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III型分泌系统的功能是将一组毒素（Yops）递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免疫机制。毒素YopJ触发感染耶尔森氏菌的巨噬细胞凋亡。YopJ在结构上与半胱氨酸蛋白酶家族相关，但其毒性活性的精确靶标仍然未知。已经显示YopJ抑制负责转录因子活化的几种信号传导途径。激活转录因子NF-kB的信号通路是YopJ的关键靶标。假设YopJ通过减少一种或多种受NF-κ B调节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是否有任何编码凋亡抑制因子的基因在感染野生型Y的巨噬细胞中以较低水平表达。鼠疫菌感染的巨噬细胞相比，鼠疫以YopJ特异性方式下调的细胞凋亡抑制剂基因将在巨噬细胞中过表达，以确定其产物是否可以防止耶尔森氏菌诱导的细胞死亡。在此R21申请中提出的实验将增加父R 01资助的具体目标3（AI 43389 -03“耶尔森氏菌对宿主信号功能的调节”）。具体目标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诱导的细胞凋亡的机制。所提出的实验与R21应用的探索性/开发性质一致，因为它们将采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.