ABSTRACTMannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme inStaphylococcus aureusmannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH fromS. aureusUSA300, a strain of community-associated methicillin-resistantS. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target.In vitrocell viability assessment of ΔmtlDknockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor ofS. aureusM1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions ofSaM1PDH at the atomic level, the crystal structure ofSaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to theSaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we proposeSaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors ofSaM1PDH based on structure-function studies.IMPORTANCEDue to the shortage of effective antibiotics against drug-resistantStaphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase ofS. aureusUSA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of usingSaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with aSaM1PDH inhibitor or knockout of the gene encodingSaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism ofSaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses ofSaM1PDH. Our results strongly support the concept that targeting ofSaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.
2P025 Structural insight into the heme-transfer mechanism between Isd proteins in Staphylococcus aureus(01B. Protein: Structure & Function,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))