Identification of novel defense factors using Luciferase expressing Listeria by RNAi screening.

天然免疫系统是植物、昆虫和哺乳动物高度保守的第一道防线系统之一。肽聚糖识别蛋白（Peptidoglycan recognition protein，PGRP）-LE是本实验室首次发现的一种在细胞内和细胞外均发挥作用的蛋白质，参与天然免疫信号通路Imd通路和黑色素合成通路的激活。此外，PGRP-LE使用称为“自噬”的一般蛋白质降解系统之一在消除细胞内病原体方面起关键作用，该系统独立于Toll和Imd途径。因此，为了进一步鉴定这种多功能PGRP-LE中涉及的新型宿主防御因子，我建立了可以通过表达组成型荧光素酶的李斯特菌菌株来监测李斯特菌生长的系统。使用这种李斯特菌菌株，我们可以看到PGRP-LE的效果的感染条件进行了仔细检查。作为一种替代策略，我还进行了DNA微阵列，并确定了一种新的抗菌素样肽命名为李斯特菌素。利用蛋白纯化、RNAi和过表达等方法对李斯特菌素进行了进一步的分析，结果表明李斯特菌素的表达受PGRP LE和JAK STAT信号通路的协同调控，其分泌形式对革兰氏阴性菌和李斯特菌具有杀菌活性。此外，使用李斯特菌素过表达的转基因果蝇的体内方法显示，它们在存活测试中表现出对李斯特菌感染的抗性。综合以上结果，我们可以证明李斯特菌素是一种新的果蝇天然免疫抗菌肽。

Innate immune system is one of the first-line defense system that is highly conserved from plants, insects and mammals. Peptidoglycan recognition protein (PGRP)-LE firstly identified in our laboratory is functioning on both intracellular and extracellular space and responsible for the activation of one of the innate immune signaling pathway, Imd pathway, and melanin synthesis pathway. Furthermore, PGRP-LE plays a crucial role to eliminate intracellular pathogens using one of the general protein degradation system called "Autophagy", which is independent on the Toll and Imd pathway. Therefore, to identify further novel host defense factors invoved in this multi-functional PGRP-LE, I set up the system where the growth of Listeria can be monitored by constitutive Luciferase expressing Listeria strains. Using this Listeria strain, the infection condition where we can see the effect of PGRP-LE was scrutinized. As an alternative strategy, I also conducted DNA microarray and identified a novel antimicrobial-like peptide named Listericin. Further analysis of Listericin using protein purification, RNAi and overexpression studies demonstrated that the expression of Listericin is cooperatively regulated by both PGRP-LE and the JAK-STAT pathway, as a secreted form, it exibits bacteriocidal activities against gram-negative bacteria and Listeria strain. Furthermore, in vivo approach using Listericin overexpressing transgenic flies showed that they exhibit resistance against Listeria infection in the surival test. Taken together with these results, we can show that Listericin is a novel antimicrobial peptide on Drosophila innate immune response.