Staphylococcus aureus HrtA is an ATPase required for protection against heme toxicity and prevention of a transcriptional heme stress response

Abstract

During systemic infection, Staphylococcus aureus acquires nutrient iron from heme, the cofactor of vertebrate myoglobin and hemoglobin. Upon exposure to heme, S. aureus up-regulates the expression of the heme-regulated transporter, HrtAB. Strains lacking hrtAB exhibit increased sensitivity to heme toxicity, and upon heme exposure they elaborate a secreted protein response that interferes with the recruitment of neutrophils to the site of infection. Taken together, these results have led to the suggestion that hrtAB encodes an efflux system responsible for relieving the toxic effects of accumulated heme. Here we extend these observations by demonstrating that HrtA is the ATPase component of the HrtAB transport system. We show that HrtA is an Mn(2+)/Mg(2+)-dependent ATPase that functions at an optimal pH of 7.5 and exhibits in vitro temperature dependence uncommon to ABC transporter ATPases. Furthermore, we identify conserved residues within HrtA that are required for in vitro ATPase activity and are essential for the functionality of HrtA in vivo. Finally, we show that heme induces an alteration in the gene expression pattern of S. aureus Delta hrtA, implying the presence of a novel transcriptional regulatory mechanism responsible for the previously described immunomodulatory characteristics of hrtA mutants exposed to heme.

FIG. 1.

HrtA purification and ATPase activity. (A) Coomassie blue-stained SDS-polyacrylamide gel showing purification of S. aureus His₆-HrtA expressed in E. coli. Lanes: M, molecular weight ladder; 1, E. coli before IPTG induction; 2, E. coli after IPTG induction; 3, total cell lysate; 4, insoluble pellet obtained after French press treatment and centrifugation; 5, soluble extract used for purification; 6 to 9, fractions obtained by elution of material bound to Ni-nitrilotriacetic acid with imidazole (10 mM, 50 mM, 100 mM, and 500 mM). (B) SDS-PAGE showing removal of the hexahistidine tag from His₆-HrtA by thrombin protease. Lanes: M, molecular weight ladder; 1, 1 μg purified His₆-HrtA; 2, 1 μg HrtA with hexahistidine tag removed by thrombin cleavage. (C) Time course analysis of ATP hydrolysis by HrtA. HrtA or heat-inactivated HrtA [HrtA (h.i.)] was incubated at 20°C in the presence of ATP, and release of inorganic phosphate was measured at the indicated time points. Error bars indicate standard deviations.

FIG. 2.

ATPase activity of HrtA is influenced by various physicochemical conditions. (A) Concentration of ATP required for saturation of enzymatic activity. HrtA (0.5 μM) was incubated with increasing concentrations of ATP at 20°C, and ATPase activity was measured. (B) ATPase activity determination at different temperatures. (C) Effect of temperature pretreatment on HrtA activity. HrtA was incubated at the indicated temperature for 20 min, and ATPase activity was determined at 20°C. (D) Influence of pH on HrtA ATPase activity. (E) Effect of divalent metal cations on HrtA activity. HrtA ATPase activity was determined with the indicated concentration of MnCl₂, MgCl₂, or CaCl₂. For all experiments, the average of triplicate ATPase activities is indicated; error bars represent one standard deviation from the mean.

FIG. 3.

Residues essential for HrtA ATPase activity. (A) Representation of a prototypical ABC transporter ATPase subunit and sequence alignment of conserved domains. Walker A, ABC signature, and Walker B motifs can be detected within HrtA orthologues and other characterized bacterial ABC transporter ATPases. Conserved residues selected for mutagenesis (Lys 45, Gly 145, and Glu 167 of S. aureus HrtA) are shown in black. (B) Coomassie blue-stained SDS-polyacrylamide gel showing recombinant purified wild-type HrtA (WT) and HrtA mutants with mutations in residues within motifs predicted to be involved in nucleotide binding or hydrolysis (G145A, G145T, K45A, E167Q) or a residue outside of such motifs (R76A). (C) Relative ATPase activities of HrtA mutants compared to the wild type. ATPase activities of recombinant HrtA and HrtA site mutants described for panel B were determined and are expressed as the percentage of ATPase activity exhibited by wild-type HrtA. The average percentage of triplicate ATPase activities is indicated; error bars represent one standard deviation from the mean.

FIG. 4.

HrtA residues essential for adaptation of S. aureus to heme toxicity. (A) Symbols for plasmids used to transform strains for panels B and C. Plasmids include an empty vector (black line), a plasmid encoding wild-type hrtA-myc (gray line with no symbol), or plasmids encoding hrtA-myc mutants (gray lines with symbols). (B) S. aureus ΔhrtA harboring the plasmids described in panel A was grown overnight in medium containing 2 μM heme and were subcultured into medium containing 10 μM heme. Bacterial growth was tracked by measuring the A₆₀₀ of triplicate cultures. Data points represent averages of triplicate measurements; error bars represent one standard deviation from the mean. (C) Wild-type S. aureus harboring the plasmids shown in panel A were grown and analyzed as for panel B. (D) Immunoblot (I.B.) against cytoplasmic extracts of S. aureus ΔhrtA expressing C-terminally c-Myc-tagged wild-type HrtA-myc or HrtA-myc mutants. Results are representative of triplicate independent experiments.