The Role of LPS and Toll-like Receptors in Plague

描述（由申请人提供）：革兰氏阴性菌鼠疫耶尔森氏菌是鼠疫的病原体，被列为NIAID A类优先生物防御剂。鼠疫菌含有一个被充分描述的III型分泌系统，该系统具有抑制宿主对细菌的反应的能力。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知之甚少。我们的目标是定义Y的角色。鼠疫菌LPS在鼠疫发展中的作用，并进一步表征疾病过程中鼠疫菌及其LPS与宿主Toll样受体（TLR）和CD14相互作用的影响。TLR和CD14在对微生物挑战的先天免疫应答中是中心的。最近有人提出，与27 ℃（跳蚤温度）相比，鼠疫耶尔森氏菌在37 ℃（宿主温度）下生长时产生效力较低的脂质A（LPS的主要生物活性组分）。我们的假设是，温度诱导Y的改变。鼠疫菌LPS使细菌能够减弱由TLR4介导的应答，从而导致感染后先天免疫应答减弱。我们将从Y中分离脂质A。鼠疫菌株KIM（其基因组序列已知）在27 ℃和37 ℃下生长，并表征详细结构。此外，我们还将分析Y.鼠疫菌（生长在27摄氏度和37摄氏度）和它的LPS与TLR信号通路的相互作用的重点，并与免疫激活潜力的结构。为了研究LPS在疾病中的具体作用，我们将通过过度表达LPS生物合成基因（来自E.co/i），使细菌在37 ℃下表达高活性LPS。我们还将产生过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 ℃和37 ℃下产生改变的LPS，我们将表征LPS结构和细胞活化潜力。确定Y.鼠疫菌LPS在体内的感染，我们将测试野生型和基因修饰的Y。鼠疫杆菌在野生型小鼠和TLR、MyD88和CD14遗传缺陷小鼠中产生免疫激活和感染的能力。这些研究的完成将为Y.鼠疫与先天免疫系统的核心要素相互作用，这一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.