INTRACELLULAR LIFESTYLE OF PSEUDOMONAS AERUGINOSA

项目总结/摘要 铜绿假单胞菌可导致严重的危及视力和生命的疾病。上皮衬里表面组织 例如眼睛、皮肤和呼吸道是最常见的目标部位。易感人群 包括患有囊性纤维化儿童、免疫功能低下的个体、烧伤患者、插管患者， 接触透镜佩戴者。铜绿假单胞菌感染的发病率正在上升;令人担忧的是，它往往是高度 具有破坏性，并伴有预后不良。众所周知，铜绿假单胞菌感染很难用 部分原因是铜绿假单胞菌拥有大量致力于生存的基因， 适应因此，迫切需要新的治疗方法。虽然已知铜绿假单胞菌可以 在感染过程中进入上皮细胞，细胞侵袭可能是发病机制的关键组成部分， 已知铜绿假单胞菌在任何细胞类型内的细胞内生活方式。本研究的目的是 了解铜绿假单胞菌使用的细胞内生存策略，并确定它们是否可以 靶向降低眼感染模型中的体内毒力。初步数据显示，铜绿假单胞菌 在上皮细胞内占据一个新的细胞内小生境;感染诱导的质膜水泡。在这些 水泡，细菌复制并表现出快速（实时可见）运动性。数据显示，第三类 分泌系统（T3 SS）是铜绿假单胞菌形成水泡龛所必需的。T3 SS突变体不能形成 而不是局限于核周空泡。与野生型细菌相比，T3 SS突变体（保留了 侵入能力）在进入上皮细胞后失去活力。T3 SS效应子和易位子 所需的运输T3 SS效应跨越宿主细胞膜都发挥作用铜绿假单胞菌 细胞内存活/运输。效应子突变体和转位子突变体各自缺乏起泡能力和运输 然而，只有效应突变体失去细胞内复制的能力。因此 数据表明T3 SS在细胞内存活中至少有两种作用：1）效应依赖性细胞内 核周空泡内复制; 2）translocon依赖的泡龛形成。假设是 目的1：在不存在T3 SS效应物的情况下，铜绿假单胞菌在溶酶体内降解，但 特异性T3 SS效应子操纵胞吞运输以使其能够在核周空泡中存活。目标2： T3 SS通过使细菌从空泡区室逃逸而参与泡龛的形成 （translocon依赖），并且T3 SS在水泡形成/向其运输中也有直接作用。 目的3：体内细胞内存活也是T3 SS依赖的，并且可以靶向操纵毒力。 目标1和2将涉及体外细胞培养感染方法、细菌突变体、活力测定和成像 使用和不使用分子事件的抑制剂/活化剂。目标3将使用一个完善的 体内角膜感染模型，目的1和2的方法，以及细菌定殖和 疾病严重程度。

Project Summary/Abstract Pseudomonas aeruginosa can cause severe sight- and life-threatening disease. Epithelial lined surface tissues such as the eye, the skin and the airways are the most commonly targeted sites. Susceptible populations include children with cystic fibrosis, immunocompromised individuals, burn victims, intubated patients, and contact lens wearers. The incidence of P. aeruginosa infection is rising; worrisome given that it is often highly destructive and associated with a poor prognosis. P. aeruginosa infection is notoriously difficult to treat using available therapies, in part because P. aeruginosa possesses a large number of genes devoted to survival and adaptation. Thus, new approaches to therapy are urgently needed. While it is known that P. aeruginosa can enter epithelial cells during infection, and that cell invasion can be a key component in pathogenesis, little is known about the intracellular lifestyle of P. aeruginosa within any cell type. The objective of this research is to understand intracellular survival strategies used by P. aeruginosa and to determine if they can be targeted to reduce virulence in vivo in an eye infection model. Preliminary data reveal that P. aeruginosa occupies a novel intracellular niche within epithelial cells; infection-induced plasma membrane blebs. In these blebs, bacteria replicate and demonstrate rapid (real-time visible) motility. The data show that the Type Three Secretion System (T3SS) is required for bleb-niche formation by P. aeruginosa. T3SS mutants fail to form blebs and instead localize to perinuclear vacuoles. In contrast to wild type bacteria, T3SS mutants (retain competency for invasion) lose viability after entering epithelial cells. The T3SS effectors and the translocon required for transporting T3SS effectors across host cell membranes both play roles in P. aeruginosa intracellular survival/trafficking. Effector mutants and translocon mutants each lack blebbing capacity and traffic to preinuclear vacuoles, however, only effector mutants lose capacity for intracellular replication. Thus, the data suggest at least two roles for the T3SS in intracellular survival; 1) effector-dependent intracellular replication in perinuclear vacuoles, and 2) translocon-dependent bleb niche formation. The hypotheses to be tested are: Aim 1: That in the absence of T3SS effectors, P. aeruginosa is degraded within lysosomes, but specific T3SS effectors manipulate endocytic trafficking to enable survival in perinculear vacuoles. Aim 2: That the T3SS participates in bleb-niche formation by enabling bacterial escape from vacuolar compartments (translocon-dependent) and that there are also direct roles for the T3SS in bleb formation/trafficking to them. Aim 3: That intracellular survival in vivo is also T3SS-dependent, and can be targeted to manipulate virulence. Aims 1 and 2 will involve in vitro cell culture infection methods, bacterial mutants, viability assays and imaging used with and without inhibitors/activators of molecular events. Aim 3 will be done using a well-established in vivo corneal infection model, methods from aims 1 and 2, and quantification of bacterial colonization and of disease severity.