Functional Impact of Stress Granules on Tick-Microbe Interactions

摘要 节肢动物的免疫力是影响媒介生物能力的关键因素。我们最近发现了一种非典型免疫 响应和限制伯氏疏螺旋体(莱姆氏疏螺旋体)的肩部硬蜱缺乏(IMD)途径 疾病)和无浆体吞噬细胞症(人粒细胞无浆体病)。宿主细胞的应激反应是 与免疫紧密交织在一起，可以增强或拮抗免疫信号。是否 应激反应途径与节肢动物免疫相交，从而影响媒介能力，目前尚不清楚。 应激颗粒(SGS)是在应激状态下细胞质中瞬间形成的RNA和蛋白质的聚集体。 细胞，例如在感染期间。SG构型在真核生物中高度保守，已知具有免疫力 在哺乳动物中的调节作用。一旦形成，SGS就成为细胞信号中心，可以拦截来自 调节细胞过程的其他途径。例如，SGS可以抑制哺乳动物的类似物 IMD途径，肿瘤坏死因子受体(TNFR)网络，通过隔离信号蛋白离开 来自免疫复合体。当eIF2α(真核细胞翻译起始因子2α)被 被压力敏感蛋白激酶，如PERK(蛋白激酶R样内质网状激酶)所磷酸化。 在这个R21应用中，我们报告了PERK-eIF2α信号促进吞噬细胞菌的定植和 在扁虱细胞中复制。这一观察与已知的eIF2α诱导的SGS的免疫抑制作用 促使我们在扁虱中寻找SG队形。我们发现了13个在唾液中诱导的SG组装基因 被吞噬细胞感染的扁虱的腺体和/或中肠。因此，我们的中心假设是津贴- EIF2α诱导的SGS拮抗硬蜱体内的免疫信号，促进病原体的定植。 这项提案的目标1将使用免疫荧光法评估细菌感染是否会诱导扁虱细胞中的SGS。 显微镜、蛋白质生物化学和药理抑制。目标2将评估SGS对以下方面的影响 用转录敲除和药理学方法研究细菌在体外的定植和壁虱免疫 调制器。目标3将调查SG机械是否影响病原体的定植和维持 在幼虫体内使用转录敲除，然后是吞噬弧菌或伯氏杆菌的挑战。 由于SG的形成是一种保守的进化机制，因此研究这种现象与 节肢动物免疫和媒介能力将为更广泛的媒介生物界提供新的见解。

Abstract Arthropod immunity is a key factor influencing vector competence. We recently identified an atypical Immune Deficiency (IMD) pathway in Ixodes scapularis ticks that responds to and restricts Borrelia burgdorferi (Lyme disease) and Anaplasma phagocytophilum (Human Granulocytic Anaplasmosis). Host cell stress-responses are closely intertwined with immunity and can function to either potentiate or antagonize immune signaling. Whether stress-response pathways intersect with arthropod immunity to influence vector competence remains unknown. Stress Granules (SGs) are aggregates of RNA and protein that transiently form in the cytoplasm of stressed cells, such as during infection. SG formation is highly conserved throughout eukaryotes and has known immune regulatory roles in mammals. Once formed, SGs become cell signaling hubs that can intercept molecules from other pathways to modulate cellular processes. For instance, SGs can inhibit the mammalian analogue to the IMD pathway, the Tumor Necrosis Factor Receptor (TNFR) network, by sequestering signaling proteins away from immune complexes. SGs are induced when eIF2α (eukaryotic translation initiation factor 2α) is phosphorylated by stress-sensing kinases, such as PERK (protein kinase R-like endoplasmic reticulum kinase). In this R21 application, we report that PERK-eIF2α signaling promotes A. phagocytophilum colonization and replication in tick cells. This observation together with known immune inhibitory roles of eIF2α-induced SGs prompted us to look for SG formation in ticks. We found 13 SG-assembly genes that are induced in the salivary glands and/or midguts of A. phagocytophilum-infected ticks. Accordingly, our central hypothesis is that PERK- eIF2α-induced SGs antagonize immune signaling in ticks, which promotes bacterial pathogen colonization. AIM 1 of this proposal will evaluate whether bacterial infection induces SGs in tick cells using immunofluorescent microscopy, protein biochemistry and pharmacological inhibition. AIM 2 will assess the impact of SGs on bacterial colonization and tick immunity in vitro using transcriptional knockdown and pharmacological modulators. AIM 3 will investigate whether SG machinery influences pathogen colonization and maintenance in vivo by using transcriptional knockdown in larvae followed by A. phagocytophilum or B. burgdorferi challenge. As SG formation is a conserved evolutionary mechanism, investigating this phenomenon as it relates to arthropod immunity and vector competence will provide novel insights for the broader vector biology community.