INTRACELLULAR LIFESTYLE OF PSEUDOMONAS AERUGINOSA

描述（申请人提供）：铜绿假单胞菌可导致严重的视力和危及生命的疾病。有上皮的表面组织，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的儿童、免疫功能低下的个体、烧伤患者、插管患者和隐形眼镜佩戴者。铜绿假单胞菌感染的发病率呈上升趋势；令人担忧的是，它通常具有高度破坏性，并伴有预后不良。众所周知，铜绿假单胞菌感染很难用现有的治疗方法治疗，部分原因是铜绿假单胞菌拥有大量致力于生存和适应的基因。因此，迫切需要新的治疗方法。虽然已知P. aeruginosa在感染期间可以进入上皮细胞，并且细胞入侵可能是发病的关键因素，但对P. aeruginosa在任何细胞类型中的细胞内生活方式知之甚少。本研究的目的是了解P. aeruginosa使用的细胞内生存策略，并确定它们是否可以在眼感染模型中靶向降低体内毒力。初步数据显示，铜绿假单胞菌在上皮细胞内占据了一个新的细胞内生态位；感染引起的质膜泡。在这些气泡中，细菌复制并表现出快速（实时可见）的运动性。数据表明，铜绿假单胞菌形成气泡生态位需要第三型分泌系统（T3SS）。T3SS突变体不能形成泡，而是局限于核周液泡。与野生型细菌相比，T3SS突变体（保留入侵能力）在进入上皮细胞后失去生存能力。T3SS效应体和跨宿主细胞膜运输T3SS效应体所需的转位子都在铜绿假单胞菌胞内存活/运输中发挥作用。效应突变体和易位突变体都缺乏泡泡能力和通往核前液泡的交通，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T3SS在细胞内存活中至少有两种作用；1)在核周液泡中依赖于效应的细胞内复制，2)依赖于跨位点的泡位形成。需要验证的假设是：目的1：在没有T3SS效应物的情况下，铜绿假单胞菌在溶酶体内被降解，但特定的T3SS效应物操纵内吞运输，使其能够在核周液泡中存活。目的2:T3SS通过使细菌从液泡室中逃逸（依赖于跨空腔）参与泡位形成，并且T3SS在泡形成/向泡位运输中也有直接作用。目的3：体内的细胞内存活也是依赖于t3ss的，并且可以靶向操纵毒力。目标1和目标2将涉及体外细胞培养感染方法、细菌突变体、活力测定和使用或不使用分子事件抑制剂/激活剂的成像。目标3将使用一个完善的体内角膜感染模型，目标1和目标2的方法，以及细菌定植和疾病严重程度的量化来完成。公共卫生相关性：铜绿假单胞菌引起的感染通常与预后不良有关，因为这种病原体具有固有的抗杀灭性，可能具有高度破坏性，易感人群包括已经因囊性纤维化、免疫功能低下、烧伤或其他损伤等现有疾病而虚弱的人群。铜绿假单胞菌可在感染期间变成细胞内，这已被证明有助于体内发病。虽然已经研究了细胞进入机制，但对铜绿假单胞菌入侵后在细胞内存活的机制几乎一无所知。研究计划的重点是研究铜绿假单胞菌在细胞内存活的策略，然后确定它们作为新疗法靶点的潜力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Infections caused by Pseudomonas aeruginosa are often associated with a poor prognosis because this pathogen is inherently resistant to killing, can be highly destructive, and susceptible populations include people already debilitated by existing conditions such as cystic fibrosis, immunocompromise, burns or other injury. P. aeruginosa can become intracellular during infection, and this has been shown to contribute to pathogenesis in vivo. While cellular entry mechanisms have been studied, almost nothing is known about mechanisms used by P. aeruginosa for survival within cells after invasion. The focus of the research plan is to study strategies used by P. aeruginosa for surviving intracellularly and then to determine their potential as targets for new therapies.