Listeria Hemolysin and Escape from a Vacuole

描述（由申请人提供）：单核细胞增生李斯特菌是一种兼性细胞内病原体，为宿主-病原体相互作用的基础研究提供了非常可靠的模型。重要的是，L。单核细胞增多症也是一种临床相关的食源性病原体，可导致孕妇和免疫功能低下者的高死亡率。L的一个初等行列式。李斯特菌溶血素O（LLO）是单核细胞增多症发病机制中的一个靶点，并且是宿主免疫应答的靶标。LLO是孔形成溶细胞素大家族的一员，主要负责介导L. LLO-突变体在动物感染模型中的毒力低5个对数。LLO活性是一把双刃剑，因为其活性必须限于酸性吞噬体，否则宿主细胞将因LLO介导的细胞死亡（称为区室化）而死亡。在本提案的目的I中，将通过使用诱变、细胞生物学和生物化学的组合来确定介导区室化的分子决定簇。LLO突变体将在磷酸化、泛素化、蛋白水解、半衰期和聚集方面进行表征。在目的II中，将检查自噬在从吞噬体逃逸和LLO分泌到宿主胞质溶胶中的命运方面所起的作用。自噬缺陷的巨噬细胞将提供一个很好的系统来研究这些问题。在目标III中，将使用新开发的基于mariner的转座子诱变系统来鉴定基因产物（而非LLO）对LLO表达、合成、分泌和毒性所起的作用。这些研究应确定与LLO和LLO mRNA相互作用以防止毒性的假设宿主蛋白，并开辟与LLO表达、合成、分泌和毒性有关的新研究领域。最后，翻译后修饰和自噬的作用将检查相对于抗原加工和呈递的MHC I类和II类途径中的LLO表位的呈递。新的体内测定将用于选择不被宿主获得性免疫识别的LLO突变体，从而提供关于导致免疫原性的外源蛋白性质的基本信息。 公共卫生相关性：由细胞内病原体引起的疾病，例如肺结核、艾滋病和疟疾，仍然是国际生物医学界面临的最大挑战之一。有关单核细胞增生李斯特菌的拟议研究，将有助深入了解与治疗和预防由细胞内病原体引起的疾病有关的分子生物学、细胞生物学和免疫学。

DESCRIPTION (provided by applicant): Listeria monocytogenes is a facultative intracellular pathogen that provides an extremely amenable model for basic studies on host-pathogen interactions. Importantly, L. monocytogenes is also a clinically relevant food-borne pathogen that causes a high rate of mortality in pregnant women and the immunocompromised. A primary determinant of L. monocytogenes pathogenesis and a target of the host's immune response is Listeriolysin O (LLO). LLO is a member of a large family of pore-forming cytolysins that is largely responsible for mediating escape of L. monocytogenes from a phagosome and for virulence; LLO-minus mutants are 5-logs less virulent in animal models of infection. LLO activity is a double-edged sword as its activity must be restricted to an acidic phagosome or the host cell will die due to LLO-mediated cell death (referred to as compartmentalization). In Aim I of this proposal the molecular determinants mediating compartmentalization will be determined by using a combination of mutagenesis, cell biology and biochemistry. LLO mutants will be characterized with respect to phosphorylation, ubiquitylation, proteolysis, half-life, and aggregation. In Aim II, the role played by autophagy will be examined with respect to escape from a phagosome and the fate of LLO secreted into the host cytosol. Macrophages that are defective for autophagy will provide an excellent system with which to examine these questions. In Aim III, a newly developed, mariner-based, transposon mutagenesis system will be used to identify the role played by gene-products, other than LLO, on LLO expression, synthesis, secretion and toxicity. These studies should identify the hypothetical host protein(s) that interact with LLO and LLO mRNA to prevent toxicity, and open up new areas of investigations pertaining to LLO expression, synthesis, secretion and toxicity. Lastly, the role of post-translational modifications and autophagy will be examined with respect to the presentation of LLO epitopes in both the MHC Class I and Class II pathways of antigen processing and presentation. Novel in vivo assays will be used to select LLO mutants that are not recognized by the host's acquired immunity, thereby providing basic information on properties of foreign proteins that leads to immunogenicity. PUBLIC HEALTH RELEVANCE: Diseases caused by intracellular pathogens, for example, tuberculosis, AIDS and Malaria, remain one of the largest challenges facing the international biomedical community. The proposed studies on Listeria monocytogenes will provide insight into the molecular biology, cell biology and immunology relevant to the treatment and prevention of diseases caused by intracellular pathogens.