Loss of GdpP function in Staphylococcus aureus confers β-lactam-specific antibiotic tolerance and promotes invasive infection

Abstract

The emergence of antibiotic tolerance in Staphylococcus aureus reduces antibiotic efficacy by allowing bacterial survival despite prolonged antibiotic exposure, the molecular basis of which remains poorly understood. Moreover, the phenotypic indistinguishability of tolerant isolates in antimicrobial susceptibility testing impedes effective diagnosis and therapy. Increased concentration of the second-messenger, cyclic-di-AMP (CDA), has recently been implicated in tolerance to β-lactams as well as other cell-wall-reactive antibiotics. Using the ScanLag assay, Tolerance-Disk test, and traditional methodologies and employing isogenic mutagenized strains, we demonstrate that loss of GdpP function, a phosphodiesterase that hydrolyzes CDA, confers tolerance specifically to β-lactam antibiotics independent of their class. The extent of β-lactam tolerance correlated directly with the intracellular CDA concentration and inversely with the inhibition of bacterial cell-wall synthesis. ΔgdpP mutants caused higher mortality than wild-type strains in the Galleria mellonella infection model upon β-lactam treatment, suggesting GdpP-mediated tolerance could lead to β-lactam treatment failure. Large-scale within-host evolution analysis demonstrated that MRSA and MSSA strains isolated from patients acquire GdpP loss-of-function mutations during invasive infections but not during nasal carriage. Overall, this study highlights the clinical relevance of gdpP mutations, frequently selected in persistent S. aureus infections, as key mediators that could promote treatment failure due to β-lactam tolerance. Importance Although GdpP loss-of-function mutations have been associated with antibiotic tolerance in MRSA and MSSA, whether they directly mediate tolerance, the antibiotic specificity of this phenotype, and how they contribute to infections remain unresolved. Our study shows that an enhanced level of cyclic-di-AMP (CDA), which results from GdpP loss-of-function mutations, promotes tolerance specifically to the β-lactam class of antibiotics in Staphylococcus aureus. The mechanism by which such tolerance is conferred is independent of the classical β-lactam resistance genes, mecA and blaZ. This report also provides evidence that GdpP loss-of-function mutations may promote persistent invasive infections in patients and points to potential future steps to re-sensitize CDA-dependent β-lactam-tolerant S. aureus strains.