Highly phosphorylated core oligosaccharides and those substituted with one O-antigen repeating unit were obtained by mild acid degradation or strong alkaline hydrolysis of lipopolysaccharide samples from 23 reference strains representing all Pseudomonas aeruginosa O-serogroups. Studies by high-resolution electrospray ionization mass spectrometry and two-dimensional NMR spectroscopy revealed both conserved and variable structural features of the lipopolysaccharides of various O-serogroups. The upstream terminal saccharide of the O-antigen, which contributes most to the immunospecificity of the bacteria, was defined in 11 from a total of 13 O-serogroups. The data obtained link together the known biosynthesis pathways, genetics and serology of the P. aeruginosa lipopolysaccharide.
This review is devoted to structural and serological characteristics of the O-antigens (O-polysaccharides) of the lipopolysaccharides of various Proteus species, which provide the basis for classifying Proteus strains to O-serogroups. The antigenic relationships of Proteus strains within and beyond the genus as well as their O-antigen-related bioactivities are also discussed.
By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.
In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely related Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic pathways were constructed by site-directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high-resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased significantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less efficient incorporation of 4-amino-4-deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS-dependent enzymatic activities of plasminogen activator and elevation of LD(50) and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and particular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague.
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