Domain Organization of the Polymerizing Mannosyltransferases Involved in Synthesis of the Escherichia coli O8 and O9a Lipopolysaccharide O-antigens

The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and β-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA^O9a contains two separable and functionally active domains, whereas WbdA^O8 possesses three. In WbdC^O9a and WbdB^O9a, substitution of the first Glu of the EX₇E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX₇E motif of the N-terminal WbdA^O9a domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA^O9a domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA^O9a possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.     

Loss of wbpL disrupts O-polysaccharide synthesis and impairs virulence of plant-associated Pseudomonas strains

Despite its importance for membrane stability and pathogenicity of mammalian pathogens, functions of the O-polysaccharide (OPS) of lipopolysaccharide (LPS) remain unclear in plant-associated bacteria. Genetic information about OPS biosynthesis in these bacteria is largely missing. Genome analysis of various plant-associated Pseudomonas strains revealed that one of the two known OPS biosynthesis clusters from Pseudomonas aeruginosa PAO1, the common polysaccharide antigen (CPA) gene cluster, is only conserved in some strains of the Pseudomonas fluorescens group. For the O-specific antigen (OSA) biosynthesis cluster, the putative genomic position could be identified, but orthologues of most functional important OSA biosynthesis enzymes could not be detected. Nevertheless, orthologues of the glycosyltransferase WbpL, required for initiation of CPA and OSA synthesis in P. aeruginosa PAO1, could be identified in the analysed Pseudomonas genomes. Knockout mutations of wbpL orthologues in Pseudomonas syringae pv. tomato DC3000 (Pst) and Pseudomonas cichorii ATCC10857/DSM50259 (Pci) resulted in strains lacking the OPS. Infection experiments of Arabidopsis thaliana plants revealed a reduced entry into the leaf apoplast after spray inoculation and a reduced apoplastic amplification of Pst ∆wbpL. Stab and spray inoculation of lettuce (Lactuca sativa) leaves with Pci ∆wbpL causes reduced infection symptoms compared to the wild-type strain. Furthermore, swarming motility was reduced in ∆wbpL mutants of Pst and Pci. This might be a possible reason for reduced bacterial titres after surface inoculation and reduced bacterial amplification in the plant. Our results imply that the presence of lipopolysaccharide OPS is required for efficient host colonization and full virulence of plant-pathogenic Pseudomonas bacteria.     

生物法合成伤寒O-糖蛋白结合疫苗及其免疫原性评估

伤寒由伤寒沙门氏菌(Salmonella Typhi)引发,至今在发展中国家仍是备受关注的重要公共卫生问题。文章通过敲除伤寒菌脂多糖合成途径中O-抗原连接酶基因,转入含脑膜炎奈瑟球菌(Neisseria meningitidis)蛋白糖基化途径中糖基转移酶的表达载体,以及改构的重组铜绿假单胞菌(Pseudomonas Aeruginosa)外毒素A(rEPA_N29)的表达载体,使细胞内能够诱导合成以伤寒O特异性多糖(O-specific polysaccharides, OPS)为目标抗原、以rEPA_N29为载体蛋白的伤寒OPS-rEPA_N29糖蛋白复合物,并对纯化所得复合物进行了免疫原性评价。ELISA测定血清抗体滴度表明,rEPA_N29作为载体蛋白能有效增加糖链的免疫原性,糖蛋白比单独的多糖能诱导产生更好的免疫应答;3次免疫、间隔3周比间隔2周IgG滴度稍有提高;而免疫过量的糖蛋白,抗O-多糖的血清抗体效价并无提升。文章为生物法制备多糖-蛋白结合疫苗提供了新思路,理论上也适用于其他革兰氏阴性菌的疫苗研发。     

Structure of the O-polysaccharide from the Azospirillum lipoferum Sp59b lipopolysaccharide

A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of the plant-growth-promoting bacterium Azospirillum lipoferum Sp59b. On the basis of sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopy, including a NOESY experiment, the following structure of the branched hexasaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text].     

The epitope structure in lipopolysaccharide recognized by the serotype 2-specific monoclonal antibody of Antinobacillus pleuropneumoniae

Abstract: The polysaccharide structure recognized by a monoclonal antibody specific to serotype 2 lipopolysaccharide of Actinobacillus pleuropneumoniae was investigated using an enzyme-linked immunosorbent assay inhibition test. Lipopolysaccharide obtained from serotype 2, strain SH-15, was hydrolysed with acetic acid to liberate the polysaccharide portion, and the polysaccharide mixture was fractionated by gel filtration. The longer polysaccharide, composed of O -antigenic polysaccharide and core, fully inhibited the binding of monoclonal antibodies to a whole cell antigen of strain SH-15, whereas the core oligosaccharide without O -polysaccharide did not. No inhibition was observed with the monosaccharides which were the components of serotype 2 LPS. Enzyme-linked immunosorbent assay inhibition ability of O -polysaccharide was completely lost only by O -deacetylation. These results demonstrate that the epitope of the serotype-specific monoclonal antibody resided in O -polysaccharide of LPS and that the O -acetyl group was essential for the epitope structure.