Rhizobial Lipopolysaccharides Essential for Infection

描述（由申请人提供）：根瘤菌-豆科植物共生涉及所有细菌-宿主相互作用的基本过程。其中包括（a）。原核-真核识别，（B.）与宿主防御机制的相互作用，以及（c.）细菌和宿主分化过程（即分别形成固氮类杆菌和根瘤）。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持续存在。这些适应涉及细胞表面多糖的变化，如脂多糖（LPS）和荚膜多糖。根瘤菌-豆科植物共生是一个很好的模型系统，以检查革兰氏阴性细菌感染和在其宿主内存活的分子基础，因为细菌和宿主细胞在整个感染过程中都是活的，并且来自入侵细菌的LPS可以被分离和表征为感染过程的函数。共生系统与形成慢性感染的许多细胞内病原体特别相关，例如布鲁氏菌属、巴尔通体属、军团菌属以及弗朗西斯菌属。我们观察到根瘤菌LPS的独特结构特征也存在于来自这些病原体的LPS上。本课题主要研究根瘤菌（R. etli和R.豆科植物;大豆和豌豆的共生体）在它们遇到应激环境（例如pH、渗透压、氧张力等的变化）时对它们的LPS的影响。的宿主细胞。我们假设根瘤菌LPS的独特结构特征对于豆科植物宿主的感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，脂质A近端葡萄糖胺残基氧化为2-氨基葡萄糖酸，以及脂质A上存在极长链脂肪酸27-羟基二十八烷酸（27 OHC 28：0）。甲基化和乙酰化改变O-链多糖，并且在共生期间发生LPS和整个根瘤菌细胞的疏水性的总体变化。我们的具体目标是：（1）表征在共生过程中对LPS的0-链多糖的修饰，（2.）产生特异性LPS突变体并表征其结构和共生表型，以及（3.）确定27 OHC 28：0缺失突变体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.