Temporal Requirements for Intracellular Pathogenesis

性状（由申请方提供）：单核细胞增生李斯特菌（L.m.）是一种细胞内细菌病原体，可导致孕妇、老年人和免疫功能低下者患上严重的食源性疾病。李斯特菌溶血素O（LLO），一种成孔细胞溶解素，和两种细菌磷脂酶，PI-PLC和PC-PLC，是介导由细菌进入和细胞内扩散引起的宿主细胞空泡裂解的发病机制的重要决定因素。LLO也在胞质内生长期间表达，并介导宿主细胞生理学的许多改变。在细胞系的体外感染期间，LLO足以促进所有液泡膜的裂解。然而，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发挥特定的作用，并且LLO的胞内产生对于最佳细胞内生长是必要的。该提案的重点是精确定义LLO在原代宿主细胞中的细胞内生长和细胞间扩散以及在小鼠感染模型中维持体内感染的时间要求。在目的I中，将确定在原代宿主细胞中细胞内生长和扩散期间对LLO表达的精确要求。这将通过使用新的遗传方法来实现，以允许在细胞内感染期间调节LLO的产生。细胞内LLO水平在原代宿主细胞感染和细菌复制期间变化，并通过显微镜分析和细胞内细菌计数确定传播。在目的II中，将确定LLO、PI-PLC和PC-PLC在细胞内扩散期间溶解液泡膜中的确切作用。L.M.允许LLO在PI-PLC和PC-PLC突变体中调节表达的菌株将用于混合宿主细胞感染。将使用细胞单层中的空斑形成、微分延时荧光显微镜和高分辨率电子显微镜评价感染进展和空泡膜溶解。在目的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小鼠将在不同时间的体内LLO诱导下感染。将通过计数器官中的细菌并与野生型和确定的L. m.变种人免疫学评估将通过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.