Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence

Listeria monocytogenes is a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches. L. monocytogenes maximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD+ while also generating a proton motive force (PMF). The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respiration. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence of L. monocytogenes independent of their role in respiration. Furthermore, we found that restoration of NAD+/NADH ratio through expression of water-forming NADH oxidase (NOX) could rescue phenotypes associated with DHNA deficiency. Here we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation in L. monocytogenes. Further, exogenous supplementation of DHNA rescues the in vitro growth and ex vivo virulence of L. monocytogenes DHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance in L. monocytogenes specifically through the recently annotated NADH dehydrogenase Ndh2, independent of the extracellular electron transport (EET) pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability by L. monocytogenes to drive energy metabolism in the absence of respiration-favorable conditions.