ABSTRACT
The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Biphenyl Compounds/pharmacology , Depsipeptides/pharmacology , Glycopeptides/pharmacology , Glycosides/pharmacology , Lipopeptides/pharmacology , Membrane Proteins/antagonists & inhibitors , Methicillin-Resistant Staphylococcus aureus/drug effects , Oligopeptides/pharmacology , Staphylococcal Infections/drug therapy , beta-Lactams/pharmacology , Animals , Anti-Bacterial Agents/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Biphenyl Compounds/chemical synthesis , Depsipeptides/isolation & purification , Drug Synergism , Drug Therapy, Combination , Female , Glycopeptides/chemical synthesis , Glycopeptides/isolation & purification , Glycosides/isolation & purification , Humans , Lipopeptides/isolation & purification , Membrane Proteins/genetics , Membrane Proteins/metabolism , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/growth & development , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Multigene Family , Oligopeptides/chemical synthesis , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Staphylococcal Infections/microbiology , beta-Lactam Resistance/drug effects , beta-Lactam Resistance/genetics , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to ß-lactam antibiotics paired with ß-lactamase inhibitors. We have adapted a Staphylococcus aureus antisense knockdown strategy to genetically identify the cell division Z ring components-FtsA, FtsZ, and FtsW-as ß-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA). We demonstrate that the FtsZ-specific inhibitor PC190723 acts synergistically with ß-lactam antibiotics in vitro and in vivo and that this combination is efficacious in a murine model of MRSA infection. Fluorescence microscopy localization studies reveal that synergy between these agents is likely to be elicited by the concomitant delocalization of their cognate drug targets (FtsZ and PBP2) in MRSA treated with PC190723. A 2.0 Å crystal structure of S. aureus FtsZ in complex with PC190723 identifies the compound binding site, which corresponds to the predominant location of mutations conferring resistance to PC190723 (PC190723(R)). Although structural studies suggested that these drug resistance mutations may be difficult to combat through chemical modification of PC190723, combining PC190723 with the ß-lactam antibiotic imipenem markedly reduced the spontaneous frequency of PC190723(R) mutants. Multiple MRSA PC190723(R) FtsZ mutants also displayed attenuated virulence and restored susceptibility to ß-lactam antibiotics in vitro and in a mouse model of imipenem efficacy. Collectively, these data support a target-based approach to rationally develop synergistic combination agents that mitigate drug resistance and effectively treat MRSA infections.
