Mutation of the Transcriptional Regulator YtoI Rescues Listeria monocytogenes Mutants Deficient in the Essential Shared Metabolite 1,4-Dihydroxy-2-Naphthoate (DHNA).

, 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 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 host macrophages. To understand the role(s) of DHNA in intracellular survival and virulence, we isolated DHNA-deficient (Δ strain) suppressor mutants that formed plaques in monolayers of fibroblasts. Five Δ suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent Δ mutant. Whole-genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator, (). Clean deletion and complementation in confirmed that loss of could restore plaquing and cytosolic survival of DHNA-deficient RNA-seq transcriptome analysis revealed five genes (, , , , and ) expressed at a higher level in the Δ strain than in the wild-type strain, whereas two genes ( and ) demonstrated lower expression in the Δ 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 Δ mutant, suggesting a critical role for regulating these metabolic programs. In conclusion, we have demonstrated that, similar to findings in select other bacteria, DHNA can act as a shared resource, and it is essential for cytosolic survival and virulence of Furthermore, we have identified a novel transcriptional regulator in and determined that its metabolic regulation is implicated in cytosolic survival of .