Microarray Analysis of Plague-Induced Apoptosis

描述（由申请人提供）：鼠疫耶尔森氏菌的质粒编码的Ill型分泌系统的功能是将一组毒素（Yops）传递到宿主真核细胞中。Yop毒素调节宿主细胞的信号通路以中和先天免疫机制。毒素YopJ触发感染耶尔森菌的巨噬细胞凋亡。YopJ在结构上与一个半胱氨酸蛋白酶家族相关，但其毒性活性的确切靶点尚不清楚。研究表明，YopJ抑制了几种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通过降低NF-kB调控的一种或多种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在感染野生型鼠疫杆菌的巨噬细胞中，与感染YopJ鼠疫杆菌的巨噬细胞相比，是否有编码凋亡抑制剂的基因表达水平较低。以yopj特异性方式下调的细胞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防止耶尔森菌诱导的细胞死亡。本R21申请中提出的实验将增强母体R01拨款（AI43389-03“Yersinia Yops调制宿主信号功能”）的特定目标3。具体目的3是通过鉴定YopJ与宿主信号通路组分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/发展性性质是一致的，因为它们将采用既定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.