Inhibition of Salmonella intracelullar replication by interferon-stimulated genes

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.