INTRACELLULAR LIFESTYLE OF PSEUDOMONAS AERUGINOSA

铜绿假单胞菌的细胞内生活方式

• 批准号: 8391254
• 负责人: Suzanne MJ FLEISZIG
• 金额: $ 35.35万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-05 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391254
• 关键词: Actins; Antibiotics; Autophagocytosis; Bacteria; Biological Assay; Bulla; Burn injury; Cell Culture Techniques; Cell membrane; Cells; Cessation of life; Contact Lenses; Cornea; Corneal Diseases; Cystic Fibrosis; Cytoplasm; Cytosol; Data; Detection; Diffusion; Disease; Epithelial; Epithelial Cells; Event; Exhibits; Exocytosis; Eye; Eye Infections; Genes; Human; Image; Immunocompromised Host; Immunohistochemistry; In Vitro; Incidence; Individual; Infection; Injury; LAMP3 gene; Life; Life Style; Lysosomes; Membrane; Methods; Microscopy; Modeling; Molecular; Needles; Outcome Measure; Pathogenesis; Pathway interactions; Patients; Play; Population; Pseudomonas aeruginosa; Publishing; Research; Resistance; Role; Severity of illness; Site; Skin; Surface; Swimming; System; Testing; Time; Tissues; Type III Secretion System Pathway; Vacuole; Virulence; Vision; abstracting; cell motility; cell type; children with cystic fibrosis; corneal epithelium; in vivo; inhibitor/antagonist; killings; late endosome; mutant; novel; novel strategies; outcome forecast; pathogen; public health relevance; residence; time use; trafficking

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.

项目摘要/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 如眼睛、皮肤和呼吸道是最常见的靶点。易感人群 包括囊性纤维化儿童、免疫功能低下者、烧伤患者、插管患者以及 隐形眼镜佩戴者。铜绿假单胞菌感染的发生率正在上升；令人担忧的是，它往往很高 破坏性的，与不良预后相关的。众所周知，铜绿假单胞菌感染很难使用 可用的治疗方法，部分是因为铜绿假单胞菌拥有大量致力于生存和 适应。因此，迫切需要新的治疗方法。虽然我们知道铜绿假单胞菌可以 在感染过程中进入上皮细胞，细胞侵袭可能是致病过程中的一个关键组成部分，但很少是 已知铜绿假单胞菌在任何细胞类型中的细胞内生活方式。这项研究的目的是 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 目的是在眼部感染模型中降低体内的毒力。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生态位；感染诱导的质膜气泡。在这些 气泡、细菌复制并表现出快速(实时可见)运动。数据显示，第三类 分泌系统(T3SS)是铜绿假单胞菌形成小泡-生态位所必需的。T3SS突变体未能形成 而不是定位于核周液泡。与野生型细菌相比，T3SS突变体(保留 侵袭能力)进入上皮细胞后丧失活力。T3SS效应器和易位子 运输T3SS效应物穿过宿主细胞膜所需的两种物质在铜绿假单胞菌中都发挥作用 细胞内生存/贩运。效应子突变体和转位突变体都缺乏气泡能力和通信量 然而，对于核前空泡，只有效应突变体失去了细胞内复制的能力。因此， 数据表明T3SS在细胞内存活中至少有两个作用：1)细胞内依赖效应器 核周空泡中的复制，以及2)转运子依赖的小泡生态位的形成。假设是 测试如下：目标1：在没有T3SS效应器的情况下，铜绿假单胞菌在溶酶体中被降解，但 特定的T3SS效应器操纵细胞内转运，使其能够在胚泡周围的空泡中存活。目标2： T3SS通过使细菌从空泡室逃逸来参与泡龛的形成 (转运子依赖)，并且T3SS在水泡的形成/向它们贩运中也有直接作用。 目的3：体内细胞内存活也是T3SS依赖的，可以作为调控毒力的靶点。 AIMS 1和2将涉及体外细胞培养感染方法、细菌突变体、活性分析和成像。 与分子事件的抑制剂/激活剂一起使用和不与其一起使用。目标3将使用一个成熟的 活体角膜感染模型，AIMS 1和2的方法，以及细菌定植和 疾病的严重性。