Ho Nathan, Kondakova Anna N, Knirel Yuriy A, Creuzenet Carole
Department of Microbiology and Immunology, Infectious Diseases Research Group, University of Western Ontario, London, Ontario, Canada.
Mol Microbiol. 2008 Apr;68(2):424-47. doi: 10.1111/j.1365-2958.2008.06163.x. Epub 2008 Feb 19.
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.
假结核耶尔森菌O:2a在其O抗原中含有6-脱氧-D-甘露庚糖。6-脱氧庚糖的生物学功能及其在毒力中的作用尚不清楚，其生物合成途径也未得到实验证实。在此，我们表明dmhA和dmhB是假结核耶尔森菌中6-脱氧庚糖生物合成所必需的。它们的缺失导致O单元中缺乏6-脱氧庚糖，并被D-甘油-D-甘露庚糖取代，从而表明参与脂多糖合成的糖基转移酶、聚合酶和连接酶的特异性有所放宽。dmhB突变体在含酮辛酸（Ko）的核心分子中含量较低，O单元的连接和聚合减少。我们还表明，Tyr128对DmhB的活性至关重要，并且基于dmhA中DmhB Y128F过表达的显性负效应，DmhB作为一种寡聚体发挥作用。此外，我们证明6-脱氧庚糖对外膜及其附属物的毒力相关功能在体外很重要，例如对胆盐、多粘菌素和新生霉素的屏障功能以及鞭毛介导的运动性。尽管两个突变体在小鼠盲肠中的定殖情况与野生型一样，但dmhB突变体在肝脏中的定殖受损，这表明DmhB是一个潜在的治疗靶点。
