INVESTIGATION OF THE EFFECTOR ROLE OF IPAD FROM THE TYPE III SECRETION SYSTEM OF SHIGELLA FLEXNERI

Abstract

Shigellosis is an infectious gastrointestinal disease caused by Shigella spp. Approximately 165 million cases of shigellosis occur every year around the world, the vast majority of them in developing countries. High levels of antibiotic resistance, an increase in multidrug-resistant Shigella isolates and the lack of a licensed vaccine are factors that situate shigellosis as a public health problem, especially among young children. Shigella is able to cause death of resident macrophages in the gut to avoid bacterial clearance early after infection. Shigella is then able to colonize the intestinal epithelium and induce inflammation, which ultimately gives rise to the symptoms of dysentery and bacterial shedding. The virulence of Shigella is intimately tied to its Type III Secretion System (T3SS) for which invasion plasmid antigen D (IpaD) is a structural element. Previous studies have established that IpaD is secreted at levels beyond what is needed for its role as the T3SS needle tip protein. Furthermore, IpaD was recently shown to induce apoptosis in B lymphocytes in conjunction with an additional unknown factor. The projects presented in this dissertation aim to identify the role of IpaD as a secreted effector in the pathogenesis of Shigella. We have studied the effect of IpaD in macrophages and epithelial cells through a multidisciplinary approach using cell biology, immunology and protein biochemistry. Our findings indicate that IpaD plays a role in the development of an apoptotic pathway in macrophages. In vitro, macrophages incubated with recombinant IpaD undergo activation of caspases, damage to mitochondria and decreased cellular integrity. Furthermore, Shigella infection of cultured macrophages showed that IpaD is responsible of a portion of the cell death caused by the bacterium. These findings allow us to conclude IpaD is responsible for a portion of the macrophage cell death during Shigella infection.