Characterization of Gut Bacterial Phospholipase Involved in Disease Associated Metabolism

Abstract

The human gut is one of the most densely populated microbial habitats on earth, collectively known as the human gut microbiota. Although some small molecules produced by this population contribute to the overall health of the host, some gut bacterial metabolites are linked to human diseases. Bacterial production of the small molecule trimethylamine (TMA) from choline has been correlated to several human disease states including non-alcoholic fatty liver disease, diabetes, colon cancer, and atherosclerosis. This thesis describes the study of alternate sources of TMA generation and the discovery that phosphatidylcholine (PC), a large component of the dietary choline pool, is metabolized into TMA through a choline intermediate via the activity of a phospholipase D (PLD) family enzyme. This activity represents a previously unrecognized role for this enzyme family in the provision of a carbon source for bacterial survival. Chapter 2 describes our efforts to characterize the ability of human commensal bacterial isolates to release free choline from various dietary and human-derived choline-containing metabolites. We identify organisms capable of this metabolism and analyze their genomes for putative responsible genes. Metabolism of only two of the molecules, PC and glycerophosphocholine (GPC) was observed. Interestingly, hydrolysis of these substrates was identified both in organisms that are capable of generating TMA from choline and those that lack the necessary genes for TMA generation. Overall, the work covered in Chapter 2 expands the known molecules that may contribute to elevated TMA concentrations. Chapter 3 details efforts to characterize the PLD in Escherichia coli MS 200-1 that is responsible the generation of choline from PC. In vivo characterization of this enzyme reveals that it is essential for the conversion of PC into choline and thereby the utilization of PC as a sole carbon source. We were able to heterologously express and purify PLD for biochemical assays. We demonstrate that this enzyme preferentially hydrolyzes PC and experimentally verify a key residue that may influence substrate specificity. Finally, Chapter 4 examines the impact of bacterial PC metabolism in the context of human disease. We identify bacterial PLDs in human stool metagenomes and observe an increase in mucosal barrier degradation in mice colonized with bacteria encoding a functional PLD. Additionally, we identify an inhibitor of E. coli PLD, 5-aminosalicylic acid. This compound is currently used in treating inflammatory bowel disease, and inhibition of bacterial PLD activity may be one mechanism by which it exerts its anti-inflammatory effects. Continued investigation is necessary to fully support this hypothesis, including mouse colonization and human studies.