Biosynthesis & Function of a Bacillus anthracis-specific cell wall polysaccharide

描述(申请人提供)：我们实验室最近报道了一种炭疽芽孢杆菌特有的多糖的结构。这种多糖(称为HF-PS)被归类为“非经典”次生细胞壁聚合物。它是用氟化氢(HF)水溶液从营养细胞的细胞壁中分离出来的，此前有报道称它能将S层蛋白SAP和EA1锚定/输出到细胞表面。我们的工作有力地支持了HF-PS在结构上是炭疽杆菌菌株所特有的，是免疫原性的，因为注射炭疽杆菌(34F2)活或死孢子的动物产生了针对HF-PS的抗体，并且关于这些抗体与甚至是密切相关的蜡状芽孢杆菌菌株的HF-PSS的结合具有免疫原性。此外，我们还发现，在炭疽芽胞中存活的恒河猴血清中含有抗HF-PS抗体。文献中的初步数据和结果支持，这些多糖可能在包括炭疽杆菌在内的致病芽孢杆菌的生长和毒力方面发挥重要作用。然而，到目前为止，直接的经验证据仍然缺乏。这项建议的目的是设计在这种HF-PS多糖的生物合成过程中受到损害的突变株，并从生长、产孢量、HF-PS结构、HF-PS与S层蛋白和各种噬菌体内毒素的相互作用等方面表征这些突变株的表型，并在小鼠模型中研究每个突变对毒力的影响。这项研究将为我们提供关于HF-PS致病功能的必要洞察力，以及它对疫苗、诊断和治疗的开发的有用性。与公共卫生相关：炭疽芽孢杆菌会引起人类的炭疽病；作为一种生物恐怖分子，这种细菌对公共健康构成威胁。我们实验室最近报道了一种炭疽杆菌的细胞壁多糖(称为HF-PS)，其结构具有这些细菌特有的结构。我们将创建在这种细胞壁多糖的生物合成中受到损害的炭疽杆菌突变体，并研究这些突变体是否在重要的细菌生长功能和疾病发展中受到损害。这项调查将为今后改进疫苗、诊断和治疗以及与炭疽病相关的新公共卫生实践的研究奠定基础。

DESCRIPTION (provided by applicant): Our laboratory has recently reported the structure of a Bacillus anthracis species-specific polysaccharide. This polysaccharide (termed HF-PS) has been classified a 'non-classical' secondary cell wall polymer. It is isolated from the cell wall of vegetative cells by treatment with aqueous hydrogen fluoride (HF), and was previously reported to function in anchoring/exporting the S-layer proteins, Sap and EA1, to the cell surface. Our work strongly supports that the HF-PS is structurally specific to B. anthracis strains, is immunogenic in that animals injected with live or dead spores of B. anthracis Sterne (34F2) produce antibodies against HF-PS, and is immunogenically specific with regard to the binding of these antibodies to the HF-PSs of even closely related B. cereus strains. Further, we have shown that sera from Rhesus macaques that survive exposure to B. anthracis spores contain IgG anti-HF-PS antibodies. Preliminary data and results in the literature support that these polysaccharides could be crucial for important functions with regard to the growth and virulence of pathogenic Bacillus species, including B. anthracis. However, to date direct empirical evidence is still missing. The aims of this proposal are to engineer mutants that are impaired in the biosynthesis of this HF-PS polysaccharide and to characterize the phenotype of these mutants with regard to their growth, sporulation, HF-PS structure, HF-PS interaction with S-layer proteins and various phage endolysins, and to study the effect of each mutation on the virulence in a mouse model. This research will provide us with necessary insight regarding the HF-PS's pathogenic functions, and its usefulness for the development of vaccines, diagnostics and therapeutics. PUBLIC HEALTH RELEVANCE: The bacterium Bacillus anthracis causes anthrax in humans; as a bioterrorism agent the bacteria are a threat to public health. Our laboratory has recently reported a cell wall polysaccharide of a B. anthracis (termed HF- PS) with a structure specific to these bacteria. We will create B. anthracis mutants impaired in the biosynthesis of this cell wall polysaccharide and investigate whether these mutants are impaired in important bacterial growth functions and disease development. This investigation will lay the groundwork for future studies into improved vaccines, diagnostics, and therapeutics, and into new anthrax-related public health practices.