Listeria monocytogenes MenI Encodes a DHNA-CoA Thioesterase Necessary for Menaquinone Biosynthesis, Cytosolic Survival, and Virulence.

is a Gram-positive, intracellular pathogen that is highly adapted to invade and replicate in the cytosol of eukaryotic cells. Intermediate metabolites in the menaquinone biosynthesis pathway are essential for the cytosolic survival and virulence of , independent of the production of menaquinone (MK) and aerobic respiration. Determining which specific intermediate metabolite(s) are essential for cytosolic survival and virulence has been hindered by the lack of an identified 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) thioesterase essential for converting DHNA-CoA to DHNA in the MK synthesis pathway. Using the recently identified DHNA-CoA thioesterase as a query, homology sequence analysis revealed a single homolog in , Genetic deletion of resulted in an ablated membrane potential, indicative of a nonfunctional electron transport chain (ETC) and an inability to aerobically respire. Biochemical kinetic analysis of LMRG_02730 revealed strong activity toward DHNA-CoA, similar to its homolog, further demonstrating that LMRG_02730 is a DHNA-CoA thioesterase. Functional analyses , , and using mutants directly downstream and upstream of revealed that DHNA-CoA is sufficient to facilitate growth in minimal medium, intracellular replication, and plaque formation in fibroblasts. In contrast, protection against bacteriolysis in the cytosol of macrophages and tissue-specific virulence requires the production of 1,4-dihydroxy-2-naphthoate (DHNA). Taken together, these data implicate LMRG_02730 (renamed MenI) as a DHNA-CoA thioesterase and suggest that while DHNA, or an unknown downstream product of DHNA, protects the bacteria from killing in the macrophage cytosol, DHNA-CoA is necessary for intracellular bacterial replication.