Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus

Abstract

Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of <i>Rhodomyrtus tomentosa</i>. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain <i>Staphylococcus aureus</i> HG001 (Rom^R) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of <i>farR</i> [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated <i>farR</i> affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (<i>farR</i>), its flanking gene <i>farE</i> (effector of FA resistance), and other global regulators such as <i>agr</i> and <i>sarA</i>. All these genes were consequently upregulated in the Rom^R clone. Particularly the upregulation of <i>agr</i> and <i>sarA</i> leads to increased expression of virulence genes rendering the Rom^R clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of <i>farE</i>. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the Rom^R clone compared to its parental strain HG001. If <i>farE</i> is deleted in the Rom^R clone, or, if native <i>farR</i> is expressed in the Rom^R strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of <i>farE</i> in the Rom^R clone, that FarR is an important regulator, and that the point mutation in <i>farR</i> (Rom^R clone) makes the clone hyper-virulent.