Mechanisms of LtxA intracellular trafficking and cell injury in human macrophages

革兰氏阴性杆菌放线菌伴生菌是人类常见的居住者 上呼吸道消化系统和局部侵袭性牙周炎(LAP)的病原体。这种细菌产生 一种RTX毒素(Ltd.A)。LtxA可以可溶性和外膜小泡(OMV)的形式输送到宿主细胞- 关联的表单。可溶性LtxA需要β2、整合素LfA-1和富含胆固醇的膜的存在 木筏对人类免疫细胞产生致命作用。最近的数据表明，可溶性的LtxA会损伤溶酶体， 导致人巨噬细胞释放促炎细胞因子并诱导细胞死亡。溶度如何的Ltd.A 被传递到巨噬细胞的溶酶体并诱导炎症反应，目前尚不清楚。这一机制 OMV携带的LitxA对细胞的损伤完全未知。这项研究的目的是确定 人巨噬细胞转运和中毒的机制。我们的中心假设是 可以通过阻止LitxA的贩运来停止LitxA的破坏性效果。因此，我们提出了一套 人可溶性与OMV相关的Ltd_xA在细胞内定位的实验研究 巨噬细胞。我们的假设将在两个具体目标上进行检验：1)确定贩运和细胞损伤 2)确定OMV相关的Ltd_xA的转运及中毒机制。在我们的 实验计划，我们将利用共聚焦成像结合免疫学和生化方法来 探讨LtxA在巨噬细胞中的胞内转运方式及其下游作用 关于炎症体的激活。毒素诱导的巨噬细胞损伤将在体外用 细菌/巨噬细胞复合体和体外使用纯化的毒素、OMV和纯化的细胞器。这个 拟议的研究与公共卫生有关，这项建议的基本原理是确定关键目标 用于防止导致巨噬细胞内细胞死亡和牙周炎的LtxA转运。这个 预计项目的完成将带来牙周炎的新型治疗和诊断方法。 重要的是，这项研究的结果将提供一个基础，可以扩展到理解致病 研究其他RTX毒素的作用机制，为疾病治疗开辟新途径。

A Gram negative bacterium Aggregatibacter actinomycetemcomitans is a common inhabitant of the human upper aerodigestive and a causative agent of localized aggressive periodontitis (LAP). The bacterium produces an RTX toxin (LtxA). LtxA can be delivered to host cells in soluble and in outer membrane vesicles (OMVs)- associated forms. Soluble LtxA requires the presence of the β2 integrin LFA-1 and cholesterol-rich membrane rafts to exert its lethal effect on human immune cells. Recent data indicate that soluble LtxA damages lysosomes, causes release of proinflammatory cytokines and induces cell death in human macrophages. How soluble LtxA is delivered to the macrophages’ lysosomes and induces inflammatory response, is not known. The mechanism of the cell injury by OMV-delivered LtxA is completely unexplored. The objective of this study is to determine the mechanism of LtxA trafficking and intoxication in human macrophages. Our central hypothesis is that the destructive effect of LtxA can be halted by preventing LtxA trafficking. Therefore, we propose a set of experiments to understand intracellular localization of soluble versus OMV-associated LtxA in human macrophages. Our hypotheses will be tested in two specific aims: 1) To determine trafficking of and cell injury by soluble LtxA; 2) To determine trafficking of OMV-associated LtxA and the mechanism of intoxication. In our experimental plan, we will utilize confocal imaging coupled with immunological and biochemical approaches to investigate the mode of LtxA endocytic trafficking in macrophages and to determine downstream effects of LtxA on inflammasome activation. The toxin-induced macrophage damage will be investigated ex vivo using bacteria/macrophages complex and in vitro employing purified toxins, OMVs, and purified organelles. The proposed research is relevant to public health and the rationale underlying this proposal is to identify key targets for preventing LtxA trafficking which induces cell death in macrophages and periodontal inflammation. The project completion is expected to give rise to new types of treatments and diagnostics for periodontitis. Importantly, the results of this study will provide a basis that can be extended to understand pathogenic mechanisms of other RTX-toxins and open new avenues for diseases therapies.