Killing of intracellular bacteria by Perforin-2

描述（由申请人提供）：致病菌进入组织后，被单核吞噬细胞和组成性表达成孔蛋白Perforin-2的粒细胞摄入并杀死。我们发现遗传缺陷或siRNA敲低穿孔素-2使专业吞噬细胞无法杀死病原体，从而导致细胞内复制。致病菌也侵入并被上皮细胞和其他组织形成细胞内吞。我们发现角质形成细胞组成性地表达Perforin-2，所有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Perforin-2。与吞噬细胞一样，组织形成细胞中穿孔素-2的siRNA敲低或遗传缺陷使病原体在穿孔素-2存在下能够在细胞内复制。进一步分析表明，活性氧、活性氮和溶酶体水解酶增强了穿孔素-2的杀菌活性，但没有穿孔素-2就无法清除细胞内病原体。小鼠中穿孔素-2的遗传缺陷导致小鼠对低剂量沙门氏菌、葡萄球菌和其他病原体的感染具有致命的易感性，而这些病原体在穿孔素-2充足的动物中被清除。穿孔素-2是一种含有MACPF结构域的整体膜蛋白；它的激活和杀伤机制非常复杂。我们发现穿孔素-2在未感染细胞中分布在整个细胞质中的膜囊中。细菌感染导致穿孔素-2在包裹细菌的吞噬体或内体膜上迅速积聚。从感染细胞中重新分离的细菌和电镜分析显示，细菌细胞壁上有100°宽的孔簇，表明致命的撞击是由穿孔素-2的聚合和成孔介导的。致病菌有逃避或破坏穿孔素-2的机制，以便在细胞内存活。在本应用中，我们将研究穿孔素-2激活和杀伤的分子机制，这对于开发增强穿孔素-2介导的杀伤的治疗方法是必要的，这可能克服抗生素耐药性。

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.