The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans. In low- income countries, poor sanitation is responsible for approximately 270 million cases of shigellosis annually, with at least 250,000 of these resulting in death. In high-income countries, shigellosis is typically associated with travel to high-risk regions (Latin America, Asia and Africa). The disease is characterized by severe bloody diarrhea and dramatic destruction of the colonic mucosa. S. flexneri is transmitted via the fecal-oral route and is extremely contagious, with a rate of attack above 90% with an infectious dose as low as 100-1000 bacteria per individual, as determined in human volunteer studies. S. flexneri is an intracellular pathogen that invades epithelial cells in the human colon. The bacteria trigger their own entry by injecting bacterial effector proteins through their type 3 secretion system (T3SS), leading to bacterial uptake into primary cellular vacuoles. The pathogen promptly escapes from primary vacuoles to gain access to the cytosolic compartment. In the cytosol, bacteria recruit components of the actin cytoskeleton and actin polymerization at the bacterial pole propel the pathogen throughout the cell. As motile bacteria reach cell-cell contacts, they form membrane protrusions that project into adjacent cells. The resolution of the formed protrusions leads to formation of double-membrane vacuoles (DMVs) in adjacent cells, from which the pathogens escape, thereby achieving cell-to-cell spread. The importance of this dissemination process in S. flexneri pathogenesis has been initially established through studies conducted in non-human primates or in human volunteers and recently demonstrated by our group in an infant rabbit model of bacillary dysentery. Although actin-based motility is now fairly well understood, the mechanisms supporting cell-to-cell spread are unclear. Our group has previously shown the importance of the T3SS in bacterial dissemination. Moreover, we have recently identified the T3SS effector protein IcsB as a critical determinant of DMV escape during cell-to-cell spread. Here we propose to explore how IcsB contribute to DMV escape in epithelial cells (Aim1) and how IcsB-mediated DMV escape contributes to pathogenesis in vivo (Aim2).
Most intracellular pathogens reside in a membrane-bound compartment after the initial invasion step. A few pathogens however, have evolved the ability to gain access to the host cell cytosol, where they manipulate the actin cytoskeleton to display actin-based motility and disseminate to adjacent cells. In this proposal, we present our plans to explore the mechanisms supporting the dissemination of the intestinal pathogen Shigella flexneri. The proposed approach will contribute to our general understanding of the mechanisms underlying microbial pathogenesis and may constitute the foundation for the rational design of preventive and therapeutic interventions against diarrheal diseases.
