The O3 antiserum bound in the same amount and pattern in ∆CPS mutant as in wild type (Figure 4) indicating that the major operon between gmhD and rjg, i. e. VP0219-0237, is not involved in O antigen synthesis. Immunoblots developed with K6 antiserum only detected the high molecular TPCA-1 weight Selleckchem SAHA polysaccharide (Figure 4) in the wild type O3:K6. The high molecular weight of the K-antigen is consistent with capsular polysaccharide. Binding of K6 antiserum was lost in the ∆CPS

mutant indicating that region B is required for K antigen biosynthesis. Stains-all/Silver-stain also showed that the high molecular weight capsular polysaccharide was lost in the ΔCPS mutant (Figure 4). Figure 4 Immunoblots and stains-all/silver-stain of V. parahaemolyticus. Whole cells lysate treated with DNase, RNase and pronase

was separated on polyacrylamide gel, transferred to PVDF membrane and probed with K6 specific antiserum (A), or O3 specific antiserum (B). Total polysaccharides were visualized by stains-all/silver-stain on polyacrylamide gel (C). lane 1, wild type VP53; lane 2, ∆CPS mutant; lane 3, ∆EPS mutant; lane 4, ∆wzabc mutant; lane 5, ∆0220 mutant; lane 6, ∆0220 mutant with trans-complementation; lane 7, ∆VP215-218 mutant. We further investigated the surface structural change in the ∆CPS mutant by immuno-gold EM using K6 antiserum (Figure 5). The EM image of wild type O3:K6 showed gold particles localized around the exterior MLN4924 clinical trial of the cell consistent with a capsule-like structure surrounding the cell. GNA12 This capsule structure was absent from ∆CPS mutant and there was no specific gold particle binding to the cell. Figure 5 Immuno-gold labeling TEM of V. parahaemolyticus with K6 antiserum. Thin sections samples were labeled with K6 antiserum, followed by gold attached secondary antibodies. Left, Wild type

VP53 (WT), right, ∆CPS mutant. Bar equals to 500 nm. K-antigen processing genes In order to have some understanding of the capsule/K-antigen biosynthesis pathway, we investigated the polysaccharide processing and assembly genes in the genome of V. parahaemolyticus. We identified a small region outside of the K-antigen genes that contains wza, wzb, and wzc genes (Region D, Figure 1). Wza, b and c together constitute an important exportation system in group 1 and group 4 capsules in E. coli. A wza gene is present in the capsule gene region in both V. vulnificus and encapsulated non-O1 V. cholerae [7, 19]. The wza gene in V. parahaemolyticus shares 75% and 64% amino acid identity to the V. vulnificus and V. cholerae wza respectively. To investigate the function of this system in V. parahaemolyticus O3:K6, we deleted all three genes in region D from V. parahaemolyticus to generate mutant Δwzabc. Δwzabc mutant did not show obvious phenotypic differences to the wild type.