Activation of multiple stress responses in Staphylococcus aureus substantially lowers the minimal inhibitory concentration when combining two novel antibiotic drug candidates

Abstract

The past few decades have been plagued by an increasing number of infections caused by antibiotic resistant bacteria. To mitigate the rise in untreatable infections, we need new antibiotics with novel targets and drug combinations that reduce resistance development. The novel β-clamp targeting antimicrobial peptide BTP-001 was recently shown to have a strong additive effect in combination with the halogenated pyrrolopyrimidine JK-274. In this study, the molecular basis for this effect was examined by a comprehensive proteomic and metabolomic study of the individual and combined effects on Staphylococcus aureus. We found that JK-274 reduced activation of several TCA cycle enzymes, likely via increasing the cellular nitric oxide stress, and BTP-001 induced oxidative stress in addition to inhibiting replication, translation, and DNA repair processes. Analysis indicated that several proteins linked to stress were only activated in the combination and not in the single treatments. These results suggest that the strong additive effect is due to the activation of multiple stress responses that can only be triggered by the combined effect of the individual mechanisms. Importantly, the combination dose required to eradicate S. aureus was well tolerated and did not affect cell viability of immortalized human keratinocyte cells, suggesting a species-specific response. Our findings demonstrate the potential of JK-274 and BTP-001 as antibiotic drug candidates and warrant further studies.

SOS genes are rapidly induced while translesion synthesis polymerase activity is temporally regulated

“…The commercial kinase inhibitor Purvalanol B used in the MIB assay has been shown to also bind to proteins that are not kinases but possess an ATP/GTP-binding motif ( Bantscheff et al, 2007 ; Petrovic et al, 2017 ). We also expect the two in-house compounds to bind to most proteins with an ATP/GTP binding motif and proteins in complex with these, based on their ability to pull down around 1,000 proteins each ( Singleton et al, 2023 ). The extracted proteins thus include kinases as well as other ATP/GTP-binding proteins and represent an enrichment of signaling proteins.…”

“…The MIB assay with on-column trypsinization was used to isolate ATP/GTP-binding proteins from 100 μL cell extract containing 1 mg/mL protein ( Petrovic et al, 2017 ). The kinase inhibitor mix was previously optimized to pull down a larger portion of the bacterial signalome, and consisted of an equal mix of Purvalanol B (Tocris, United Kingdom), L-1 and L-3 (in-house) ( Singleton et al, 2023 ). Samples were stored at −20°C until analysis.…”

“…Unlike looking at the whole proteome, signalomics can capture changes in protein activation and deactivation that occur much faster than translation by binding to exposed ATP/GTP-binding motifs. Signalomics was performed by selective enrichment for activated proteins and protein complexes using the recently established multiplexed kinase inhibitor bead (MIB) assay for prokaryotes ( Singleton et al, 2023 ). Finally, metabolomics revealed how DNA damage caused by ciprofloxacin changes the metabolite pools and suggests which pathways have increased or decreased flux.…”