Inhibition of Salmonella intracelullar replication by interferon-stimulated genes

鼠伤寒沙门氏菌是全世界食源性和水源性胃肠炎的主要原因(1)。感染通常是自限性的，但可能对婴儿和免疫系统造成重大威胁，非伤寒沙门氏菌每年在全球造成约155,000人死亡(2，3)。一旦被摄入，它会被M细胞和树突状细胞吞噬，并能侵袭非吞噬细胞，包括上皮细胞(4，5)。突破上皮屏障后，细菌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)。侵袭非吞噬细胞的能力需要沙门氏菌致病岛1(Spi1)编码的三型分泌系统(T3SS)。效应器相互协作以促进膜结合的大针体的完成，也被称为含有空泡的沙门氏菌(SCV)(6-8)。在几个小时内，营养耗竭和pH的下降导致另一种T3SS，Spi2的表达上调，这赋予了鼠伤寒沙门氏菌在SCV内生存的能力(9-13)。已知这两种T3SS的效应器蛋白都会干扰宿主信号传递，奥登达尔实验室最近的工作表明，另一种肠道病原体志贺氏菌编码的T3SS效应器的表达会阻止干扰素信号的传递。革兰氏阴性沙门氏菌的宿主识别是通过模式识别受体(PRRs)实现的。PRRs的结扎激活了主要的促炎反应，促进了病原体的清除。干扰素(IFN)是一组典型的抗病毒细胞因子。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，表达一组部分重叠的数百个基因，统称为干扰素刺激基因(ISGs)。ISGs包括JAK、STAT1/2和PRRs，它们的上调使细胞变得敏感，从而增强进一步的抗菌反应。ISGS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，干扰素对细菌感染的影响是复杂的，有时对细菌是有益的(15-18)。由于它们作为一个群体被激活，单个ISG的作用还没有被很好地理解，特别是在细菌感染期间。已经进行了大规模的筛查，以调查超过380个ISG在一组病毒感染中的作用(19-21)，但还没有对细菌感染进行这种规模的筛查。在轮换项目中，干扰素治疗导致侵袭性显著降低，初步数据表明细胞内复制也受到抑制，然而生物重复之间的高水平变异意味着这将需要进一步的研究。干扰素的前处理似乎是限制细胞内复制和侵袭所必需的，因为仅在感染时处理的样本两者都没有显示出明显的减少。这可能表明干扰素的作用是由ISGs介导的，而ISGs需要时间才能在适当的水平上表达。收集的数据为拟议的PHD项目提供了几条有趣的线索。该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。主要研究内容如下：1.研究SPI1和SPI2效应蛋白是否影响上皮细胞中的干扰素信号转导通路。描绘了干扰素-b介导的抑制侵袭和细胞内复制的具体步骤。确定影响鼠伤寒沙门氏菌感染上皮细胞的特定ISGs。

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.