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。这些包括(a)原核-真核生物识别，(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，可以研究革兰氏阴性菌在其宿主内感染和存活的分子基础，因为细菌和宿主细胞在整个感染过程中都是有活力的，并且入侵细菌的lps可以被分离出来并表征为感染过程的一个功能。共生系统与许多形成慢性感染的细胞内病原体特别相关，例如布鲁氏菌、巴尔通体菌、军团菌以及弗朗西斯菌。我们观察到根瘤菌lps的独特结构特征也存在于这些病原体的lps上。我们的项目重点研究根瘤菌（r.e etli和r.e leguminosarum；分别是豆类和豌豆的共生体）在遇到宿主细胞的应激环境（如pH、渗透压、氧张力等变化）时对其lps的适应。我们假设根瘤菌lps的独特结构特征对豆科寄主的感染和持久性至关重要。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-氨基葡萄糖酸盐，以及在脂质-a上存在很长链脂肪酸27-羟基八羧酸（27OHC28:0）。在共生过程中，o链多糖发生甲基化和乙酰化变化，LPS和整个根瘤菌细胞的疏水性发生全面变化。我们的具体目标是：(1)表征共生过程中对LPS的o链多糖的修饰，(2)创建特异性LPS突变体并表征其结构和共生表型，以及(3)确定27ohc28:0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.