The major role of Listeria monocytogenes folic acid metabolism during infection is the generation of N-formylmethionine

ABSTRACT Folic acid and its derivatives (folates) play central roles in one-carbon metabolism, necessary for the synthesis of purines, pyrimidines, and some amino acids. Antifolate drugs are widely used as broad-spectrum antibiotics for bacterial infections, including listeriosis, a disease caused by the facultative intracellular pathogen Listeria monocytogenes. However, folate-derived metabolites required during bacterial infection are poorly understood. Here, we report that L. monocytogenes encodes two enzymes, methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (FolD) and formyltetrahydrofolate synthetase (Fhs), that catalyze the formation of N10-formyltetrahydrofolate, a critical intermediate in folate metabolism. N10-formyltetrahydrofolate is an essential carbon donor for biosynthesis of purines and N-formylmethionine, the amino acid used during initiation of bacteria translation. While L. monocytogenes mutants lacking Fhs had no observable defects and mutants lacking FolD had moderate virulence defects, mutants lacking both were highly attenuated in mice, especially in the liver. We compared the growth and virulence of mutants that were unable to synthesize folates with mutants unable to generate folate downstream products, including purines, thymidine, and N-formylmethionine. Mutants unable to synthesize N-formylmethionine behaved almost identically to FolD/Fhs double mutants during growth in broth or macrophages, but most importantly, were similarly attenuated to mutants unable to make folates, both of which had approximately 4-log10 less colony-forming units in the livers compared with wild-type L. monocytogenes. The purine auxotrophic mutants were only 1.5-log10 less virulent, and the thymidine mutants were fully virulent in the mouse infection model. These results strongly suggest that the main role of L. monocytogenes folates during infection is the generation of N-formylmethionine. Importance Folic acid is an essential vitamin for bacteria, plants, and animals. The lack of folic acid leads to various consequences such as a shortage of amino acids and nucleotides that are fundamental building blocks for life. Though antifolate drugs are widely used for antimicrobial treatments, the underlying mechanism of bacterial folate deficiency during infection is unclear. This study compares the requirements of different folic acid end-products during the infection of Listeria monocytogenes, a facultative intracellular pathogen of animals and humans. The results reveal the critical importance of N-formylmethionine, the amino acid used by bacteria to initiate protein synthesis. This work extends the current understanding of folic acid metabolism in pathogens and potentially provides new insights into antifolate drug development in the future.