INTRACELLULAR LIFESTYLE OF PSEUDOMONAS AERUGINOSA

铜绿假单胞菌的细胞内生活方式

• 批准号: 7743826
• 负责人: Suzanne MJ FLEISZIG
• 金额: $ 47.99万
• 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/7743826
• 关键词: Actins; Antibiotics; Autophagocytosis; Bacteria; Biological Assay; Bulla; Burn injury; Cell Culture Techniques; Cell Nucleus; 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; 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

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.

描述（由申请人提供）：铜绿假单胞菌会引起严重的观察和威胁生命的疾病。上皮衬里的表面组织，例如眼睛，皮肤和气道是最常见的部位。易感人群包括患有囊性纤维化的儿童，免疫受损的个体，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。令人担忧的是，它通常具有很高的破坏性，并且预后不良。众所周知，铜绿假单胞菌感染很难使用可用的疗法治疗，部分原因是铜绿假单胞菌具有大量用于生存和适应性的基因。因此，迫切需要新的治疗方法。虽然众所周知，铜绿假单胞菌可以在感染过程中进入上皮细胞，并且该细胞浸润可能是发病机理中的关键成分，但对于任何细胞类型中铜绿假单胞菌的细胞内生活方式知之甚少。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以减少眼部感染模型中的体内毒力。初步数据表明，铜绿假单胞菌在上皮细胞内占据了一个新颖的细胞内细胞裂市场。感染引起的质膜泡沫。在这些泡沫中，细菌复制并表现出快速（实时可见）运动。数据表明，铜绿假单胞菌需要三型分泌系统（T3SS）。 T3SS突变体无法形成泡沫，而是本地化为核周液泡。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效应子和跨宿主细胞膜中T3SS效应子所需的转运剂在铜绿假单胞菌内生存/运输中起着作用。但是，效应子突变体和转运突变体每个都缺乏临界前液泡的流量和流量，只有效应子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表明了两个角色。 1）钙核液泡中的效应子依赖性细胞内复制，以及2）易转基因依赖性的泡泡形成。要测试的假设是：AIM 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，但是特定的T3SS效应器会操纵内吞运输以实现多环液泡的生存。 AIM 2：T3SS通过使细菌逃离液泡隔室（转运依赖性）来参与BLEB-niche形成，并且T3SS在BLEB形成/运输中也有直接的作用。 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，可活力测定和与分子事件的抑制剂/活化剂一起使用的成像。 AIM 3将使用良好的体内角膜感染模型，AIM 1和2的方法以及细菌定殖和疾病严重程度的定量。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，因为这种病原体固有地抵抗杀害，可能具有很高的破坏性，并且易感人群包括已经因囊性纤维化，囊性纤维化，免疫症状，烧伤或其他伤害而衰弱的人。铜绿假单胞菌在感染过程中可能会在细胞内变成细胞内，这已被证明有助于体内发病机理。虽然已经研究了细胞进入机制，但铜绿假单胞菌在侵袭后生存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使用的策略，用于细胞内生存，然后确定其作为新疗法的靶标的潜力。