Unique ADP-ribosylating and vacuolating properties of Mycoplasma pneumoniae CARDS toxin trigger airway inflammation and disease progression

摘要 肺炎支原体是儿童细菌性肺炎的主要原因， 社区获得性肺炎（CAP）和成人细菌性CAP的第二大常见原因， 美国的持续性和重复性M。肺炎感染导致严重的呼吸系统疾病， 包括哮喘和COPD，以及一系列肺外病变。直到最近，人们才知道 呼吸道病原体，如M。肺炎，诱导细胞病理学和夸大的炎症 导致气道损伤、功能障碍和重塑的反应。我们发现了一个新的M。肺炎ADP- 核糖基化和空泡化毒素，称为社区获得性呼吸窘迫综合征 毒素单用肉毒毒素可引起特征性气道炎症、肺组织病理学、细胞免疫学改变。 中毒啮齿类和灵长类动物中的空泡化、粘液化生和肺功能障碍， 在感染M.肺炎。全长（FL）肉毒毒素的氨基末端结构域 (i.e., N-ATP）保留ADP-核糖基转移酶（ART）活性。N-腺苷二磷酸选择性核糖基化NLRP 3 的NLRP 3炎性体复合物，导致炎性体活化和随后的IL-10的释放。 1β，一种有效的促炎细胞因子。在初步结果中，我们还表明， ADP-核糖基化丝氨酸羟甲基转移酶（SHMT 2），其参与一碳代谢，和 EF 1 γ，其参与氨酰-tRNA向核糖体的转移。FL独特的羧基区域 肉毒毒素（即，C-β）选择性结合受体表面活性蛋白-A（SP-A）、膜联蛋白A2（AnxA 2） 以及磷脂、磷脂酰胆碱（PC）和鞘磷脂（SM）。FL毒素的内化 随后受体介导的结合，随后宿主靶蛋白的ADP-核糖基化，空泡化， 过度炎症和细胞/组织组织病理学和损伤。有趣的是，单独的C-淀粉酶会导致 人类细胞中的空泡形成和幼稚小鼠中的嗜酸性粒细胞炎症，导致哮喘样 表型在这个建议中，我们打算确定ART和空泡化特性是如何触发 人WT、沉默或敲除细胞和WT中的促炎和病理学应答， 敲除小鼠基于我们的初步结果，我们假设ADP-核糖基化和空泡化 这些活性有助于肉毒毒素启动和维持疾病发病机制的总体能力。我们 我计划通过以下方法来验证这一假设：a）研究抗逆转录病毒药物如何启动炎症通路 和细胞病理学，B）阐明受体结合在肉毒毒素介导的炎症中的作用，c） 表征空泡化活性如何促进气道炎症和损伤，以及d）分析体内 选择ART和受体靶点参与触发阿托伐他汀介导的气道炎症， 肺病理学了解ART和肉毒毒素空泡化活性的机制 调节宿主反应应导致治疗干预和改善社会福祉。

Abstract Mycoplasma pneumoniae is the leading cause of bacterial pneumonia in children hospitalized with community acquired pneumonia (CAP) and the second most common cause of bacterial CAP in adults in the United States. Persistent and recurring M. pneumoniae infection leads to severe respiratory disorders, including asthma and COPD, and a range of extrapulmonary pathologies. Until recently, it was unknown how a respiratory pathogen, like M. pneumoniae, induces cytopathology and exaggerated inflammatory responses that cause airway injury, dysfunction and remodeling. We identified a novel M. pneumoniae ADP- ribosylating and vacuolating toxin designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. CARDS toxin alone elicits the characteristic airway inflammation, lung histopathology, cellular vacuolation, mucus metaplasia and pulmonary dysfunction in intoxicated rodents and primates that are observed during infection with M. pneumoniae. The amino terminal domain of full length (FL) CARDS toxin (i.e., N-CARDS) retains ADP-ribosyltransferase (ART) activity. N-CARDS selectively ADP-ribosylates NLRP3 of the NLRP3 inflammasome complex, resulting in inflammasome activation and subsequent release of IL- 1β, a potent pro-inflammatory cytokine. In preliminary results, we also show that CARDS toxin selectively ADP-ribosylates serine hydroxymethyltransferase (SHMT2), which is involved in one carbon metabolism, and EF1γ, which is involved in the transfer of aminoacyl-tRNAs to the ribosome. The unique carboxyl region of FL CARDS toxin (i.e., C-CARDS) selectively binds to receptors surfactant protein-A (SP-A), annexin A2 (AnxA2) and phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM). Internalization of FL CARDS toxin follows receptor-mediated binding, with subsequent ADP-ribosylation of host target proteins, vacuolation, hyperinflammation and cell/tissue histopathology and injury. Interestingly, C-CARDS alone causes both vacuole formation in human cells and eosinophilic inflammation in naïve mice, leading to an asthma-like phenotype. In this proposal, we intend to identify how ART and vacuolating properties of CARDS toxin trigger pro-inflammatory and pathologic responses in human WT, silenced or knockout cells and in WT and knockout mice. Based on our preliminary results, we hypothesize that both ADP-ribosylating and vacuolating activities contribute to the overall ability of CARDS toxin to initiate and sustain disease pathogenesis. We plan to test this hypothesis by – a) studying how CARDS toxin ART activities initiate inflammatory pathways and cytopathology, b) elucidating the role of receptor binding in CARDS toxin-mediated inflammation, c) characterizing how vacuolating activity promotes airway inflammation and injury, and d) analyzing the in vivo involvement of select ART and receptor targets in triggering CARDS toxin-mediated airway inflammation and lung pathology. Understanding the mechanisms by which ART and vacuolating activities of CARDS toxin regulate host response should lead to therapeutic interventions and improved societal well-being.