Molecular dissection of Pseudomonas aeruginosa Exotoxin T virulence functions

 描述（由申请人提供）：铜绿假单胞菌（PA）是医院获得性肺炎和呼吸衰竭的最常见原因，是接受癌症药物患者菌血症和脓毒症的主要原因，也是免疫功能低下患者的杀手。PA具有许多细胞相关的和分泌的毒力因子。其中最主要的是III型分泌系统（T3 SS）装置，其充当导管，允许PA将一组效应外毒素（即ExoS、ExoT、ExoU和ExoY）直接转运到目标宿主细胞质中，在那里它们改变宿主细胞过程并促进PA感染。ExoT是迄今为止检查的所有铜绿假单胞菌分离株中存在的唯一T3 SS效应毒素，表明该毒力因子在PA发病机制中具有更重要的作用。最近，我们证明，PA抑制伤口愈合的方式，主要是依赖于其T3 SS效应毒素。我们发现ExoT在创伤修复的T3 SS依赖性抑制中起关键作用。我们和其他人已经进一步证明，ExoT破坏肌动蛋白细胞骨架，阻止细胞迁移，干扰细胞-基质接触，并在多个步骤中阻断胞质分裂。最近，我们已经证明ExoT阻断与T3 SS装置插入靶宿主膜相关的坏死毒性，并将细胞重新路由为凋亡性细胞死亡。我们认为，虽然T3 SS是必不可少的PA感染，其活动触发主机炎症反应是有害的PA发病机制。为了避免这些有害事件，我们假设PA采用ExoT作为抗炎剂来处理感染期间T3 SS的这种不良副作用。在本提案中，我们将确定ExoT抑制T3 SS诱导的促炎相关细胞毒性的机制（目的1），并确定ExoT在体内伤口感染背景下作为铜绿假单胞菌中的抗炎剂的作用（目的2）。这些研究的结果对于确定ExoT在PA发病机制中的独特作用至关重要，可以用于治疗性干预以在免疫受损患者的情况下治疗PA和/或利用ExoT诱导非炎性细胞凋亡的强大能力用于癌症治疗。

 DESCRIPTION (provided by applicant): Pseudomonas aeruginosa (PA) is the most common cause of hospital-acquired pneumonia and respiratory failure, a leading cause of bacteremia and sepsis in patients receiving cancer drugs, and a killer of immunocompromised patients. PA possesses a number of cell-associated and secreted virulence factors. Chief amongst them is a Type III Secretion System (T3SS) apparatus which functions as a conduit, allowing PA to directly translocate a set of effector exotoxins, namely ExoS, ExoT, ExoU, and ExoY, into the target host cytoplasm where they modify host cellular processes and advance PA infections. ExoT is the only T3SS effector toxin that is present in all P. aeruginosa isolates examined thus far, suggesting a more fundamental role for this virulence factor in PA pathogenesis. Recently, we demonstrated that PA inhibits wound healing in a manner that is primarily dependent on its T3SS effector toxins. We found that ExoT playes a pivotal role in the T3SS-dependent inhibition of wound repair. We and others have further demonstrated that ExoT disrupts the actin cytoskeleton, prevents cell migration, interferes with cell-substratum contacts, and blocks cytokinesis at multiple steps. More recently, we have demonstrated that ExoT blocks the necrotic toxicity associated with T3SS apparatus insertion into target host membrane and reroutes the cell into apoptotic cell death. We propose that although T3SS is essential for PA infection, its activity triggers host inflammatory responses which are harmful to PA pathogenesis. To circumvent these harmful events, we hypothesize that PA employs ExoT as an anti-inflammatory agent to deal with this undesirable side effect of T3SS during infection. In this proposal, we will determine the mechanism(s) by which ExoT inhibits T3SS-induced pro-inflammatory associated cytotoxicity (Aim 1) and determine the role of ExoT as an anti-inflammatory agent in Pseudomonas aeruginosa in the context of wound infection in vivo (Aim 2). Findings from these studies will be essential to define a unique role of ExoT in PA pathogenesis, one that can be exploited for therapeutic intervention to treat PA in the setting of immune compromised patients and/or exploit the powerful ability of ExoT to induce none inflammatory apoptosis for cancer therapy.