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

Abstract

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 host macrophages. To understand the role(s) of DHNA in L. monocytogenes intracellular survival and virulence, we isolated DHNA-deficient (ΔmenD strain) 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 transcriptome analysis revealed five genes (lmo0944, lmo1575, lmo1577, lmo2005, and lmo2006) expressed at a higher level in the ΔytoI strain than in the wild-type strain, whereas two 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 findings in 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 determined that its metabolic regulation is implicated in cytosolic survival of L. monocytogenes.

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

“…(menI) is a putative DHNA-CoA thioesterase required for menaquinone biosynthesis Previous reports have highlighted the essentiality of MK biosynthetic intermediates in the survival and virulence of L. monocytogenes; however, the lack of a fully annotated pathway (Fig. 1A) has prevented the identification of the metabolite(s) responsible for the observed phenotypes 15,16 . Recently, the final unknown gene in the MK pathway, encoding a DHNA-CoA thioesterase, has been identified in the Gram-negative organism Escherichia coli, referred to as ydiI 18 .…”

“…as a shared resource in complex communities [22][23][24] . L. monocytogenes can both secrete and scavenge DHNA 16 . Consistent with previous reports, cell-free supernatants from aerobically grown cultures of wild-type or ΔmenA mutants in defined minimal media were able to rescue the growth of ΔmenB in minimal defined media (Fig.…”

“…The ability of L. monocytogenes to adapt to life within the cytosol is achieved through multiple mechanisms including, but not limited to, modulation of specific metabolic processes, tight regulation of virulence factors to maintain the integrity of its intracellular niche, and avoidance of immune detection and host defense mechanisms [12][13][14] . Menaquinone (MK) biosynthesis is an integral metabolic pathway and intermediate metabolite(s) in this pathway are required for L. monocytogenes cytosolic survival, independent of their known role in the production of MK 15,16 . MK acts as the sole lipid mediator of electron transport in L. monocytogenes and is required for a functional electron transport chain (ETC) 17 .…”

Listeria monocytogenesMenI encodes a DHNA-CoA thioesterase necessary for menaquinone biosynthesis, cytosolic survival, and virulence

“…intermediate metabolite(s) in this pathway are required for L. monocytogenes cytosolic survival, independent of their known role in the production of MK 15,16 . MK acts as the sole lipid mediator of electron transport in L. monocytogenes and is required for a functional electron transport chain (ETC) 17 .…”

“…Previous reports have highlighted the essentiality of MK biosynthetic intermediates in the survival and virulence of L. monocytogenes; however, the lack of a fully annotated pathway ( Fig. 1A) has prevented the identification of the metabolite(s) responsible for the observed phenotypes 15,16 . Recently, the final unknown gene in the MK pathway, encoding a DHNA-CoA thioesterase, has been identified in the Gram-negative organism Escherichia coli, referred to as ydiI 18 .…”