title: The biosynthesis and biological role of 6-deoxyheptose in the lipopolysaccharide O-antigen of Yersinia pseudotuberculosis.
abstract: Yersinia pseudotuberculosis O:2a harbours 6-deoxy-d-manno-heptose in its O-antigen. The biological function of 6-deoxyheptose and its role in virulence is unknown and its biosynthetic pathway has not been demonstrated experimentally. Here, we show that dmhA and dmhB are necessary for 6-deoxyheptose biosynthesis in Y. pseudotuberculosis. Their disruption resulted in the lack of 6-deoxyheptose in the O-unit and its replacement by d-glycero-d-manno-heptose, thus indicating relaxed specificity of the glycosyltransferases, polymerase and ligase involved in lipopolysaccharide synthesis. The dmhB mutant exhibited a lower content in ketooctonic acid (Ko)-containing core molecules and reduced ligation and polymerization of the O-unit. We also show that Tyr128 is essential for activity of DmhB, and that DmhB functions as an oligomer, based on the dominant negative effect of overexpression of DmhB Y128F in dmhA. Moreover, we demonstrate that 6-deoxyheptose is important for virulence-related functions of the outer membrane and its appendages in vitro, such as barrier function against bile salts, polymyxin and novobiocin, and flagella-mediated motility. Although both mutants colonized the mouse ceacum as well as the wild type, the dmhB mutant was impaired for colonization of the liver, suggesting that DmhB represents a potential therapeutic target.
authors.affiliation: Department of Microbiology and Immunology, Infectious Diseases Research Group, University of Western Ontario, London, Ontario, Canada.
authors.forename: Nathan
authors.initials: N
authors.lastname: Ho
authors.forename: Anna N
authors.initials: AN
authors.lastname: Kondakova
authors.forename: Yuriy A
authors.initials: YA
authors.lastname: Knirel
authors.forename: Carole
authors.initials: C
authors.lastname: Creuzenet
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001647:Bile Acids and Salts / Q000494:pharmacology; D053898:Biosynthetic Pathways; D002432:Cecum / Q000382:microbiology; D015169:Colony Count, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006539:Heptoses / Q000096:biosynthesis*; D008099:Liver / Q000382:microbiology; D008124:Locomotion; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D016254:Mutagenesis, Insertional; D009675:Novobiocin / Q000494:pharmacology; D019081:O Antigens / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011112:Polymyxin B / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D013154:Spleen / Q000382:microbiology; D015011:Yersinia pseudotuberculosis / Q000737:chemistry* / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D015012:Yersinia pseudotuberculosis Infections / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D006539:Heptoses; D019081:O Antigens; D009675:Novobiocin; D011112:Polymyxin B