Microarray Analysis of Plague-Induced Apoptosis

描述(申请人提供)：鼠疫耶尔森氏菌的质粒编码型Ill分泌系统的功能是将一组毒素(YOPs)输送到宿主真核细胞中。YOP毒素调节宿主细胞中的信号通路，以中和先天性免疫机制。YopJ毒素可触发感染耶尔森氏菌的巨噬细胞的凋亡。YopJ在结构上与半胱氨酸蛋白酶家族有关，但其毒力的确切靶点(S)尚不清楚。已有研究表明，YopJ抑制了几个负责转录因子激活的信号通路。激活转录因子NF-kB的信号通路是YopJ的关键靶点。据推测，YopJ通过减少一个或多个受NF-kB调控的凋亡抑制基因的表达来促进巨噬细胞死亡。我们将使用微阵列分析来确定与感染YopJ鼠疫杆菌的巨噬细胞相比，野生型鼠疫杆菌感染的巨噬细胞中是否有编码凋亡抑制物的基因表达水平较低。以YopJ特异性方式下调的凋亡抑制基因将在巨噬细胞中过度表达，以确定其产物是否可以预防耶尔森氏菌诱导的细胞死亡。在该R21申请中提出的实验将增加父R01授权的特定目标3(AI43389-03“Yersinia YOPs的主机信号功能的调制”)。具体目的3是通过确定YopJ与宿主信号通路组件之间的功能相互作用来阐明YopJ诱导细胞凋亡的机制。拟议的实验与R21应用的探索性/发展性是一致的，因为它们将使用已建立的基因组方法来表征全基因组转录反应并促进基因发现。

DESCRIPTION (provided by applicant): The plasmid-encoded type Ill secretion system of Yersinia pestis functions to deliver a set of toxins (Yops) into host eukaryotic cells. The Yop toxins modulate signaling pathways in host cells to neutralize innate immune mechanisms. The toxin YopJ triggers apoptosis in macrophages infected with Yersinia. YopJ is structurally related to a family of cysteine proteases, but the precise target(s) of its toxic activity remain unknown. It has been shown that YopJ inhibits several signaling pathways that are responsible for activation of transcription factors. The signaling pathway that activates the transcription factor NF-kB is a key target of YopJ. It is hypothesized that YopJ promotes macrophage death by reducing expression of one or more apoptosis inhibitor genes that are regulated by NF-kB. We will use microarray analysis to determine if any genes encoding apoptosis inhibitors are expressed at lower levels in macrophages infected with wild-type Y. pestis as compared to macrophages infected with YopJ Y. pestis. Apoptosis inhibitor genes that are down regulated in a YopJ-specific manner will be overexpressed in macrophages to determine if their products can protect against Yersinia-induced cell death. The experiments proposed in this R21 application will augment specific aim 3 of the parent R01 grant (AI43389-03 "Modulation of Host Signaling Functions by Yersinia Yops"). Specific aim 3 is to elucidate the mechanism of YopJ-induced apoptosis by identifying functional interactions between YopJ and components of host signaling pathways. The proposed experiments are consistent with the exploratory/developmental nature of the R21 application because they will employ established genomic approaches to characterize genome-wide transcriptional responses and to facilitate gene discovery.