The Role of LPS and Toll-like Receptors in Plague

描述(申请人提供)：革兰氏阴性菌鼠疫耶尔森氏菌是鼠疫的病原体，被归类为NIAID A类优先生物防御剂。鼠疫杆菌含有一种众所周知的III型分泌系统，它能够抑制宿主对细菌的反应。然而，关于外膜成分如脂多糖(LPS)的免疫激活，这些成分如何与宿主免疫系统相互作用，以及它们在疾病进展中的作用，人们知之甚少。我们的目标是明确鼠疫杆菌脂多糖在鼠疫发生发展中的作用，并进一步表征鼠疫杆菌及其脂多糖与宿主Toll样受体(TLRs)和CD14在疾病过程中相互作用的影响。TLRs和CD14在对微生物攻击的先天免疫反应中起核心作用。最近有人提出，鼠疫杆菌在37摄氏度(宿主温度)与27摄氏度(跳蚤温度)下生长时产生的类脂A(脂多糖的主要生物活性成分)的效力较低。我们的假设是，温度诱导的鼠疫杆菌内毒素的变化使细菌能够钝化TLR4介导的反应，导致感染后先天免疫反应减弱。我们将从27xC和37xC生长的鼠疫耶尔森菌菌株Kim(其基因组序列已知)中分离出类脂A，并对其详细结构进行表征。此外，我们将分析鼠疫杆菌(在27℃和37℃下生长)及其内毒素的免疫激活能力，重点分析与TLR信号通路的相互作用，并将结构与免疫激活潜力联系起来。为了研究内毒素在疾病中的具体作用，我们将通过过量表达E.Co/I的内毒素生物合成基因，使细菌在37摄氏度表达高活性的内毒素。我们还将产生过度表达鼠疫杆菌基因的突变体，并使细菌缺乏相同的基因。这些细菌将在27摄氏度和37摄氏度产生变化的内毒素，我们将表征内毒素的结构和细胞激活潜力。为了确定鼠疫杆菌脂多糖在体内感染中的作用，我们将测试野生型和转基因鼠疫杆菌在野生型小鼠和TLRs、MyD88和CD14基因缺陷小鼠中的免疫激活和感染能力。这些研究的完成将提供有关鼠疫杆菌如何与先天免疫系统的核心成分相互作用的新信息，这些知识将有助于开发鼠疫和相关感染的新疗法。

DESCRIPTION (provided by applicant): The Gram-negative bacteria Yersinia pestis is the causative agent of plague, and is classified as an NIAID category A priority biodefense agent. Y.pestis contains a well-described type III secretion system that has the ability to repress the host responses to the bacteria. However, less is know about immune activation by outer membrane components such as lipopolysaccharide (LPS), how these components interact with the host immune system, and their role in disease progression. Our goal is to define the role of Y. pestis LPS in the development of plague, and furthermore to characterize the impact of interactions by Y.pestis and its LPS with host Toll-like receptors (TLRs) and CD14 during the course of the disease. TLRs and CD14 are central in the innate immune response to microbial challenge. It has recently been suggested that Y.pestis produces a lipid A (main biologically active component of LPS) of lower potency when grown at 37 degrees C (host temperature) compared to 27 degrees C (flea temperature). Our hypothesis is that temperature induced alterations in Y. pestis LPS enable the bacteria to blunt responses mediated by TLR4, contributing to the diminished innate immune responses following infection. We will isolate lipid A from Y. pestis strain KIM (for which the genomic sequence is known) grown at 27xC and 37xC, and characterize the detailed structures. Furthermore, we will analyze the immune activation ability of Y. pestis (grown at 27 degrees C and 37 degrees C) and its LPS with an emphasis on interactions with the TLR signaling pathways, and relate structures to immune activation potential. To investigate the specific role of LPS in disease, we will make bacteria expressing a highly active LPS at 37 degrees C, by over-expressing LPS biosynthesis genes from E.co/i. We will also generate mutants over-expressing the Y.pestis genes, and make bacteria deficient in the same genes. These bacteria will produce altered LPS at both 27 degrees C and 37 degrees C, and we will characterize LPS structure and cell activation potential. To establish the role of Y. pestis LPS in infection in vivo, we will test wild-type and the genetically modified Y. pestis for ability to mount immune activation and infection in wildtype mice and mice genetically deficient for TLRs, MyD88 and CD14. The completion of these studies will provide new information on how Y. pestis interacts with central elements of the innate immune system, knowledge that would be helpful in the development of new therapies for plague and related infections.