Study of virulence in Shigella using a bar-coded collection of mutants

Shigella is a devastating bacterial pathogen that causes severe diarrheal disease, resulting in the deaths of a million people per year. Most of the victims are children under the age of 5 in the developing world. The disease caused by Shigella, known as shigellosis, is characterized by high fever and bloody diarrhea as a result of massive destruction of the colonic tissues. There is no animal model of shigellosis that accurately reflects the disease in humans since Shigella only infects humans and closely related primates. As a result, there are many gaps in our understanding of how Shigella causes disease. The human gut has a surveillance system, known as the innate immune system that senses the presence of bacteria. Shigella must overcome this first line of defense in order to cause disease and has provided several key insight to how the innate immune system functions. The ability of Shigella to cause disease (virulence) has received intense investigation at the molecular level. We now know many of the genes required for virulence, how these genes are turned on and off, their location and DNA sequence. However, functions for most of these genes remain unknown. One way to assign function to genes is to inactivate them and compare the activity of this mutant organism to that of the non-mutant parental strain. In recent years, there have been breakthroughs in technology that allow rapid and accurate construction of mutants in bacteria such as Shigella. As a result, a systematic approach to the study of virulence gene function is now feasible. In the proposed research, we will inactivate each virulence gene in Shigella creating a resource that will be useful to the research community. We will identify genes involved in the ability to enter human cells, those required for Shigella to infect neighboring cells, and analyze the effect of each mutant to alter the innate immune system. These studies will provide a baseline for the understanding of virulence in Shigella.