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

通过 RNAi 筛选，使用表达李斯特菌的荧光素酶鉴定新型防御因子。

• 批准号: 21780088
• 负责人: GOTO Akira
• 金额: $ 2.91万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2010
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21780088/
• 关键词: 細胞応答; 自然免疫; 抗菌ペプチド; DNAマイクロアレイ; RNAi; ショウジョウバエ; 細胞内寄生細菌; レポーターアッセイ; コロニー形成法; リステリア菌; DNAマイクロアレイ法

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.

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，我建立了一个系统，其中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的生长。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。

项目成果

Cooperative regulation of the induction of the novel antibacterial Listericin by PGRP-LE and JAK-STAY pathway

PGRP-LE 和 JAK-STAY 通路协同调控新型抗菌李斯特菌素的诱导

• 发表时间: 2010
• 作者: Akira Goto;Shoichiro Kurata
• 通讯作者: Shoichiro Kurata

Cooperative regulation of the induction of the novel antibacterial Listericin by PGRP-LE and JAK-STAT pathway.

PGRP-LE 和 JAK-STAT 途径协同调节新型抗菌李斯特菌素的诱导。

• 发表时间: 2010
• 作者: Goto;A.;Kurata;S.
• 通讯作者: S.

Novel Drosophila antimicrobial peptide-like gene Listericin regulated by JAK-STAT pathway has bacteriocidal activities against gram-negative bacteria

JAK-STAT通路调控的新型果蝇抗菌肽样基因李斯特菌素对革兰氏阴性菌具有杀菌活性

• 发表时间: 2009
• 作者: Akira Goto;Tamaki Yano;Jun Terashima;Shoichiro Kurata
• 通讯作者: Shoichiro Kurata

Novel roles of receptor-type Guanylyl Cyclase Gyc76C on Drosophila innate immune responses.

受体型鸟苷酸环化酶 Gyc76C 对果蝇先天免疫反应的新作用。

• 发表时间: 2010
• 作者: Goto;A.;Iwashita;S.;Nabe;K.;Fukuzaki;M.;Oshima;Y.;Kurata;S.
• 通讯作者: S.

Cooperative regulation of the induction of the novel antibacterial Listericin by PGRP-LE and JAK-STAY pathway.

PGRP-LE 和 JAK-STAY 途径协同调节新型抗菌李斯特菌素的诱导。

• 发表时间: 2010
• 作者: Akira Goto;Tamaki Yano;Jun Terashima;Shinzo Iwashita;Yoshiteru Oshima;Shoichiro Kurata.
• 通讯作者: Shoichiro Kurata.