Proteomics and Transcriptomics Uncover Key Processes for Elasnin Tolerance in Methicillin-Resistant Staphylococcus aureus.

Elasnin is a new antibiofilm compound that was recently reported to have excellent activity against methicillin-resistant Staphylococcus aureus (MRSA) biofilms. In this study, we established that elasnin also has antibacterial activity against growing S. aureus planktonic cells. To explore elasnin's potential as an antibiotic, we applied adaptive laboratory evolution (ALE) and produced evolved strains with elevated elasnin tolerance. Interestingly, they were more sensitive toward daptomycin and lysostaphin. Whole-genome sequencing revealed that all of the evolved strains possessed a single point mutation in a putative phosphate transport regulator. Subsequently, they exhibited increased intracellular phosphate (P i ) and polyphosphate levels. Inhibition of the phosphate transport regulator gene changed the phenotype of the wild type to one resembling those observed in the evolved strains. Proteomics and transcriptomics analyses showed that elasnin treatment resulted in the downregulation of many proteins related to cell division and cell wall synthesis, which is important for the survival of growing exponential-phase cells. Other downregulated processes and factors were fatty acid metabolism, glycolysis, the two-component system, RNA degradation, and ribosomal proteins. Most importantly, transport proteins and proteins involved in oxidative phosphorylation and the phosphotransferase system were more upregulated in the evolved strain than in the ancestral strain, indicating that they are important for elasnin tolerance. Overall, this study showed that elasnin has antibacterial activity against growing S. aureus cells and revealed the altered processes due to elasnin treatment and those associated with its tolerance. IMPORTANCE Besides the excellent antibiofilm properties of elasnin, we discovered that it can also kill growing methicillin-resistant Staphylococcus aureus (MRSA) planktonic cells. We subjected MRSA cells to an in vitro evolution experiment, and the resulting evolved strains exhibited increased elasnin tolerance, reduced growth rate, loss of pigmentation, and an increased proportion of small-colony formation, and they became more sensitive toward daptomycin and lysostaphin. Through multiomics analysis, we uncovered the affected processes in growing S. aureus planktonic cells following elasnin treatment, including the downregulation of cell wall synthesis, cell division, and some genes/proteins for the two-component system. These findings suggest that elasnin suppressed processes important for the cells' survival and adaptation to environmental stresses, making it an ideal drug adjuvant candidate. Overall, our study provides new insights into the mechanism of elasnin in S. aureus planktonic cells and pointed out the potential application of elasnin in clinics.