guanosine-diphosphate and Staphylococcal-Infections

Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to β-lactam antibiotics paired with β-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 β-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA). We demonstrate that the FtsZ-specific inhibitor PC190723 acts synergistically with β-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 β-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 β-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.

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; beta-Lactams; Cell Division; Crystallography, X-Ray; Cytoskeletal Proteins; Disease Models, Animal; Drug Resistance, Bacterial; Drug Synergism; Gene Regulatory Networks; Guanosine Diphosphate; Imipenem; Methicillin-Resistant Staphylococcus aureus; Mice; Microbial Sensitivity Tests; Mutation; Protein Structure, Secondary; Protein Transport; Pyridines; Staphylococcal Infections; Thiazoles; Virulence

Neutrophils are first responders rapidly mobilized to inflammatory sites by a tightly regulated, nonredundant hierarchy of chemoattractants. These chemoattractants engage neutrophil cell surface receptors triggering heterotrimeric G-protein Gα(i) subunits to exchange GDP for GTP. By limiting the duration that Gα(i) subunits remain GTP bound, RGS proteins modulate chemoattractant receptor signaling. Here, we show that neutrophils with a genomic knock in of a mutation that disables regulator of G-protein signaling (RGS)-Gα(i2) interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a nonlethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gα(i) activation, helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent.

Topics: Animals; Bone Marrow; Chemotaxis; Down-Regulation; Enzyme Activation; Gene Knock-In Techniques; GTP-Binding Protein alpha Subunit, Gi2; Guanosine Diphosphate; Guanosine Triphosphate; Inflammation; Mice; Mice, Transgenic; Mutation; Neutrophils; Receptors, Interleukin-8B; RGS Proteins; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Time Factors