Mobile-Genetic-Element-Encoded Hypertolerance to Copper Protects Staphylococcus aureus from Killing by Host Phagocytes

Figures

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  FIG 1. Electrophoretic mobility shift assay analysis of recombinant CsoR variants binding to the putative promoters upstream of copA (PcopA), copB (PcopB), and mco (Pmco). (A) Schematic representation of the copA-copZ and copB-mco operons with putative promoter sequences. (B to D) Recombinant wild-type (WT) CsoRWT or the CsoRCHC mutant (CsoR C41A/H66A/C70A [CHC]) were purified and tested for binding to PCR products containing the DNA sequences (∼200 bp) upstream of the respective start codon, and a control DNA fragment of nonspecific DNA sequence (nsDNA). The concentrations of protein (in micromolar) are shown above the lanes. (B) Incubation of PcopA DNA with wild-type apo-CsoR, but not Cu(I)-CsoR, retards the migration of PcopA. (C) CsoR CHC retards migration of PcopA in both the presence and absence of Cu(I). (D) CsoR retards migration of PcopA and PcopB, but not migration of Pmco.

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  FIG 2. Enhanced growth in subinhibitory concentrations of copper chloride requires expression of copper tolerance genes. Growth of S. aureus 14-2533T and MRSA252 variants was measured in TSB supplemented with subinhibitory (4 mM) concentrations of copper chloride (A and C) or TSB broth alone (B and D). Growth curves representing data obtained from at least three independent experiments are presented. (E) Fold change in expression of copB and mco in S. aureus cultured in TSB versus TSB with copper chloride (4 mM). The ΔΔCT method was used to determine the relative expression levels of the copB and mco genes normalized to gyrB. Values are means plus standard deviations (SD) (error bars) from three independent experiments, with statistical significance determined by analysis of variance (ANOVA). Values that are statistically significantly different by ANOVA are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Values that are not significantly different are indicated by bars labeled ns.

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  FIG 3. Hypertolerance to copper increases resistance of S. aureus to macrophage killing. Mouse macrophage cell line (RAW264.7) was suspended in DMEM supplemented with mouse IFN-γ (40 ng/ml) and CuSO4 (40 µM) and seeded in the wells of 24-well plates at 2 × 106 cells per ml for 18 h at 37°C in 5% CO2. (A and B) S. aureus strain 14-2533T (A) or MRSA252 (B) and derivatives were grown overnight in RPMI 1640 and then inoculated into the wells at a multiplicity of infection (MOI) of 10 in DMEM allowing phagocytosis for 30 min followed by killing of extracellular bacteria with gentamicin/lysostaphin for 30 min. Macrophages were then lysed at this time point (time zero [T0]) and after 3 h of incubation (T3), and viable bacteria were counted to determine the levels of bacterial survival. The mutants expressing CsoR C41A/H66A/C70A (CHC) are indicated. Values are means plus SD from three independent experiments. Statistical significance is indicated as follows: **, P < 0.005; *, P < 0.05; ns, not significant.

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  FIG 4. Intracellular expression of copB and mco. (A and B) Fold change in expression of copB and mco by wild-type S. aureus relative to CsoR CHC mutants of either S. aureus 14-2533T(pSCBU) (A) or MRSA252 (B). RAW264.7 macrophages were activated with IFN-γ (50 µg/ml) and copper chloride (40 µM) for 18 h. Infections of the macrophage monolayer were performed with S. aureus grown in RPMI 1640 at an MOI of 20. Extracellular bacteria were killed by treatment with gentamicin/lysostaphin following by washing of the monolayers with phosphate-buffered saline (PBS). Three hours postinfection, RNA was isolated from infected macrophages and for RT-qPCR. The ΔΔCT method was used to determine the relative expression levels of the mco and copB genes in WT and CHC mutants normalized to gyrB. Values are means plus SD from three independent experiments. Statistical significance was determined by ANOVA and Bonferroni’s multiple-comparison posttest and indicated as follows: *, P < 0.5; **, P < 0.05; ***, P < 0.005; ns, not significant.

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  FIG 5. Increased survival of copper-hypertolerant S. aureus in human blood. S. aureus (ca. 1 × 104 CFU/ml) strains were inoculated into freshly drawn human blood (A and B) or plasma (C and D) and incubated for 3 h at 37˚C. Viable counts were used to determine the numbers of bacteria in blood or plasma. The number of CFU after 3 h is expressed as a percentage of the original input CFU at 0 h. Horizontal lines represent the means ± SD from at least three independent experiments. Statistical significance was determined by ANOVA following Dunnett’s multiple-comparison test (A) or by an unpaired t test (B) and indicated as follows: for panel A, **, P < 0.01; ***, P < 0.001; for panel B, **, P = 0.0052.

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  FIG 6. Maximum likelihood trees of CC22, CC30, and CC398 showing distribution of copA, copB, mco, and copL genes. Trees are rooted in the longest branch (CC22 to S. aureus 08 01492; CC398 to S. aureus SO1977; CC30 to S. aureus MRSA252). The four rings show the presence of copA, copB, mco, copL as blue, purple, yellow, and red, respectively.