Defining the Mechanism of Intoxication of the Clostridioides difficile Transferase

About This Grant

PROJECT SUMMARY The leading cause of hospital-acquired, infectious diarrhea in the United States is a gastrointestinal pathogen known as Clostridioides difficile. C. difficile can be found in the guts of many healthy individuals but typically only becomes problematic after disruption of the gut microbiota and is thus often associated with the administration of broad-spectrum antibiotics. The pathology that accompanies infection varies from one person to the next, ranging from relatively mild symptoms like diarrhea to severe, and potentially life-threatening conditions, known as pseudomembranous colitis and toxic megacolon. All these symptoms are the product of three toxins secreted by the bacterium during infection known as Toxin A, Toxin B, and the C. difficile Transferase (CDT). While Toxins A and B have historically been considered the drivers of the disease that accompanies infection, recent studies have suggested a potential synergy among all three toxins in the most severe cases. These observations have underscored the need to develop a better understanding of how CDT contributes to disease. The goal of this proposal is to expand our understanding of the intoxication mechanism governing CDT activity by addressing two key questions: 1) how is toxin assembly regulated and triggered? and 2) what is the mechanism underlying CDT delivery into host cells? We will use a combination of cryogenic electron microscopy (Cryo-EM), biochemical/biophysical assays, and cellular biology to describe these two points in molecular detail thereby generating a framework that can be used to deduce the precise role of CDT during infection. We also anticipate our analysis will uncover new structures and activities that will be exploited in future studies aimed at developing CDT inhibitors. As a third goal, we aim to leverage the toolkit we have assembled to probe the activity of two extrachromosomal CDT variants. Our plan is to use our full array of biochemical/biophysical assays, cellular biology, and Cryo-EM to generate a detailed description of the activity of these variants to better understand their potential to cause disease. Our work will thereby inform future studies that seek to understand how variation among toxins leads to different clinical outcomes. Together, this proposal will develop a thorough understanding of the function of CDT, an often overlooked, yet problematic, virulence factor associated with a notorious pathogen.