Discovery and Characterization of Immunomodulatory Gut Microbiome-Derived Small Molecules

Abstract

The gut microbiome has long been associated with human physiology, but relatively little is known about the molecular mechanisms underlying the gut bacteria-host immune system connection. To deconvolute these two notoriously intricate systems, we can examine individual disease-associated species from the gut microbiome in relation to individual immune response pathways. This allows identification of specific molecules of known origin that have physiological relevance. In this thesis, I present three projects that apply activity-guided fractionation and chemistry to discover novel small molecules from the gut microbiome that may impact human health. In Chapter 1 I outline the foundation of host-microbe interactions and describe the challenges in interpreting the metabolomics data widely used for investigation of the gut microbiome. This chapter also details the history of activity-guided fractionation in natural product discovery and how this approach may benefit studies of gut microbial species. Then, I provide precedents for the use of activity-guided fractionation to identify and characterize immunomodulatory molecules from the gut microbiome. I conclude the chapter by examining the importance of demystifying interactions between gut bacteria and the host immune system in the context of disease. Chapter 2 describes my use of activity-guided fractionation on two microbial species of interest: Bifidobacterium breve and Fusobacterium nucleatum. After culturing both species anaerobically on a large scale under various growth conditions, I report that fractionation of Bifidobacterium breve produces a glycolipid that induces TNF- production, while Fusobacterium nucleatum produces a fraction that results in increased production of TNF-, a key inflammatory cytokine. Further investigation of the active fraction from Fusobacterium nucleatum reveals a previously unreported cardiolipin unique to Fusobacterium nucleatum that may contribute to the increased inflammation and colorectal cancer progression correlated with high levels of Fusobacterium nucleatum. This molecule from Fusobacterium nucleatum demonstrates the key role microbial lipids play in immune response. In Chapter 3, I describe testing of antibody specificity towards cardiolipins and related phospholipid compounds, as well as cardiolipin synthesis methods to thoroughly probe the relationship between cardiolipin structure and immune system activation. Testing of these cardiolipins in an anti-cardiolipin assay reveals that antibody recognition of cardiolipins is selective to the identity and position of the lipid chains. I report that some cardiolipins may be synthesized using the enzyme phospholipase D (PLD) and commercially available phosphatidylcholine. In Chapter 4, I describe the optimization and application of a cell-based screen to identify a novel small molecule that induces autophagy, which expands the scope of activity-guided fractionation to additional cellular processes. Through imaging of a GFP-tagged version of the LC3 protein that forms part of autophagosome membranes, induction of autophagy by challenge compounds can be visualized and quantified. Additionally, localization of transcription factor TFE3 to the nucleus during autophagy can be observed in response to challenge compounds through TFE3 antibodies. By optimizing and combining readouts of both of these known markers of autophagy, I develop a screen that can be used on microbial fractions to identify inducers or inhibitors of autophagy. After application of this screen to multiple species, I identify a single fraction that potently induces autophagy. We report ongoing work to characterize this small molecule as a thelephantin. This represents an expansion in the capabilities of activity-guided fractionation to reveal further mechanisms of host-microbe interaction. Together, the projects in this thesis demonstrate how activity-guided fractionation can be used to further understanding of gut microbe-derived molecules in the context of the host immune system. The findings reported show the importance of lipids in molecular signaling within the immune system.