Mycoplasma Model of Infection-mediated Sterile Inflammation

描述(申请人提供)：与通常轻微的支原体病不同，鳄支原体感染会导致易感宿主的致命性多系统炎症性疾病。我们的总体目标是了解其分泌的糖苷酶在其生态和显著毒力中的作用，这种糖苷酶在支原体中是史无前例的。在其他感染期间，表现病原体相关分子模式(PAMPs)的细菌来源的外源配体通过TLR信号刺激宿主促炎细胞因子的产生。自我限制反应可以清除病原体，但不受控制的TLR激活会导致夸大的炎症，这对宿主是毁灭性的。无菌炎症不是对外源性PAMPs的反应，而是对从受损宿主组织中释放出来的内源性TLR激动剂的反应。因此，具有讽刺意味的是，感染期间分泌的细菌糖苷酶降解的ECM成分可能会介导无菌炎症。感染介导的不孕不育性炎症仍未被研究，这是因为存在难以解决的内在细菌PAMPs，如肽聚糖或脂多糖。它们使得很难将对PAMP的反应与无菌炎症区分开来，以便评估它们在细菌感染中的相对重要性。然而，像鳄鱼支原体这样的模型可以避免这种混淆，因为它自然缺乏相关的PAMP，但能够通过其糖苷酶活性从宿主ECM酶促释放内源性TLR激动剂，如降解的透明质酸低聚糖(HA)。我们将用野生型和透明质酸酶基因敲除的鳄鱼分支杆菌感染原代肺成纤维细胞：1)鉴定感染后主动诱导的TLR信号转导，重点是血凝素的激活和关键的TLR适配器蛋白MyD88的信号转导；2)确定鳄鱼分支杆菌感染过程中TLR信号的下游效应，重点是核因子？B的激活和肿瘤坏死因子？？分泌物作为关键事件的标志，机械地将TLR激活与无菌炎症联系起来。预期的结果是证明细菌糖苷酶可以积极地诱导参与无菌炎症的TLR信号。 这项工作意义重大，因为它将区分感染介导的不孕症炎症中的TLR信号与细菌PAMP的反应。一旦这成为可能，就可以更有效地确定感染介导的体内无菌炎症过程中的完整级联事件。除了提高我们对鳄鱼分枝杆菌毒力的理解外，这一区别有望为TLRs和/或糖苷酶拮抗剂作为治疗干预措施管理许多种类细菌引起的人和动物感染提供循证理由的垫脚石。

DESCRIPTION (provided by applicant): In contrast to usually subtle mycoplasmosis, Mycoplasma alligatoris infection causes lethal multisystemic inflammatory disease of susceptible hosts. Our overall objective is to understand the roles of its secreted glycosidases, which are unprecedented among mycoplasmas, in its ecology and remarkable virulence. During other infections, exogenous ligands of bacterial origin that display pathogen-associated molecular patterns (PAMPs) stimulate host pro-inflammatory cytokine production via TLR signaling. Self-limiting responses can clear pathogens, but uncontrolled TLR activation results in exaggerated inflammation that is devastating to the host. Sterile inflammation is a response not to exogenous PAMPs but to endogenous TLR agonists liberated from damaged host tissues. Thus, ironically, ECM components degraded by bacterial glycosidases secreted during infection may mediate sterile inflammation. Infection-mediated sterile inflammation remains unexplored because of the inextricably confounding presence of intrinsic bacterial PAMPs like peptidoglycan or lipopolysaccharide. They make it difficult to partition responses to PAMPs from sterile inflammation, in order to assess their relative significance in bacterial infections. However, a mycoplasma model like M. alligatoris can avoid that confounding because it naturally lacks the relevant PAMPs, but is capable of liberating endogenous TLR agonists like degraded hyaluronan oligosaccharides (HA) from host ECM enzymatically through its glycosidase activity. We will infect primary pulmonary fibroblasts with wild type and hyaluronidase-knockout M. alligatoris to: 1) Identify TLR signaling actively elicited by infection, with focus on activation by HA and signal transduction via the key TLR adapter protein MyD88; and 2) Determine downstream effectors of TLR signaling during M. alligatoris infection, with focus on NF?B activation and TNF-? secretion as markers of the key events that mechanistically link TLR activation to sterile inflammation. The expected outcome is proof that a bacterial glycosidase can actively elicit TLR signaling involved in sterile inflammation. The work is significant because it will distinguish TLR signaling in infection-mediated sterile inflammation from responses to bacterial PAMPs. Once this becomes possible, the complete cascade of events during infection-mediated sterile inflammation in vivo can be determined much more effectively. Beyond improving our understanding of M. alligatoris' virulence, this distinction can be expected to provide a stepping stone toward evidence-based rationales for antagonists of TLRs and/or glycosidases as therapeutic interventions for management of human and animal infections caused by many species of bacteria.