INTRACELLULAR LIFESTYLE OF PSEUDOMONAS AERUGINOSA

铜绿假单胞菌的细胞内生活方式

• 批准号: 8196921
• 负责人: Suzanne MJ FLEISZIG
• 金额: $ 37.61万
• 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 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8196921
• 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 铜绿假单胞菌会引起严重的观察和威胁生命的疾病。上皮表面组织 例如眼睛，皮肤和气道是最常见的位置。易感人群 包括具有囊性纤维化的儿童，免疫功能低下的个体，烧伤受害者，插管患者和 隐形眼镜佩戴者。铜绿假单胞菌感染的发生率正在上升。鉴于它通常很高，令人担忧 破坏性和预后不良。众所周知，铜绿假单胞菌感染很难治疗 可用的疗法，部分是因为铜绿假单胞菌拥有大量用于生存的基因 适应。因此，迫切需要新的治疗方法。众所周知，铜绿假单胞菌可以 在感染过程中输入上皮细胞，该细胞侵袭可能是发病机理中的关键成分，几乎没有IS 有关铜绿假单胞菌在任何细胞类型中的细胞内生活方式已知。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 靶向减少眼部感染模型中体内毒力。初步数据表明，铜绿假单胞菌 占据上皮细胞内的新型细胞内生态位；感染引起的质膜泡沫。在这些 泡沫，细菌复制并表现出快速（实时可见）运动。数据显示三型 铜绿假单胞菌需要分泌系统（T3SS）。 T3SS突变体无法形成 爆炸，而是本地化为核周液泡。与野生型细菌相反，T3SS突变体（保留 入侵能力）进入上皮细胞后失去活力。 T3SS效应子和转运器 跨宿主细胞膜运输T3SS效应子的所需 细胞内生存/贩运。效应子突变体和转运突变体每个都缺乏流动能力和流量 然而，对于核可吸收液泡，只有效应突变体失去了细胞内复制的能力。因此， 数据表明T3S在细胞内存活中至少有两个角色。 1）依赖效应器的细胞内 核周液泡中的复制和2）易转克依赖性的泡沫形成。假设是 测试是：目标1：在没有T3SS效应子的情况下，铜绿假单胞菌在溶酶体内降解，但 特定的T3SS效应子操纵内吞运输以使周围液泡的生存。目标2：那 T3SS通过使细菌从液泡隔间逃脱来参与BLEB-NIHICH形成 （依赖性依赖性），并且T3S在BLEB形成/贩运中也有直接的作用。 AIM 3：体内细胞内存活也依赖于T3SS，并且可以针对操纵毒力。 目标1和2将涉及体外细胞培养感染方法，细菌突变体，生存力测定和成像 与分子事件的抑制剂/活化剂一起使用。 AIM 3将使用良好的 体内角膜感染模型，目标1和2的方法，以及细菌定植的定量和 疾病的严重程度。