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

描述（由申请人提供）：我们实验室最近报道了一种炭疽芽孢杆菌物种特异性多糖的结构。这种多糖（称为HF-PS）被归类为“非经典”次级细胞壁聚合物。它是通过氟化氢水溶液（HF）处理从营养细胞的细胞壁中分离出来的，以前报道过它的功能是将s层蛋白Sap和EA1锚定/输出到细胞表面。我们的研究结果有力地支持了该病毒在结构上对炭疽芽孢杆菌菌株具有特异性，具有免疫原性，因为注射了炭疽芽孢杆菌（34F2）的活孢子或死孢子的动物会产生针对该病毒的抗体，并且这些抗体与密切相关的蜡样芽孢杆菌菌株的HF-PS结合具有免疫原性特异性。此外，我们已经证明，从恒河猴暴露于炭疽芽孢杆菌存活的血清含有IgG抗hf - ps抗体。初步数据和文献结果表明，这些多糖可能对包括炭疽芽孢杆菌在内的致病性芽孢杆菌的生长和毒力具有重要作用。然而，迄今为止仍然缺乏直接的经验证据。本提案的目的是设计在这种HF-PS多糖的生物合成中受损的突变体，并表征这些突变体的表型，包括它们的生长，产孢，HF-PS结构，HF-PS与s层蛋白和各种噬菌体内溶素的相互作用，并研究每种突变对小鼠模型中毒力的影响。这项研究将为我们提供有关HF-PS的致病功能及其对开发疫苗、诊断和治疗方法的有用性的必要见解。公共卫生相关性：炭疽芽孢杆菌引起人类炭疽；作为一种生物恐怖制剂，这种细菌对公众健康构成威胁。我们的实验室最近报道了一种炭疽杆菌的细胞壁多糖（称为HF- PS），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损的炭疽芽孢杆菌突变体，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损。这项调查将为今后研究改进的疫苗、诊断和治疗方法以及与炭疽有关的新公共卫生做法奠定基础。

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.