Role of Carbon Metabolism and Virulence of Shigella flexneri

PROJECT SUMMARY (See instructions): The long-term goal of the proposed research is to understand the adaptation of the invasive pathogen Shigella to the human gut environment. Shigella species are associated with bacillary dysentery, a potentially fatal diarrheal disease. With the rise in drug resistance and no effective vaccine available, the global health burden of shigellosis, already significant, is increasing. During the course of an infection, Shigella must transit the gastrointestinal tract, penetrate the mucous layer, and invade and replicate within intestinal epithelial cells. To adapt these diverse environments, Shigella senses the availability of carbon sources or products of carbon metabolism. Our hypothesis is that Shigella uses these signals to regulate the expression of genes, including virulence genes, to maximize survival and growth in a particular niche. A mechanistic understanding of these regulatory pathways is needed to design effective methods for treatment and prevention of Shigella infection. We have the tools and expertise to carry out these studies. Our work has demonstrated the importance of the alarmone (p)ppGpp, a signaling molecule produced in response to carbon starvation, for Shigella invasion and spread in cultured epithelial cells. We will move these studies into a model that more closely resembles the gut epithelium during natural infection. Human intestinal organoids, or "mini-intestines", which contain multiple differentiated cell types and display an innate immune response, will be used to study the effects of ppGpp on Shigella pathways leading to adaptation to the host environment and virulence gene expression. Using Dual RNA-Seq, we will determine both the Shigella and the host genes that are regulated in response to ppGpp during organoid infection, and this will give insights into the host-pathogen interplay and guide the construction of mutants to ascertain the relevance of both host and pathogen regulatory networks for the disease outcome. We recently discovered that the Type I interferon beta (IFN-Beta) innate immune signaling pathway plays a role in Shigella infection. While IFN-Beta is somewhat effective in preventing Shigella from entering host cells, the bacteria that do get in spread more rapidly and cause larger lesions. This indicates that Shigella has a mechanism(s) for not only overcoming IFN-Beta-induced protective responses but also hijacking the host innate immune response to increase its virulence. We propose to use dual RNA-Seq and genetic analysis of Shigella-infected intestinal cells to investigate how this pathogen subverts the host interferon response.