A. Actinomycetemcomitans Cdt Induces Pro-Inflammatory Innate Immune Responses

牙周病是一种由多种微生物感染引起的慢性炎症性疾病。发病机制 这种疾病的发生涉及先天性和适应性免疫应答的激活和可能的干扰。超过 我们的研究过程中，我们已经证明，Aggregatibacteractinomycetemcomitans（Aa）， 与局部侵袭性牙周炎（localized aggressive periodontitis，简称牙周炎）的发病机制有关的一种假定病原体， 免疫毒素，细胞致死膨胀毒素（Cdt）。Cdt是一种异源三聚体全毒素， 毒素：细胞结合（B）单位由CdtA和CdtC亚基和活性（A）亚基CdtB组成。 我们已经证明CdtB作为5 '-磷脂酰肌醇（PI）三磷酸酶发挥功能，因为它 将PI-3 K信号传导脂质PI-3，4，5-三磷酸（PIP 3）降解为PI-3，4P 2;该作用导致阻断PI-3 K信号传导。 信号通路人巨噬细胞中PI-3 K信号传导的阻断导致促炎细胞因子 涉及典型和非典型炎性小体激活的反应。Cdt还诱导产生 衍生自花生四烯酸的炎性介质，包括PGE 2和血栓烷。最近，我们 观察到Cdt处理的巨噬细胞中的吞噬缺陷与改变的吞噬-溶酶体成熟一致。我们 提出Cdt扰乱巨噬细胞功能，从而导致炎症和持续感染。 我们的总体假设是，Aa Cdt有助于改变局部宿主防御， 使其他微生物能够逃避宿主的防御。我们研究的目的是扩大我们的调查 并推进我们对PI-3 K信号传导阻断与下游前体蛋白的分子事件的认识， 炎症反应和改变的囊泡运输和融合。具体来说，我们提出CdtB诱导 GSK 3-依赖性HSP 90激活，进一步说，HSP 90通过其对NLRP 3、考克斯-2和可能的ESCRT的作用， 蛋白质是导致成熟细胞因子和类花生酸释放的事件中的关键中间体（特异性目的 1）。我们计划进一步了解Cdt激活非典型炎性小体的作用， 其涉及gasdermin D（GSDMD）孔形成和焦亡。我们的重点将是确定CdtB如何 活化胱天蛋白酶-4并有助于GSDMD孔形成;后面的研究将集中在PI-3，4P 2和孔 通过ESCRT系统进行修复（特定aim 2）。在具体的目标3中，我们将重点关注囊泡运输的扰动 和吞噬溶酶体形成。我们认为，部分由于CdtB介导的生产，PI分布改变， 4P 2促进CdtB的逆行转运并调节吞噬溶酶体的形成。长期 我们研究的目的是将我们对控制Cdt毒性的分子事件的理解转化为，反过来， 产Cdt生物体的致病性。这些研究具有特别重要的意义，因为Cdt的生产不是 不仅是Aa，还有许多其他导致慢性感染和炎症性疾病的病原体。洞察力 通过这项调查获得的信息将促进我们对潜在的生物学和其他生物学事件的理解。 研究由产生Cdt的生物体引起的疾病，并确定治疗干预的新途径。 .

Periodontal disease is a chronic inflammatory disorder driven by polymicrobial infection. The pathogenesis of this disorder involves activation, and possibly perturbation, of both innate and adaptive immune responses. Over the course of our investigation we have demonstrated that Aggregatibacter actinomycetemcomitans (Aa), a putative pathogen implicated in the pathogenesis of localized aggressive periodontitis (LAP), produces an immunotoxin, the cytolethal distending toxin (Cdt). Cdt is a heterotrimeric holotoxin which functions as an AB2 toxin: the cell binding (B) units are comprised of the CdtA and CdtC subunits and the active (A) subunit, CdtB. We have demonstrated that CdtB functions as a 5'-phosphatidylinositol (PI) triphosphate phosphatase as it degrades the PI-3K signaling lipid, PI-3,4,5-triphosphate (PIP3) to PI-3,4P2; this action leads to blockade of the signaling pathway. Blockade of PI-3K signaling in human macrophages leads to a pro-inflammatory cytokine response involving both canonical and noncanonical inflammasome activation. Cdt also induces production of inflammatory mediators derived from arachidonic acid including PGE2 and thromboxanes. Most recently, we have observed phagocytic defects in Cdt-treated macrophages consistent with altered phago-lysosome maturation. We propose that Cdt perturbs macrophage function thereby contributing to both inflammation and sustained infection. Our overarching hypothesis is that the Aa Cdt contributes to altered local host defense which facilitate Aa survival and enables other microbes to evade host defense. The goal of our study is to extend our investigation and advance our knowledge of the molecular events that link PI-3K signaling blockade to downstream pro- inflammatory responses and altered vesicular trafficking and fusion. Specifically, we propose that CdtB induces GSK3â-dependent HSP90 activation and further that HSP90, via its effects on NLRP3, Cox-2 and possibly ESCRT proteins is a critical intermediary in events leading to release of mature cytokines and eicosanoids (specific aim 1). We plan to advance our understanding of the role that Cdt plays activating the noncanonical inflammasome which involve gasdermin D (GSDMD) pore formation and pyroptosis. Our focus will be to determine how CdtB activates caspase-4 and contributes to GSDMD pore formation; the latter studies will focus on PI-3,4P2 and pore repair via the ESCRT system (specific aim2). In specific aim 3 we will focus on perturbation of vesicular transport and phago-lysosome formation. We propose that altered PI distribution due in part to CdtB-mediated production of PI3,4P2 facilitates CdtB retrograde transport and modulates the formation of phago-lysosomes. The long-term goals of our study are to translate our understanding of the molecular events that govern Cdt toxicity and, in turn, the pathogenicity of Cdt-producing organisms. These studies are of particular significance as Cdt is produced not only by Aa but many other pathogens that contribute to chronic infectious and inflammatory disorders. Insight gained through this investigation will advance our understanding of the molecular events underlying LAP and other diseases caused by Cdt-producing organisms and identify new avenues for therapeutic intervention. .