Molecular Basis of Rhodomyrtone Resistance in Staphylococcus aureus

Rhodomyrtone (Rom) is a plant-derived broad-spectrum antibiotic active against many Gram-positive pathogens. A single point mutation in the regulatory farR gene (farR*) confers resistance to Rom in Staphylococcus aureus (RomR). The mutation in farR* alters the activity of the regulator, FarR*, in such a way that not only its own gene, farR*, but also the divergently transcribed farE gene and genes controlled by the global regulator, agr, are highly upregulated. Here, we show that mainly the upregulation of the fatty acid efflux pump FarE causes the RomR phenotype, as farE deletion in either the parent or the RomR strain (RomR ΔfarE) yielded hypersensitivity to Rom. Comparative lipidome analysis of the supernatant (exolipidomics) and the pellet fraction revealed that the RomR strain excreted about 10 times more phospholipids (PGs) than the parent strain or the ΔfarE mutants. Since the PG content in the supernatant (2,244 ng/optical density [OD]) was more than 100-fold higher than that of fatty acids (FA), we assumed that PG interacts with Rom, thereby abrogating its antimicrobial activity. Indeed, by static and dynamic light scattering (SLS and DLS) and isothermal titration calorimetry (ITC) analyses, we could demonstrate that both PG and Rom were vesicular and reacted with each other in milliseconds to form a 1:1.49 [Rom-PG(32:0), where PG(32:0) is PG with C32:0 lipids] complex. The binding is entropically driven and hence hydrophobic and of low specificity in nature. Our results indicate that the cytoplasmic membrane is the actual target of Rom, which is also in agreement with Rom’s induced rapid collapse of the membrane potential and decreased membrane integrity.