Listeria Hemolysin and Escape from a Vacuole

描述(由申请人提供)：单核细胞增生性李斯特菌是一种兼性的细胞内病原体，为宿主-病原体相互作用的基础研究提供了一个极其顺从的模型。重要的是，单核细胞增多性李斯特菌也是一种临床相关的食源性病原体，会导致孕妇的高死亡率和免疫功能受损。Listeriolysin O(LLO)是单核细胞增多性李斯特菌致病的主要决定因素，也是宿主免疫反应的靶标。LLO是形成孔的溶细胞素大家族中的一员，该家族主要负责介导单核细胞增多性李斯特菌从吞噬小体中逃逸和毒力；在动物感染模型中，LLO-突变体的毒力较低。LLO活性是一把双刃剑，因为它的活性必须限制在酸性吞噬小体上，否则宿主细胞将因LLO介导的细胞死亡(称为区隔作用)而死亡。在这项提议的目标I中，将通过结合诱变、细胞生物学和生物化学来确定介导区隔作用的分子决定因素。LO突变体将在磷酸化、泛素化、蛋白分解、半衰期和聚集方面进行表征。在AIM II中，将研究自噬在从吞噬小体逃逸以及LLO分泌到宿主细胞质中的命运方面所起的作用。具有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AIM III中，将使用一种新开发的基于水手的转座子突变系统来鉴定LLO以外的基因产物对LLO的表达、合成、分泌和毒性所起的作用。这些研究应确定与LLO和LLO mRNA相互作用的假想宿主蛋白(S)以防止毒性，并开辟与LLO的表达、合成、分泌和毒性相关的新领域。最后，将研究翻译后修饰和自噬在抗原处理和递呈的MHC I类和II类途径中LLO表位呈递方面的作用。新的体内检测方法将被用来选择不被宿主获得性免疫识别的LLO突变体，从而提供有关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，例如结核病、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。关于单核细胞增多性李斯特菌的拟议研究将有助于深入了解与细胞内病原体引起的疾病的治疗和预防相关的分子生物学、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.