Causal Determination of Microbiome Induced Phenotypes

About This Grant

Project Summary The human gut microbiome is a complex network of microorganisms living inside the gastrointestinal tract. There have been many connections made between the gut microbiome and many aspects of human health, including allergies, infections, and autoimmune diseases; most relevant to this proposal is the connection between the gut microbiome and enteric infection with the pathogen Salmonella enterica serovars typhimurium (hereafter Salmonella). First, I have established a new mouse model to causally examine the role of the gut microbiome in modulating various phenotypes, such as Salmonella infection. In this model, germ-free mice are first colonized with microbiomes from healthy human subjects, transferred via frozen fecal samples. These colonized mice are then allowed to mate and produce offspring, which receive the human microbiome from their parents. Importantly, the new generations grow up in the presence of a microbiome, which allows their immune system to develop properly. Throughout this process, the mice will be kept in gnotobiotic conditions to prevent contamination from environmental bacteria. Through this approach, I have established four ‘surrogate colonies’ of mice, with each colony colonized with a unique donor microbiome. I subjected age-matched male and female mice from each surrogate colony to Salmonella infection and used Specific Pathogen Free (SPF) mice as controls. I found that mice from one colony had significantly increased susceptibility to infection, quantified by greater mortality using log-rank analysis. This project will identify the mechanism that underlies this difference in survival. In aim 1, I will investigate microbiome-induced immune changes that led to increased susceptibility. I will do this by using immunophenotyping to identify immune changes and then using confirmatory experiments to demonstrate that the putative mechanisms are responsible. Based on preliminary data, I predict that I will find causal innate immune differences. Next, I will repeat the original infection experiment with germ-free mice colonized after weaning. This will allow me to identify deficits in immune maturation and function due to no microbiome present early in life. In Aim 2, I will use ex-vivo culture methods to characterize differences in colonization resistance between healthy human microbiomes. I will then use sub-culture techniques to isolate either a strain or smaller consortium capable of recapitulating the original colonization resistance. From here, I will characterize the mechanism of colonization resistance. I predict that this will be either through a bactericidal compound, which I will characterize, or through nutrient competition, which I will confirm by demonstrating that addition nutrients restore Salmonella growth. As a whole, this project will establish a new model for characterizing the causal impact of the microbiome on clinically relevant phenotypes, with Salmonella infection being a strong initial example. Through this project, as well as the curriculum and excellent training environment of the Robert Wood Johnson Medical School – Princeton University MD/PhD program, I will gain the skills necessary to allow me to match into a research-track residency, and eventually become a practicing physician-scientist.