Rhizobial Lipopolysaccharides Essential for Infection

描述(由申请人提供)：根瘤菌-豆类共生涉及对所有细菌-宿主相互作用至关重要的过程。这些措施包括(A)原核-真核识别，(b.)与寄主防御机制的相互作用，以及(C)细菌和寄主的分化过程(即分别形成固氮细菌和根瘤)。与动物病原体一样，入侵的根瘤菌必须适应宿主的环境，以避免破坏并在宿主细胞中生存。这些适应包括细胞表面多糖的变化，如脂多糖(LPS)和囊膜多糖。根瘤菌-豆类共生是研究革兰氏阴性菌侵染和在宿主内存活的分子基础的一个很好的模式系统，因为革兰氏阴性菌和宿主细胞在整个感染过程中都是活的，入侵细菌的LPS可以被分离出来并表征为感染过程的函数。共生系统与形成慢性感染的一些细胞内病原体特别相关，例如布鲁氏菌、巴通氏菌、军团菌以及方济氏菌。我们观察到，这些病原菌的LPS上也存在根瘤菌LPS的独特结构特征。我们的研究重点是根瘤菌(R.etli和R.legumosarum，分别是豆类和豌豆的共生体)在遇到胁迫环境(如pH、渗透压、氧分压等)时对其LPS的适应。宿主细胞的。我们推测，根瘤菌LPS独特的结构特征对于豆科植物寄主的侵染和持久性是必不可少的。独特的结构特征包括用半乳糖醛酸残基取代磷酸盐，将脂-A近端的氨基葡萄糖残基氧化成2-氨基葡萄糖酸，以及脂-A上存在超长链脂肪酸27-羟基二十八碳酸(27uch28：0)。在共生过程中，O链多糖的甲基化和乙酰化发生了变化，脂多糖和整个根瘤细胞的疏水性发生了全面的变化。我们的具体目标是：(1)表征共生过程中内毒素O链多糖的修饰情况，(2.)创建特定的内毒素突变体并鉴定其结构和共生表型，以及(3)确定27uch28：0缺失的突变体acpXL：：Kan如何能够激活宿主细胞内的替代机制。

DESCRIPTION (provided by applicant): Rhizobium-legume symbioses involve processes that are fundamental to all bacterial-host interactions. These include (a.) prokaryotic-eukaryotic recognition, (b.) interaction with the host defense mechanism, and (c.) bacterial and host differentiation processes (i.e. formation of nitrogen-fixing bacteroids and root nodules, respectively). As with animal pathogens, the invading rhizobia must adapt to the environment of the host to avoid destruction and persist in the host cell. These adaptations involve changes to cell surface polysaccharides, such as lipopolysaccharides (LPSs) and capsular polysaccharides. Rhizobium-legume symbiosis is an excellent model system to examine the molecular bases by which Gram-negative bacteria infect and survive within their host since both the bacteria and the host cells are viable throughout the infection process, and the LPSs from the invading bacteria can be isolated and characterized as a function of the infection process. The symbiosis system is particularly relevant to a number of intracellular pathogens that form chronic infections such as species of Brucella, Bartonella, Legionella, as well as Francisella. We have observed that unique structural features of Rhizobium LPSs are also present on LPSs from these pathogens. Our project focuses on the adaptation that rhizobia (R. etli and R. leguminosarum; symbionts of bean and pea, respectively) make to their LPSs as they encounter the stressful environment (e.g. changes in pH, osmolarity, oxygen tension, etc.) of the host cell. We hypothesize that unique structural features of rhizobial LPSs are essential for infection and persistence in the legume hosts. Unique structural features include a replacement of phosphate with galacturonic acid residues, oxidation of the lipid-A proximal glucosamine residue to 2-aminogluconate, and the presence of the very long chain fatty acid, 27- hydroxyoctacosaonic acid (27OHC28:0), on the lipid-A. Methylation and acetylation changes to the O-chain polysaccharide and an overall change in the hydrophobicity of the LPS and the whole rhizobial cell occur during symbiosis. Our Specific Aims are: (1.) characterize the modifications to the O-chain polysaccharide of the LPS during symbiosis, (2.) create specific LPS mutants and characterize their structural and symbiotic phenotypes, and (3.) determine how a 27OHC28:0-lacking mutant, acpXL::kan, is able to activate an alternative mechanism within the host cell.