Killing of intracellular bacteria by Perforin-2

描述（由申请方提供）：病原菌进入组织后，被组成型表达孔形成蛋白穿孔蛋白-2的单核吞噬细胞和粒细胞摄入并杀死。我们表明穿孔蛋白-2的遗传缺陷或siRNA敲低使专职吞噬细胞不能杀死病原体，导致细胞内复制。病原菌也侵入并被上皮细胞和其他组织形成细胞内吞。我们表明，角质形成细胞组成型表达穿孔蛋白-2，并且所分析的所有其他上皮和组织形成细胞通过干扰素或细菌入侵诱导表达穿孔蛋白-2。如在吞噬细胞中，组织形成细胞中穿孔蛋白-2的siRNA敲低或遗传缺陷使得病原体能够在穿孔蛋白-2存在下被杀死的细胞内复制。进一步分析揭示，活性氧和氮物质以及溶酶体水解酶增强穿孔蛋白-2的杀菌活性，但在没有穿孔蛋白-2的情况下不能清除细胞内病原体。小鼠中穿孔蛋白-2的遗传缺陷导致对低剂量沙门氏菌、葡萄球菌和其他病原体感染的致命易感性，所述低剂量沙门氏菌、葡萄球菌和其他病原体在穿孔蛋白-2充足的动物中被清除。穿孔蛋白-2是一种含有MACPF结构域的膜蛋白，其激活和杀伤机制非常复杂。我们表明，穿孔蛋白-2定位于膜囊泡，分布在整个未感染细胞的胞质溶胶。细菌感染引起穿孔蛋白-2在包围细菌的吞噬体或内体膜中的快速积累。从感染细胞中重新分离细菌和电子显微镜分析揭示了细菌细胞壁上100 μ宽的孔簇，表明致命的打击是由穿孔蛋白2的聚合和孔形成介导的。致病细菌具有逃避或破坏穿孔蛋白-2以在细胞内存活的机制。在本申请中，我们将研究穿孔蛋白-2活化和杀伤的分子机制，这是开发增强穿孔蛋白-2介导的杀伤的治疗所必需的，所述治疗可以克服抗生素抗性。

DESCRIPTION (provided by applicant): After entering tissues, pathogenic bacteria are ingested and killed by mononuclear phagocytes and by granulocytes that constitutively express the pore-forming protein Perforin-2. We show that genetic deficiency or siRNA knock down of Perforin-2 disables killing of the pathogen by professional phagocytes resulting in intracellular replication. Pathogenic bacteria also invade and are endocytosed by epithelial cells and other tissue forming cells. We show that keratinocytes express Perforin-2 constitutively and that all other epithelial and tissue forming cells analyzed are induced to express Perforin-2 by interferons or bacterial invasion. As in phagocytes, siRNA knock down or genetic deficiency of Perforin-2 in tissue forming cells enables intracellular replication of the pathogen which is kille in the presence of Perforin-2. Further analysis reveals that reactive oxygen and nitrogen species and lysosomal hydrolases enhance the bactericidal activity of Perforin-2 but are unable to clear intracellular pathogens without Perforin-2. Genetic deficiency of Perforin-2 in mice causes lethal susceptibility to infection with low doses of Salmonella, Staphylococcus and other pathogens that are cleared in Perforin-2 sufficient animals. Perforin-2 is a MACPF domain containing, integral membrane protein; its activation and killing mechanisms is highly complex. We show that Perforin-2 is localized in membrane vesicles that are distributed throughout the cytosol in uninfected cells. Bacterial infection causes rapid accumulation of Perforin-2 in the phagosomal or endosomal membrane enclosing the bacterium. Re-isolation of bacteria from infected cells and electron microscopic analysis reveals clusters of 100 � wide pores on bacterial cell walls suggesting that the lethal hit is mediated by polymerization and pore-formation by Perforin-2. Pathogenic bacteria have mechanisms to evade or subvert Perforin-2 in order to survive inside cells. In this application we will study the molecular mechanisms of Perforin-2 activation and killing, which is requisite for development of treatments to enhance Perforin-2 mediated killing that may overcome antibiotic resistance.