Inhibition of Salmonella intracelullar replication by interferon-stimulated genes

肠炎沙门氏菌血清型鼠伤寒沙门氏菌是世界范围内食源性和水源性肠胃炎的主要原因(1)。感染通常是自限性的，但可能对婴儿和免疫功能低下者构成重大威胁，非伤寒沙门氏菌每年在全球造成约15.5万人死亡（2,3）。一旦被消耗，它被M细胞和树突状细胞吞噬，并可以侵入包括上皮细胞在内的非吞噬细胞（4,5）。突破上皮屏障后，它在上皮细胞和巨噬细胞内繁殖，细菌可以从基底外侧侵入其他细胞(6)。沙门氏菌入侵非吞噬细胞的能力需要沙门氏菌致病性岛1 （Spi1）上编码的3型分泌系统（T3SS）。效应物合作促进膜结合大酶体的完成，也称为含沙门氏菌液泡（SCV）（6-8）。在几个小时内，营养物质的消耗和pH的降低导致另一种T3SS Spi2的表达上调，这赋予了鼠伤寒沙门氏菌在SCV中生存的能力（9-13）。已知这两种t3ss的效应蛋白都会干扰宿主信号传导，Odendall实验室最近的研究表明，由另一种肠道病原体志贺氏菌编码的t3ss效应蛋白的表达会阻断IFN信号传导。革兰氏阴性沙门氏菌的宿主识别是通过模式识别受体（PRRs）进行的。PRRs的结扎激活了主要的促炎反应，并促进了病原体的清除。干扰素（ifn）是一类典型的抗病毒细胞因子（14）。它们由三个家族组成，通过各自的受体以自分泌和旁分泌的方式发出信号，激活JAK/STAT信号级联，表达部分重叠的数百个基因，统称为干扰素刺激基因（ISGs）。isg包括JAK、STAT1/2和PRRs，它们的上调使细胞敏感，从而增强进一步的抗菌反应。isg干扰病毒生命周期的所有阶段，并已被证明影响细菌感染。然而，ifn对细菌感染的影响是复杂的，有时ifn对细菌有益（15-18）。由于它们作为一个群体被激活，个体isg的作用尚不清楚，特别是在细菌感染期间。已经进行了大规模筛选，以调查380多个isg在一组病毒感染中的作用（19-21），但这种规模的筛选尚未对细菌感染进行。在轮换项目中，IFN-处理导致入侵显著减少，初步数据表明细胞内复制也被抑制，然而生物重复序列之间的高水平差异意味着这需要进一步研究。用IFN预处理似乎是限制细胞内复制和入侵所必需的，因为仅在感染时处理的样品在这两方面都没有显着减少。这可能表明IFN-的作用是由isg介导的，而isg需要时间才能表达到适当的水平。收集到的数据为拟议的博士项目提供了几个有趣的研究方向。这个项目旨在描述干扰素和肠道沙门氏菌感染之间的相互作用。研究将进行如下：1。研究Spi1和Spi2效应蛋白是否影响上皮细胞中IFN信号通路。描述ifn -b介导的抑制侵袭和细胞内复制的具体步骤。鉴定影响鼠伤寒沙门氏菌上皮细胞感染的特异性isg。

Salmonella enterica serovar Typhimurium is a leading cause of food- and water-borne gastroenteritis worldwide(1). Infections are usually self-limiting but can pose a significant threat to infants and the immunocompromised, with non-typhoidal Salmonella causing approximately 155,000 deaths globally each year(2, 3). Once consumed it is phagocytosed by M cells and dendritic cells and can invade non-phagocytic cells including epithelial cells(4, 5). After breaching the epithelial barrier it multiplies within epithelial cells and macrophages, and bacteria can invade other cells from the basolateral side(6). The ability to invade non-phagocytic cells requires the type three secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (Spi1). Effectors cooperate to promote the completion of a membrane-bound macropinosome, also known as the Salmonella containing vacuole (SCV)(6-8). Within a few hours, nutrient depletion and a decrease in pH lead to the upregulation of expression of another T3SS, Spi2, which confers the ability of S. Typhimurium to survive within the SCV(9-13). Effector proteins of both T3SSs are known to interfere with host signalling and recent work by the Odendall lab has shown that expression of effectors of T3SSs encoded by another enteric pathogen, Shigella, blocks IFN signalling. Host recognition of Gram-negative Salmonella occurs via pattern recognition receptors (PRRs). Ligation of PRRs activates a primarily pro-inflammatory response and promotes clearance of the pathogen. Interferons (IFNs) are a group of archetypically antiviral cytokines(14). They consist of three families and signal in an autocrine and paracrine manner through their respective receptors, which activate JAK/STAT signalling cascades to express a partly-overlapping set of hundreds of genes, known collectively as interferon-stimulated genes (ISGs). ISGs include JAK, STAT1/2, and PRRs, whose upregulation sensitises cells to enhance further antimicrobial responses. ISGs interfere with all stages of the viral life cycle and have been demonstrated to influence bacterial infections. However the effect of IFNs on bacterial infections is complex, with IFNs sometimes being beneficial to the bacteria(15-18). As they are activated as a group, the roles of individual ISGs are not well understood, particularly during bacterial infections. Large-scale screens have been performed to investigate the role of over 380 ISGs in a panel of viral infections(19-21), but screens of this scale have not been carried out for bacterial infections.During the rotation project, IFN- treatment resulted in a significant decrease in invasion, and initial data suggests that intracellular replication was also inhibited, however a high level of variation between biological repeats means this will require further investigation. The pre-treatment with IFN appeared to be required for the restriction of intracellular replication and invasion as samples that were treated only at the time of infection did not exhibit a significant reduction in either. This may indicate that the effects of IFN- are mediated by ISGs, which require time to be expressed at appropriate levels. The data collected have provided several interesting lines of enquiry for the proposed PhD project.This project aims to characterise the interaction between interferons and Salmonella enterica infections. The research will be carried out as follows:1. Investigate whether Spi1 and Spi2 effector proteins affect IFN signalling pathways in epithelial cells.2. Delineate the specific steps of IFN-b-mediated inhibition of invasion and intracellular replication.3. Identify specific ISGs that affect S. Typhimurium infections of epithelial cells.