Temporal Requirements for Intracellular Pathogenesis

描述（由申请人提供）：单核增生李斯特菌（l.m.）是一种细胞内细菌病原体，可引起孕妇、老年人和免疫功能低下个体的严重食源性疾病。李斯特菌溶素O （LLO）是一种形成孔的细胞溶素，以及两种细菌磷脂酶PI-PLC和PC-PLC是介导细菌进入和细胞内扩散引起的宿主细胞液泡裂解的发病机制的重要决定因素。LLO也在胞浆内生长过程中表达，并介导宿主细胞生理的许多变化。在细胞系的体外感染过程中，LLO足以促进所有液泡膜的溶解。而PI-PLC和PC-PLC提高了膜裂解的效率。据推测，这些决定因素在每个液泡膜的溶解中起着特定的作用，而细胞内LLO的产生对于最佳的细胞内生长是必要的。本研究的重点是在小鼠感染模型中精确定义LLO对原代宿主细胞内生长和细胞间传播以及维持体内感染的时间要求。在Aim I中，将确定原代宿主细胞内生长和扩散过程中对LLO表达的确切需求。这将通过使用一种新的遗传方法来实现在细胞内感染期间允许调节LLO的产生。细胞内LLO水平在原代宿主细胞感染和细菌复制和传播过程中会发生变化，这是通过显微镜分析和细胞内细菌计数确定的。在Aim II中，将确定LLO， PI-PLC和PC-PLC在细胞内扩散过程中液泡膜溶解中的确切作用。允许在PI-PLC和PC-PLC突变体中调控LLO表达的L.m.菌株将用于混合宿主细胞感染。细胞单层斑块的形成、差分延时荧光显微镜和高分辨率电子显微镜将用于评估感染的进展和液泡膜的溶解。在Aim III中，我们将评估LLO表达对维持体内感染和获得性免疫发展的要求。BALB/c小鼠将在体内不同时间的LLO诱导下感染。感染的进展将通过从器官中枚举细菌并与野生型和定义的L.m.突变体的感染进行比较来评估。免疫评估将通过ELISPOT分析和对野生型细菌攻击的保护来确定。

DESCRIPTION (provided by applicant): Listeria monocytogenes (L.m.) is an intracellular bacterial pathogen that causes serious food-borne illness in pregnant women, the elderly and immunocompromised individuals. Listeriolysin O (LLO), a pore-forming cytolysin, and two bacterial phospholipases, PI-PLC and PC-PLC, are essential determinants of pathogenesis that mediate lysis of host cell vacuoles resulting from bacterial entry and intracellular spread. LLO is also expressed during intracytosolic growth and mediates numerous alterations in host cell physiology. During in vitro infection of cell lines, LLO is sufficient to facilitate lysis of all vacuolar membranes. Yet, PI-PLC and PC-PLC increase the efficiency of membrane lysis. It is hypothesized that these determinants play a specific role in the dissolution of each vacuolar membrane and the intracytosolic production of LLO is necessary for optimal intracellular growth. The focus of this proposal is to precisely define the temporal requirement of LLO for intracellular growth and cell-to-cell spread in primary host cells and for the maintenance of in vivo infection in a mouse infection model. In Aim I, the precise requirement for LLO expression during intracellular growth and spread in primary host cells will be determined. This will be accomplished by using a novel genetic approach to allow regulated production of LLO during intracellular infection. Intracellular LLO levels will be varied during infection of primary host cells and bacterial replication and spread determined by microscopic analysis and enumeration of intracellular bacteria. In Aim II, the precise roles of LLO, PI-PLC and PC-PLC in dissolution of vacuolar membranes during intracellular spread will be identified. L.m. strains allowing regulated expression of LLO in PI-PLC and PC-PLC mutants will be used in mixed host cell infections. Plaque formation in cell monolayers, differential time-lapse fluorescence microscopy and high-resolution electron microscopy will be used to evaluate progression of infection and dissolution of vacuolar membranes. In Aim III, we will evaluate the requirement of LLO expression for maintenance of in vivo infection and the development of acquired immunity. BALB/c mice will be infected under varying times of in vivo LLO induction. Progression of infection will be evaluated by enumerating bacteria from organs and comparing to infection of wild-type and defined L.m. mutants. Immunological assessment will be determined by ELISPOT analysis and protection from wild-type bacterial challenge.