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(申请人提供)：铜绿假单胞菌可导致严重危及视力和生命的疾病。上皮衬里的表面组织，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，它往往具有极强的破坏性，并与不良预后有关。众所周知，使用现有的治疗方法很难治疗铜绿假单胞菌感染，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。虽然已经知道铜绿假单胞菌在感染过程中可以进入上皮细胞，而且细胞侵袭可能是致病过程中的一个关键组成部分，但对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，并确定在眼感染模型中，这些策略是否可以靶向降低体内的毒力。初步数据显示，铜绿假单胞菌在上皮细胞中占据了一个新的细胞内生态位；感染诱导的质膜气泡。在这些气泡中，细菌复制并表现出快速(实时可见)的运动。结果表明，铜绿假单胞菌形成泡-生态位需要T3SS分泌系统。T3SS突变体不能形成气泡，而是定位于核周液泡。与野生型细菌不同，T3SS突变体(保持侵袭能力)在进入上皮细胞后失去活力。T3SS效应子和转运T3SS效应子穿过宿主细胞膜都在铜绿假单胞菌的细胞内存活/运输中发挥作用。效应器突变体和易位突变体都缺乏气泡能力和到核前空泡的运输能力，然而，只有效应器突变体失去了细胞内复制的能力。因此，这些数据表明，T3SS在细胞内存活中至少有两个作用：1)核周空泡中依赖效应器的细胞内复制，2)依赖转位的水泡生态位的形成。需要检验的假设是：目的1：在缺乏T3SS效应器的情况下，铜绿假单胞菌在溶酶体内被降解，但特定的T3SS效应器操纵内吞转运，使其能够在胞周空泡中存活。目的2：T3SS通过使细菌从空泡室逃逸(转运子依赖)来参与泡龛的形成，T3SS在泡泡的形成/运输中也有直接的作用。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、活性分析以及使用和不使用分子事件抑制/激活剂的成像。目标3将使用已建立的体内角膜感染模型、目标1和目标2中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：由铜绿假单胞菌引起的感染通常与预后不良有关，因为这种病原体天生对杀戮具有抵抗力，可能具有极强的破坏性，易感人群包括已经因囊性纤维化、免疫功能受损、烧伤或其他伤害等现有疾病而虚弱的人。铜绿假单胞菌在感染过程中可进入细胞内，这已被证明有助于体内的发病。虽然已经研究了细胞进入机制，但几乎对铜绿假单胞菌入侵后在细胞内存活的机制知之甚少。研究计划的重点是研究铜绿假单胞菌在细胞内生存所使用的策略，然后确定它们作为新疗法靶点的潜力。