzz gene specifies synthesis of an Oag of a characteristic length. Salmonella enterica serovar Typhimurium possesses two Wzz proteins: Wzz ST , which confers a modal range of 16 to 35 RUs, and Wzz fepE , which confers a very long modal length estimated to be Ͼ100 RUs (20). Mutation of both of the corresponding genes resulted in a smooth, unregulated Oag phenotype characterized by predominantly shortlength Oag chains and significantly compromised virulence and complement resistance (20,21).The identification of wzz genes in many pathogenic bacterial species (18) suggests that the control of Oag length confers distinct advantages. Two functional wzz genes have been identified in Shigella flexneri (11), with the product of each wzz gene fulfilling a distinct role in pathogenesis (11,17,29). The interest in the role of S. enterica serovar Typhimurium Oag length is further increased by recent findings that this property is under regulatory control in vivo (2, 15, 21).Several approaches have been used to investigate the importance of Salmonella Oag length for pathogenic potential, but each has had its limitations. This study describes a new approach through complementation of a Salmonella enterica serovar Typhimurium C5 double wzz mutant with heterologous wzz genes. The resulting panel of strains was used to examine new and revisit previously investigated relationships between Oag length and virulence. Our results indicate that Oag modal length impacts upon S. enterica serovar Typhimurium virulence-related properties.Constru...

BackgroundKilled oral cholera vaccines (OCVs) have been licensed for use in developing countries, but protection conferred by licensed OCVs beyond two years of follow-up has not been demonstrated in randomized, clinical trials.Methods/Principal FindingsWe conducted a cluster-randomized, placebo-controlled trial of a two-dose regimen of a low-cost killed whole cell OCV in residents 1 year of age and older living in 3,933 clusters in Kolkata, India. The primary endpoint was culture-proven Vibrio cholerae O1 diarrhea episodes severe enough to require treatment in a health care facility. Of the 66,900 fully dosed individuals (31,932 vaccinees and 34,968 placebo recipients), 38 vaccinees and 128 placebo-recipients developed cholera during three years of follow-up (protective efficacy 66%; one-sided 95%CI lower bound = 53%, p<0.001). Vaccine protection during the third year of follow-up was 65% (one-sided 95%CI lower bound = 44%, p<0.001). Significant protection was evident in the second year of follow-up in children vaccinated at ages 1–4 years and in the third year in older age groups.Conclusions/SignificanceThe killed whole-cell OCV conferred significant protection that was evident in the second year of follow-up in young children and was sustained for at least three years in older age groups. Continued follow-up will be important to establish the vaccine's duration of protection.Trial RegistrationClinicalTrials.gov NCT00289224.

The role of the flagellar structure in the in vitro adherence and in vivo colonization of Vibrio cholerae was studied by comparing the behavior of pairs of related motility variants. Although the presence of the flagellum seemed essential for in vitro attachment, the property of motility was neither necessary nor sufficient. Since it was possible to demonstrate independently both motility and binding capacities associated with this structure, it was concluded that the flagellum functions as the carrier of the moieties that promote adherence. Studies using the infant mouse cholera model unequivocally demonstrated the in vivo significance of a functional flagellum and suggested that this structure enhances virulence by facilitating the initial colonization of the small bowel.