Killing of intracellular bacteria by Perforin-2

描述(申请人提供)：病原菌进入组织后，被单核吞噬细胞和粒细胞吞噬并杀死，这些细胞组成地表达穿孔蛋白穿孔素-2。我们表明，遗传缺陷或siRNA击倒穿孔素-2使专业吞噬细胞无法杀死病原体，从而导致细胞内复制。病原菌也入侵并被上皮细胞和其他组织形成细胞吞噬。我们发现角质形成细胞结构性地表达穿孔素-2，所有被分析的其他上皮细胞和组织形成细胞都被干扰素或细菌入侵诱导表达穿孔素-2。与吞噬细胞一样，组织形成细胞中穿孔素-2的siRNA敲除或遗传缺陷使病原体能够在细胞内复制，而在穿孔素-2存在的情况下，病原体是KILL。进一步的分析表明，活性氧和氮物种以及溶酶体水解酶增强了穿孔素-2的杀菌活性，但不能清除没有穿孔素-2的细胞内病原体。在小鼠中，穿孔素-2的遗传缺陷会导致对低剂量沙门氏菌、葡萄球菌和其他病原体的致命易感性，而在穿孔素-2充足的动物中，这些病原体被清除了。穿孔素-2是一个含有MACPF结构域的完整膜蛋白，其激活和杀伤机制非常复杂。我们发现穿孔素-2定位于膜小泡中，这些小泡分布在未感染细胞的整个胞浆中。细菌感染会导致穿孔素-2在包裹细菌的吞噬小体或内小体膜中迅速积累。从感染细胞中重新分离细菌和电子显微镜分析显示，细菌细胞壁上有100�宽的毛孔集群，这表明致命的打击是由穿孔素-2介导的聚合和成孔。病原菌具有逃避或颠覆穿孔素-2的机制，以便在细胞内生存。在这项应用中，我们将研究穿孔素-2激活和杀伤的分子机制，这是开发治疗方法以增强穿孔素-2介导的杀伤作用以克服抗生素耐药性所必需的。

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.