Casey Theriot

In the Theriot laboratory we apply cutting-edge technology and high-throughput methods to analyze the gut microbiome and metabolome using a range of experimental techniques and animal models. We leverage many approaches that span diverse fields including bacterial genetics, bacterial physiology, protein engineering, biochemistry, and apply a variety of omic approaches (microbiomics, transcriptomics, proteomics, and metabolomics) in vitro and in vivo to define the mechanisms behind how the gut microbiota provides colonization resistance against C. difficile.

We will test PID-X and PID-4-122 (using the same single dose ��� 10mpk as we did previously) in the relapse model using a dose range and dosing schedule that will be established in 2(c) above for PID-X. In addition to the requisite control groups, we will test three doses and different dosing schedules. We will increase the number of mice to 8 mice per group to better power this study and establish robust statistics for the effects. Histology of sectioned intestinal samples will be quantified in a blinded fashion for each treatment group.

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated colitis and is responsible for significant morbidity, mortality and increased healthcare costs. Antibiotics disrupt the indigenous gut microbiota, reducing resistance to C. difficile colonization. Our knowledge of the mechanism(s) by which the gut microbiota confers resistance to CDI is incomplete, presenting a significant roadblock to improving preventative and therapeutic approaches against this pathogen. My long-term goal is to understand how the gastrointestinal tract microbiota mediates colonization resistance against enteric pathogens, including C. difficile. The overall objective of this application is to define members of the gut microbiota that are able to alter bile acids and consume sugars which are required for C. difficile colonization and pathogenesis. Based on preliminary studies the central hypothesis is that the production and consumption of specific metabolites (secondary bile acids and sugars) by the indigenous gut microbiota contribute to colonization resistance against C. difficile. The rationale that underlies the proposed research is that the targeting of metabolites required for C. difficile colonization has the potential to improve directed therapeutic approaches for this infection. Guided by strong preliminary data, this hypothesis will be tested by exploring the following key questions: 1) Can restoring microbial-mediated secondary bile acid metabolism in the large intestine restore colonization resistance against C. difficile? and 2) Can restoring bacteria that are able to compete for the same nutrients (sugars) as C. difficile requires for growth reestablish colonization resistance against C. difficile? To answer the first key question, we will select for and characterize bacteria that are capable of secondary bile acid metabolism. Genetic engineering of bacterial strains for efficient enzyme delivery to the gastrointestinal tract will be evaluated in vitro and in vivo, in a mouse model of CDI. Under the second key question, we will screen and characterize bacteria that are able to compete for the same nutrients as C. difficile. Bacteria will be evaluated by competition assays in vitro and in vivo, in a mouse model of CDI. Both approaches will explore how these bacterial strains alter C. difficile colonization resistance in the gastrointestinal tract as well as how they alter the surrounding environment including the microbiome, metabolome and host response. The proposed research program in this application is innovative because it represents a departure from the status quo, namely in the approach of using a targeted bacterial therapy to restore secondary bile acids and competition, ultimately restoring colonization resistance against C. difficile. The proposed research is significant, because it will lead to the identification of bacteria and new-targeted approaches to be used for therapeutic interventions to prevent or treat CDI, and potentially other metabolic diseases.

Dr. Casey Theriot and members of her lab will be responsible for running mouse models of Clostridium difficile infection (CDI) with potential therapeutics against C. difficile, provided by Locus Biosciences. She will be responsible for running the relapse model of CDI with potential therapeutics and assessing disease based on weight loss, bacterial load, toxin activity and histopathological changes to the mouse large intestine. She will also help to interpret the results of the mouse models. It is expected that she and her team will meet once a month with Dr. David Ousterout of Locus Biosciences for a conference call to discuss research progress, benchmarks, and strategy. She will continue to provide additional data that are relevant for these approaches as they become available. She will work with the team to draft publications and to present data from this proposal at local and national meetings.

The microbiota-derived bile acid pool in the gastrointestinal (GI) tract is linked to many human diseases including obesity, diabetes, cancer, and GI disorders including Clostridium difficile infection. These diseases are associated with significant morbidity, mortality, and increased healthcare costs. Despite the importance of bile acids to host metabolism and colonization resistance against enteric pathogens, our knowledge of how the gut microbiota modulates the bile acid pool remains rudimentary, presenting a significant roadblock to improving preventative and therapeutic approaches against the above-described GI and metabolic diseases. The objective of this application is to demonstrate that specific members of the gut microbiota are capable of deconjugation and dehydroxylation of primary bile acids into secondary bile acids, which are important for host health and colonization resistance against C. difficile. Based on our preliminary data we hypothesize that members of the gut microbiota capable of deconjugation (Lactobacillus spp.) and dehydroxylation (non-pathogenic Clostridium spp.) of primary bile acids into secondary bile acids contribute to colonization resistance against C. difficile. The rationale that underlies the proposed research is by altering the bile acid pool with candidate microbes there is potential to improve targeted therapeutic approaches for C. difficile infection and other human diseases. Guided by strong preliminary data, this hypothesis will be tested by exploring the following specific aims: 1) To determine whether Lactobacillus and non-pathogenic Clostridium species from the gut microbiota are capable of deconjugation and dehydroxylation of primary bile acids into secondary bile acids; and 2)Demonstrate that modulation of the bile acid composition correlates with decreased disease susceptibility of C. difficile in mice. The approach is innovative, because it represents a departure from the status quo, namely in the approach of using a targeted bacterial therapy to restore the population of secondary bile acids, ultimately restoring colonization resistance against C. difficile. The proposed research is significant, because it will lead to the identification of specific bacterial strains used for therapeutic interventions to rationally alter the bile acid pool in the gut, modulating CDI, and potentially other metabolic diseases.

Aim 1. Identify metabolites in the gastrointestinal tract that contribute to C. difficile colonization and pathogenesis. Cecal and fecal contents of mice treated with broad-spectrum antibiotics and challenged with C. difficile will be analyzed by unbiased liquid chromatography-mass spectrometry to determine the identity and levels of metabolites throughout infection. Using novel mass spectrometry techniques and state of the art instruments we will define the gut metabolome associated with different disease states of C. difficile infection (CDI). Aim 2. Determine the physiological concentrations of gut metabolites that modulate C. difficile pathogenesis. Gastrointestinal metabolites that correlate with the C. difficile lifecycle: germination, outgrowth, and toxin production, will be investigated by using both targeted metabolomics (GC- and LC/MS), ex vivo and in vitro studies.