Abstract
ABSTRACTThe type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro. These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB. Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo.
Highlights
Discovery of novel antibiotics has become an important goal for biomedical research in both academia and industry, mainly driven by the growing problem of widespread antibiotic resistance [1]
We found that resistance to this inhibitor occurs with high frequency in vitro and is mediated by robust overexpression of a putative ABC transporter that results from loss-of-function mutations in cro/cI, a homolog of the lambda phage transcriptional repressor cro gene
This study reveals a novel bacterial resistance mechanism that led to the discovery of an alternative system for cleavage and secretion of signal peptide-containing proteins that counteracts the essentiality of SpsB in vitro but not in vivo
Summary
Discovery of novel antibiotics has become an important goal for biomedical research in both academia and industry, mainly driven by the growing problem of widespread antibiotic resistance [1]. We found that resistance to this inhibitor occurs with high frequency in vitro and is mediated by robust overexpression of a putative ABC transporter that results from loss-of-function mutations in cro/cI, a homolog of the lambda phage transcriptional repressor cro gene. The overexpression of this ABC transporter prevented bacterial lethality caused by disruption of the spsB gene. This was associated with secretion of a subset of proteins that are normally cleaved by SpsB and were cleaved at a site distinct from the canonical SpsB cleavage site. This study reveals a novel bacterial resistance mechanism that led to the discovery of an alternative system for cleavage and secretion of signal peptide-containing proteins that counteracts the essentiality of SpsB in vitro but not in vivo
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