Mutation of the transcriptional regulator YtoI rescues Listeria monocytogenes mutants deficient in the essential shared metabolite 1,4-dihydroxy-2-naphthoate (DHNA)

Listeria monocytogenes, a Gram-positive, facultative intracellular pathogen, survives and replicates in the cytosol of host cells. Synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an intermediate of menaquinone biosynthesis, is essential for cytosolic survival of L. monocytogenes independent from its role in respiration. Here we demonstrate that DHNA is essential for virulence in a murine model of Listeriosis due to both respiration dependent and independent functions. In addition, DHNA can be both secreted and utilized as an extracellular shared metabolite to promote cytosolic survival inside of host macrophages. To understand the role(s) of DHNA in L. monocytogenes intracellular survival and virulence, we isolated DHNA-deficient (ΔmenD) suppressor mutants that formed plaques in monolayers of fibroblasts. Five ΔmenD suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent ΔmenD mutant. Whole genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator ytoI (lmo1576). Clean deletion and complementation in trans confirmed that loss of ytoI could restore plaquing and cytosolic survival of DHNA deficient L. monocytogenes. RNA-seq analysis revealed five genes (lmo0944, lmo1575, lmo1577, lmo2005, and lmo2006) expressed at a higher level in the ΔytoI compared to the wild-type strain, whereas 2 genes (lmo1917 and lmo2103) demonstrated lower expression in the ΔytoI mutant. Intriguingly, the majority of these genes are involved in controlling pyruvate flux. Metabolic analysis confirmed that acetoin, acetate and lactate flux were altered in a ΔytoI mutant, suggesting a critical role for regulating these metabolic programs. In conclusion, we have demonstrated that, similar to select other bacteria, DHNA can act as a shared resource and it is essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, we have identified a novel transcriptional regulator in L. monocytogenes and implicated its metabolic regulation in cytosolic survival of L. monocytogenes.