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 Rhodomyrtus tomentosa. 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 Staphylococcus aureus 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 farR [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 farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the Rom^R clone. Particularly the upregulation of agr and sarA 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 farE. 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 farE is deleted in the Rom^R clone, or, if native farR 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 farE in the Rom^R clone, that FarR is an important regulator, and that the point mutation in farR (Rom^R clone) makes the clone hyper-virulent.

Keywords: Gram-positive bacteria; Staphylococcus; antibiotic; membrane active; rhodomyrtone.

FIGURE 1

Rom-resistance phenotype, genetic analysis and construction of deletion mutants and clones. (A) The Rom^R clone shows no inhibition halo in the agar diffusion method with 100 μg Rom loaded on the filter disks. (B) Genetic organization of the divers oriented farR and farE genes and location of the single point mutation (^∗) in farR leading to an amino acid exchange (Cys116/Arg) in the FarR regulator protein in the Rom^R clone. (C) Illustration of farE deletion construction in HG001 and the Rom^R clone. For homologous double-cross recombination, pBASE6-ΔfarE containing approximately 1 kb upstream and downstream DNA sequences were used; ΔfarE positive clones were controlled by PCR and sequencing. (D) pCX30::farR is the farR complementation plasmid in which the transcription of the gene is xylose-inducible.

FIGURE 2

Transcriptome analysis (RNA-seq) of genes differently expressed in Rom^R clone compared to HG001 wild type strain. (A) The Venn diagrams show numbers of genes that are at least fourfold up- or downregulated after 4 and 8 h of growth in BM in the Rom^R clone. (B) Percentage of genes being at least fourfold up (red bars) or downregulated (blue bars) in each of cluster orthologous groups (COG) based on functional categories. Designations of functional categories: C, energy production and conversion; D, cell division and chromosome partitioning; E, amino acid metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, coenzyme metabolism; I, lipid metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, DNA replication, recombination, and repair; M, cell wall structure and biogenesis, outer membrane; N, Cell motility and chemotaxis; O, post-translational modification, protein turnover, chaperone functions; P, Inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general functional prediction; T, signal transduction mechanisms; U, secretion; V, defense mechanisms.

FIGURE 3

Comparison of release of PSMα and the cytoplasmic proteins FbaA and GAPDH. (A) (Right) PSMα1 expression of HG001, Rom^R and ΔfarE (Rom^RΔfarE) were determined by Real-Time PCR. The strains were aerobically cultured in TSB medium for 16 h. The pellets were used for total RNA extraction. qPCR experiments were carried out to determinate psmα1 and gyrB was used as housekeeping gene. Fold changes were calculated using ΔΔCt method and relativized to the HG001 expression. Error bars indicate standard error. Statistical significances between mutant clones Rom^R, ΔfarE (Rom^R ΔfarE) with the wild type HG001 were analyzed by 1-way ANOVA: not significant P > 0.05, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. (Left) Release of PSMα1 into the supernatant of HG001, Rom^R clone and Rom^RΔfarE cultured in TSB for 16 h was determined by HPLC; the relative amount of PSMα was calculated by comparing the peak-area in the samples. All experiments were performed in triplicate. Error bars indicate standard error. Statistical significances between mutant clones Rom^R, ΔfarE (Rom^R ΔfarE) with the wild type HG001 were analyzed by Student t-test: not significant P > 0.05, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. (B) Comparative release of FbaA (aldolase) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) over time in HG001, Rom^R clone and Rom^RΔfarE clone. Only TSB was used as control.

FIGURE 4

Comparison of release of lipids and total FAs into the supernatant. (A) Release of lipids. The S. aureus clones (Rom^R clone, Rom^RΔfarE, and Rom^RΔfarR) were aerobically cultured in TSB medium for 8 and 16 h. The supernatants were filtrated and 100 μl of each sample were mixed with FM5-95 to a final concentration of 5 μg/ml. Fluorescence was measured with a Tecan microplate reader using excitation at 565 nm and emission at 660 nm. All experiments were performed in triplicate. The relative lipids in the supernatant were relativized to the HG001 value. Error bars indicate standard error. Statistical significances between mutant clones Rom^R, ΔfarE (Rom^R ΔfarE), farR [Rom^R (pCX-farR)] with the wild type HG001 were analyzed by 1-way ANOVA: not significant P > 0.05, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. (B) Comparison of FAMEs (C15 to C20). The FAs in the supernatants of 16 h cultures of HG001, Rom^R clone, ΔfarE (Rom^RΔfarE), and farR [Rom^R (pCX-farR)] were qualitatively and quantitatively analyzed by GC-MS. The FAs from C15 to C20 were assigned different colors. The total FAs for each clone are shown in the inserted graph. Experiments were performed independently in duplicate. Error bars indicate standard error. Statistical significances between mutant clones Rom^R, ΔfarE (Rom^R ΔfarE), farR [Rom^R (pCX-farR)] with the wild type HG001 were analyzed by 1-way ANOVA: not significant P > 0.05, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 5

Comparison of cytotoxicity and mouse pathogenicity. (A) Comparison of the cytotoxicity of HG001, Rom^R clone, and Rom^RΔfarE mutant in various host cells A495, HEK, and HaCaT. Experiments were performed in triplicate. Error bars indicate standard error. Statistical significances between mutant clones Rom^R, ΔfarE (Rom^R ΔfarE) with the wild type HG001 were analyzed by 1-way ANOVA: not significant P > 0.05, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001. (B,C) Comparison of HG001 and Rom^R clone in 48 h intranasal mouse infection model (pneumonia model). The Rom^R clone shows higher CFU values in the lungs after 48 h of infection (B), and a slightly stronger weight loss than the wild type HG001 (C). Significant differences (^∗P = 0.0313) in bacterial burden were noted between Rom^R and the wt HG001. Data were analyzed using Mann–Whitney one tailed test.

FIGURE 6

Model for FarE mediated resistance to Rom in the Rom^R clone. In the Rom^R clone farR is inactivated by the point mutation that leads to an amino acid exchange (Cys116/Arg) in the FarR regulator protein. FarR, acts not only as a repressor of its own farR gene but also represses farE. The mutation in farR indicated by (^∗) leads to inactivation of FarR and consequently to derepression of farE and many other genes (including farR, agr, sarA, hla, psm-α,β, hld, geh). Derepression of farE causes over-expression of FarE. We propose that FarE acts as an exporter of lipids that interact with Rom thus causing neutralization of Rom and leading to high Rom resistance.