Temporal Requirements for Intracellular Pathogenesis

描述(申请人提供)：单核细胞增多性李斯特菌(L.M.)是一种细胞内的细菌病原体，会导致孕妇、老年人和免疫功能受损的人患上严重的食源性疾病。Listeriolysin O(LLO)是一种致孔细胞溶血素，它和两种细菌磷脂酶PI-PLC和PC-PLC是致病的重要决定因素，它们介导了细菌进入和细胞内扩散导致的宿主细胞空泡的溶解。LIO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。在体外感染细胞系的过程中，LLO足以促进所有空泡膜的溶解。但PI-PLC和PC-PLC提高了膜裂解效率。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，胞内LLO的产生是细胞内最佳生长所必需的。这项建议的重点是精确定义LLO对原代宿主细胞内生长和细胞间传播的时间要求，以及在小鼠感染模型中维持体内感染的时间要求。在目标I中，将确定在原代宿主细胞内生长和扩散过程中对LLO表达的精确要求。这将通过使用一种新的遗传方法来实现，该方法允许在细胞内感染期间调节LLO的产生。在原代宿主细胞感染和细菌复制和传播过程中，细胞内LLO水平会发生变化，这是由细胞内细菌的显微镜分析和计数确定的。在AIM II中，将确定LLO、PI-PLC和PC-PLC在细胞内扩散过程中对液泡膜溶解的确切作用。L.M.在PI-PLC和PC-PLC突变体中允许LLO可调控表达的菌株将用于混合宿主细胞感染。细胞单层中的斑块形成、差分时移荧光显微镜和高分辨率电子显微镜将被用来评估感染的进展和空泡膜的溶解。在目标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需求。BALB/c小鼠在不同的体内LLO诱导时间下会被感染。感染的进展将通过计数器官中的细菌并与野生型和限定L.M.的感染进行比较来评估。变种人。免疫学评估将通过ELISPOT分析和保护免受野生型细菌攻击来确定。

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.