Systems Analysis of the Host Response to Clostridium Difficile Toxins

Toxins A and B, two highly potent protein toxins, are the essential virulence factors of C. difficile, a bacterium that infects 300,000+ people in the US every year. Since the extent to which a person’s body overreacts to the toxins determines disease severity, controlling the host response is critical for improving treatments. Given the manifestations of diarrhea and colitis, most research to date has predictably focused on known inflammatory pathways and related cellular responses. However, 40 years after the toxins’ discovery, the number of fatalities has continued to rise. A different approach is needed. In this dissertation, I present a holistic approach, profiling the physiological and transcriptional changes of host cells to toxins in vitro and in vivo. I determine the most appropriate statistical methods for identifying genes and pathways affected by toxins, leading to discovery of an unrecognized cell-cycle disruption of epithelial cells treated with toxins. I then extend the approach to investigate epithelial-layer cells in intoxicated mice, identifying pathways altered only in vivo. These pathways offer new therapeutic targets, as is shown by antibody neutralization experiments in which the levels of two cytokines are predictive of survival. I again extend the systems approach to analyze the dynamics of toxin-induced morphological changes of many cell types in vitro. Sensitivities of macrophages, epithelial cells, and endothelial cells indicate that epithelial cells may not be the critical cell type for initiating disease and show that the most well-studied toxin molecular activity (glucosylation) is not required for all toxin effects. This dissertation has therefore introduced new ways of thinking about host responses to C. difficile toxins, revealing complexities that mediate disease and can be controlled or detected for improved treatments and diagnoses.