Mechanisms of LtxA intracellular trafficking and cell injury in human macrophages

一种革兰氏阴性细菌伴放线聚集杆菌是人类的常见居民， 上呼吸消化道疾病和局部侵袭性牙周炎（牙周炎）的病原体。所述细菌产生 RTX毒素（LtxA）。LtxA可以在可溶性和外膜囊泡（OMV）中递送至宿主细胞。 相关形式。可溶性LtxA需要β2整联蛋白LFA-1和富含胆固醇的膜的存在 筏发挥其对人体免疫细胞的致命作用。最近的数据表明，可溶性LtxA损害溶酶体， 引起促炎细胞因子的释放并诱导人巨噬细胞中的细胞死亡。LtxA的可溶性 被递送到巨噬细胞的溶酶体并诱导炎症反应，这是未知的。机制 由OMV递送的LtxA引起的细胞损伤是完全未探索的。本研究的目的是确定 人巨噬细胞中LtxA运输和中毒的机制。我们的中心假设是， LtxA的破坏性作用可以通过防止LtxA运输来停止。因此，我们提出一套 了解可溶性与OMV相关LtxA在人体内定位的实验 巨噬细胞我们的假设将在两个特定的目标进行测试：1）确定运输和细胞损伤 2）研究OMV相关LtxA的转运及中毒机制。在我们 实验计划，我们将利用共聚焦成像结合免疫学和生物化学方法， 研究LtxA在巨噬细胞内吞运输的模式，并确定LtxA的下游效应 炎性小体激活毒素诱导的巨噬细胞损伤将使用 细菌/巨噬细胞复合物，并在体外使用纯化的毒素，OMV和纯化的细胞器。的 拟议的研究与公共卫生有关，提出这一建议的理由是确定关键目标 用于防止诱导巨噬细胞中的细胞死亡和牙周炎症的LtxA运输。的 预计项目完成后将产生新型牙周炎治疗和诊断方法。 重要的是，这项研究的结果将提供一个基础，可以扩展到了解致病性 其他RTX毒素的机制，并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.