UPR determinants of Brucella virulence

项目摘要 布鲁氏菌病仍然是世界范围内最流行的人畜共患病， 冲击目前还没有安全的人类疫苗。最常与人类疾病有关的物种是B。梅利滕西斯 被证明难以根除摄入或吸入后，布鲁氏菌侵入巨噬细胞和其他细胞， 吞噬细胞，并在空泡内运输到内质网（ER），在那里它们建立一个复制系统。 利基在此过程中，布鲁氏菌会诱导宿主产生一种应激反应，称为“未折叠蛋白反应” （普遍定期审议）。UPR使细胞能够通过暂时减少蛋白质产生和扩增来科普ER应激 ER能力。然而，如果ER应激是长期的或深刻的，UPR启动细胞凋亡。我们注意到 UPR诱导程度与人毒力之间存在显著相关性。与B相反。流产和 疫苗株S19、B.羊种（毒性最强的种）诱导UPR靶标显著上调 基因C/EBP同源蛋白（CHOP）在小鼠和人巨噬细胞。在某些细菌感染中， CHOP增强毒力。在布鲁氏菌鼠模型中，CHOP对细胞凋亡和流产有重要作用。 CHOP还刺激可能导致人类疾病的促炎细胞因子（例如IL-23）的产生 表现为脊椎关节炎。目前，CHOP在布鲁氏菌感染巨噬细胞和 脊椎关节炎是未知的，也没有适当的疾病模型来解决这个具体的问题。我们 假设B. Melitensis有助于毒力， 该物种的致病性。为了更好地了解布鲁氏菌与宿主之间的关键相互作用， UPR和关节炎的发展，我们提出以下目的：目的1：检验假设，CHOP 有助于B的毒力。通过更大的细菌复制和诱导特异性 宿主炎症反应。我们将证实我们的初步发现，物种特异性的普遍定期审议诱导， 通过比较由B诱导的巨噬细胞基因表达来扩展这些结果。Melitensis vs. B.流产 此外，ddit 3（CHOP）-/-巨噬细胞将描绘由于CHOP引起的差异基因表达的部分。 我们将确定CHOP是否调节B的细胞凋亡、复制或细胞间扩散。melitensis。最后 B中的CHOP要求。将在体内测定羊种的复制和宿主的炎症反应。 目的2：建立布鲁氏菌脊椎关节炎易感小鼠模型。 近年来，已经开发了几种小鼠脊柱关节炎模型，其需要细胞因子 或感染性触发物，即SKG-ZAP-70突变模型和B10.RIII小鼠。我们将系统地测试这些 小鼠品系，以建立一个强大的，易于处理的模型，布鲁杆菌诱导的脊柱关节炎， 我们的总体假设。越来越多的病原体已经被认识到操纵 普遍定期审议。这项工作将有助于了解普遍定期审议如何介导细菌之间的关系， 入侵者、宿主反应和疾病表现。

PROJECT SUMMARY Brucellosis remains the most prevalent zoonosis worldwide, incurring significant human morbidity and economic impact. No safe human vaccine exists. The species most often implicated in human disease, B. melitensis has proven refractory to eradication. Following ingestion or inhalation, Brucella invade macrophages and other phagocytes, and traffic within vacuoles to the endoplasmic reticulum (ER) where they establish a replicative niche. During this process, Brucella induce a host stress response known as the “Unfolded Protein Response” (UPR). The UPR enables cells to cope with ER stress by transiently decreasing protein production and expanding ER capacity. However, if ER stress is prolonged or profound, the UPR initiates apoptosis. We have noted a striking correlation between extent of UPR induction and human virulence. In contrast to B. abortus and the vaccine strain S19, B. melitensis (the most virulent species) induces marked upregulation of the UPR target gene C/EBP homologous protein (CHOP) in murine and human macrophages. In certain bacterial infections, CHOP enhances virulence. CHOP critically contributed to apoptosis and abortion in a Brucella murine model. CHOP also stimulates the production of pro-inflammatory cytokines (e.g. IL-23) that could lead to human disease manifestations such as spondyloarthritis. Currently, the role of CHOP in Brucella infection of macrophages and spondyloarthritis is unknown, and there are no appropriate disease models to address this specific question. We hypothesize that the induction of a more extensive UPR by B. melitensis contributes to the virulence and pathogenicity of this species. To gain a greater understanding of the critical interactions between Brucella, host UPR and the development of arthritis, we propose the following aims: Aim 1: Test the hypothesis that CHOP contributes to the virulence of B. melitensis via greater bacterial replication and induction of specific host inflammatory responses. We will confirm our preliminary findings of species-specific UPR induction and expand upon these results by comparing macrophage gene expression induced by B. melitensis vs. B. abortus. Further, ddit3 (CHOP)-/- macrophages will delineate the portion of differential gene expression due to CHOP. We will determine if CHOP regulates apoptosis, replication or cell-cell spread of B. melitensis. Finally, the requirement for CHOP in B. melitensis replication and host inflammatory responses will be determined in vivo. Aim 2: Establish a mouse model of Brucella spondyloarthritis using spondyloarthritis-susceptible mice. Within recent years, several murine spondyloarthritis models have been developed that require either cytokine or infectious triggers, namely the SKG-ZAP-70 mutant model and B10.RIII mice. We will systematically test these mice strains to establish a robust, tractable model of Brucella-induced spondyloarthritis that will enable testing of our overarching hypothesis. An increasing number of pathogens have been recognized to manipulate the UPR. This work will contribute to the understanding of how the UPR mediates the relationships between bacterial invaders, host responses and disease manifestations.