Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus

Abstract

In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J., Joachmiak, A., Missiakas, D. M., and Schneewind, O. (2003) Science 299, 906-909. The heme-binding NEAT domains of Isd proteins IsdA, IsdB (domain 2), IsdC, and HarA/IsdH (domain 3), and the heme-binding IsdE protein, were overexpressed and purified in apo (heme-free) form. Absorption and magnetic circular dichroism spectral data, together with electrospray ionization mass spectrometry were used to unambiguously identify that heme transfers from NEAT-A through NEAT-C to IsdE. Heme transfer was demonstrated to occur in a unidirectional fashion in the sequence NEAT-B2 --> NEAT-A --> NEAT-C --> IsdE or, alternatively, initiating from NEAT-H3 instead of NEAT-B2: NEAT-H3 --> NEAT-A --> NEAT-C --> IsdE. Under the conditions of our experiments, only NEAT-H3 and NEAT-B2 could transfer bidirectionally, which is in the reverse direction as well, and only with each other. Whereas apo-IsdE readily accepted heme from holo-NEAT-C, it would not accept heme from holo-NEAT-A. Heme transfer to IsdE requires the presence of holo-NEAT-C, in agreement with the proposal that IsdC serves as the central conduit of the heme transfer pathway. These experimental findings corroborate the heme transfer model first proposed by the Schneewind group. Our data show that heme transport from the wall-anchored IsdH/IsdB proteins proceeds directly to IsdE at the membrane and, for this to occur, we propose that specific protein-protein interactions must take place.

FIGURE 1.

ESI-MS charge state spectra for four Isd NEAT domains and IsdE studied as the heme-free, apo-NEAT-A, -NEAT-C, -NEAT-B2, -NEAT-H3, and -IsdE species (A, C, E, G, I) and the heme-bound, holo-NEAT-A, -NEAT-C, -NEAT-B2, -NEAT-H3, and -IsdE species (B, D, F, H, J). The charge states provide information about the change in conformation associated with heme binding. The close similarities in charge state distribution between the apo-and holo-pairs of data show that the proteins do not significantly change in folding following heme binding. Each charge state arises from the same protein mass, as shown in Fig. 2. The peak marked with * between +6 and +5 in G arises from an unknown identified species that is not seen in the holo-NEAT-H3 (H); the lack of corresponding mass in the deconvoluted spectra in Fig. 2G suggests a low molecular mass species.

FIGURE 2.

Deconvoluted ESI-mass spectra of the heme-free Isd apo-NEAT-A, -NEAT-C, -NEAT-B2, -NEAT-H3, and -IsdE species (A, C, E, G, I) and Isd heme-bound-NEAT-A, -NEAT-C, -NEAT-B2, -NEAT-H3, and -IsdE species (B, D, F, H, J). The data show that only one heme binds to each of these species. The mass difference between the pairs of data for the apo- and holo-species is ∼616 Da, the mass of a single heme.

FIGURE 3.

Absorption and MCD spectra recorded for heme transfer from Isd holo-NEAT-A to Isd apo-NEAT-C and for a mixed solution of Isd holo-NEAT-A and apo-IsdE. A, changes in the spectra when apo-NEAT-C is added to holo-NEAT-A in steps of 0.3 mol eq. Lines are shown for additions to: 0, 0.3, 0.6, 0.9, and 1.2 mol eq. apo-IsdE. The spectra have been corrected for dilution effects. In the absorption spectrum, the only significant change is the increase in absorbance at 390 nm and the slight blue shift from 405 to 402 nm; as marked by arrows. There is more change in the MCD spectrum because the MCD spectrum of the holo-NEAT-C product is slightly blue-shifted when compared with the holo-NEAT-A reactant, and also exhibits a more prominent negative signal at 390 nm, a more positive signal at 408, and a less negative signal at 424 nm than holo-NEAT-C resulting in a significant change in the intensities of the bands at these wavelengths as the reaction proceeds. B, absorption and MCD spectra recorded when heme-free, apo-IsdE was added to a solution of heme-containing, Isd holo-NEAT-A. Three sets of spectra are shown: with a total of 0, 0.6, and 1.2 mol. eq. IsdE added. Whereas there is little change in the absorption spectrum, addition of apo-IsdE results in intensification of the Soret MCD band envelope centered on 416 nm, a sign of increased low spin contribution in the ferric heme. There is no indication of the distinctly different absorption and MCD spectra of heme-containing, holo-IsdE. The changes are a result of interactions between the two proteins. Mass spectral data in Fig. 7 also clearly show that no heme transfer took place and that the solution after mixing contains only holo-NEAT-A and apo-IsdE.

FIGURE 4.

Changes in the absorption and magnetic circular dichroism spectra when heme-free, apo-IsdE is added to a solution of heme-containing, Isd holo-NEAT-A, and apo-NEAT-C (the solution used in Fig. 3A). Isosbestic change in spectral properties indicates the formation of heme-containing, holo-IsdE that was transferred from the holo-NEAT-A, to the apo-NEAT-C, then to the apo-IsdE. Spectra are shown for solutions with a total of: 0, 0.3, 0.6, 0.9, 1.2, and 2.1 mol eq apo-IsdE added. The lines show the direction of the spectral changes. The final absorption and MCD spectra closely resemble those of heme-loaded-IsdE (holo-IsdE).

FIGURE 5.

Mass spectral data for solution of mixed proteins showing heme transfer from the Isd holo-NEAT domains (A, B2, and H3) to apo-NEAT-C. A, charge state and deconvoluted mass spectra measured for a mixture of heme-containing, Isd holo-NEAT-A, and apo-NEAT-C. Excess heme-free, apo-NEAT-C was added. Three species coexist in solution and are identified in the charge state spectra with the abbreviations for apo-NEAT-C (a-C; with a mass of 14,442), apo-NEAT-A (a-A; with a mass of 14,626), and holo-NEAT-C (h-C; with a mass of 15,059). B, charge state and deconvoluted mass spectra measured for a mixture of heme-containing, Isd holo-NEAT-B2, and apo-NEAT-C. Excess heme-free, apo-NEAT-C was added. Three species coexist in solution and are identified in the charge state spectra with the abbreviations for apo-NEAT-B2 (a-B2; with a mass of 14,345), apo-NEAT-C (a-C; with a mass of 14,444), and holo-NEAT-C (h-C; with a mass of 15,060). C, charge state and deconvoluted mass spectra measured for a mixture of heme-containing, Isd holo-NEAT-H3 and apo-NEAT-C. Excess heme-free, apo-NEAT-C was added. Three species coexist in solution and are identified in the charge state spectra with the abbreviations for apo-NEAT-H3 (a-H3; with a mass of 14,640), apo-NEAT-C (a-C; with a mass of 14,444), and holo-NEAT-C (h-C; with a mass of 15,060). In each case, heme transfer to Isd NEAT-C was observed, resulting in a mixture of the heme-free-Isd-NEAT domain of NEAT-A, NEAT-B2, NEAT-H3) and heme-free apo-NEAT-C and heme-bound-holo-NEAT-C. No heme-bound-holo-NEAT-A (at 15,245 Da), NEAT-B2 (at 14,960 Da), or NEAT-H3 (at 15,256 Da) was observed in their respective mass spectra following mixing with apo-NEAT-C. The difference in relative %-abundances of the proteins depends on the relative efficiencies of the charged proteins reaching the detector in competition with the other ions. Closely similar ratios of protein were used in each of the spectral data shown here. In each case, excess (∼1.5×) of the heme-acceptor protein was used, so that there was also apo-heme-free protein remaining.

FIGURE 6.

Heme transfer from holo-NEAT-C to apo-IsdE. A, charge state and deconvoluted mass spectra of the solution made by mixing holo-NEAT-C and apo-IsdE. Excess apo-IsdE was added. B, absorption spectra of holo-NEAT-C before (indicated by arrows) and following the addition of apo-IsdE. The significant change in the Soret band maxima following heme transfer to the apo-IsdE (indicated by arrows from the legend'holo-IsdE + apo-NEAT-C') means that for this heme transfer experiment, absorption spectra can be used to monitor the progress of the reaction. The absorption spectrum of holo-IsdE is quite different from that of either apo-NEAT-C or holo-NEAT-C.

FIGURE 7.

Demonstration that no heme transfer takes place from holo-NEAT-A to apo-IsdE. A, ESI-MS spectra (charge states and deconvolution) for a mixture of holo-NEAT-A and apo-IsdE showing the presence of only holo-NEAT-A and apo-IsdE; there is no indication of the presence of either apo-NEAT-A or holo-IsdE showing that no heme transfer took place. B, the absorption spectra recorded before and after addition of the apo-IsdE are characteristic of holo-NEAT-A for both solutions. There is no indication of formation of holo-IsdE, which has a significantly different absorption spectrum.

FIGURE 8.

Heme transfer from holo-NEAT-B2 and holo-NEAT-H3 to apo-IsdE. Charge state and deconvoluted mass spectra and absorption spectra measured for mixtures of A holo-NEAT-B2 with apo-IsdE as a heme acceptor and B holo-NEAT-H3 with apo-IsdE as a heme acceptor. In A, the data show that holo-NEAT-B2 transfers heme to apo-IsdE. In each mass spectrum are peaks that correspond to both the holo-and apo-proteins: peaks that correspond to apo-IsdE (+11 to +13) at 30,218 Da, peaks that correspond to holo-IsdE (+11 to +13) at 30,832 Da and peaks that correspond to apo-NEAT-B2 (+7 to +8) at 15,272 Da. The absorption spectra represent the holo-NEAT-B2 before the addition of IsdE (indicated by the arrow from the legend holo-NEAT-B2) and following mixing with IsdE for 25 min (indicated by the arrows from the legend after mixing with apo-IsdE). The holo-NEAT-B2 was formed by binding heme to heme-free, apo-NEAT-B2 (construct 2), which was made using a different cloning strategy than that used to make NEAT-B2 which was used for the data in Figs. 1, 2, and 5. Residues in the His tag region (non-Isd) differ between the two constructs (see “Experimental Procedures”). The experimental results were unchanged by the use of the second construct. In B, the data show that holo-NEAT-H3 transfers heme to apo-IsdE. In each mass spectrum are peaks that correspond to both the holo-and apo-proteins: peaks that correspond to apo-IsdE (+11 to +13) at 30,250 Da, peaks that correspond to holo-IsdE (+11 to +13) at 30,865 Da and peaks that correspond to apo-NEAT-H3 (+7 to +8) at 14,156 Da. The absorption spectra of the holo-NEAT-H3 before the addition of IsdE (indicated by arrows from the legend holo-NEAT-H3) and following mixing with apo-IsdE for 25 min (indicated by arrows from the legend after mixing with apo-IsdE). The final spectrum corresponds to that of heme-containing, holo-IsdE. The apo-NEAT-H3 (construct 2) was made using a different cloning procedure than that used for the NEAT-H3 which was used to generate the data shown in Figs. 1, 2, and 5. Residues in the His tag region (non-Isd) were altered between the two constructs (see “Experimental Procedures”). The experimental results were unchanged by the use of the second construct.

FIGURE 9.

Heme transfer from holo-NEAT-B2 to apo-NEAT-A, and holo-NEAT-H3 to apo-NEAT-A and NEAT-B2. Charge state and deconvoluted mass spectra, and absorption spectra measured for mixtures of (A) holo-NEAT-B2 with apo-NEAT-A as a heme acceptor, (B) holo-NEAT-H3 with apo-NEAT-A as a heme acceptor, and (C) holo-NEAT-H3 with apo-NEAT-B2 as heme acceptor. In A, the data show that holo-NEAT-B2 transfers heme to apo-NEAT-A. In each mass spectrum are peaks that correspond to both the holo-and apo-proteins: peaks in the mass spectra that correspond to apo-NEAT-A (a-A: +7 and +8) at 14,630 Da, peaks that correspond to holo-NEAT-A (h-A: +7 and +8) at 15,245 Da and peaks that correspond to apo-NEAT-B2 (a-B2: +7 to +8) at 14,347 Da. In B, the data show that holo-NEAT-B2 transfers heme to apo-NEAT-A. In each mass spectrum are peaks that correspond to both the holo-and apo-proteins: peaks that correspond to apo-NEAT-A (a-A: +7 and +8) at 14,630 Da, peaks that correspond to holo-NEAT-A (h-A: +7 and +8) at 15,245 Da and peaks that correspond to apo-NEAT-H3 (a-H3: +7 to +8) at 14,640 Da. In C, the data show that holo-NEAT-H3 transfers heme to apo-NEAT-B2. In each mass spectrum are peaks that correspond to both the holo- and apo-proteins: peaks that correspond to apo-NEAT-B2 (a-B2: +7 and +8) at 14,346 Da, peaks that correspond to holo-NEAT-B2 (h-B2: +7 and +8) at 14,962 Da, and peaks that correspond to holo-NEAT-H3 (h-H3: +7) at 15,256 Da, and to apo-NEAT-H3 (a-H3: +7 to +8) at 14,640 Da.

FIGURE 10.

A heme transport model based on analysis of the magnetic circular dichroism and mass spectral data obtained from this study. Overall, heme transfer is from membrane distal IsdH/HarA (studied using NEAT domain H3) and IsdB (studied using the NEAT domain B2) proteins through to the membrane-proximal IsdE. There is unidirectional heme transfer from NEAT-A to NEAT-C to IsdE. There is no transfer from NEAT-A to IsdE. The mass spectral data to support the model are in agreement with the model first proposed by Schneewind and co-workers (10). The absorption and MCD data of the equilibrated solutions fully support the conclusions reached from analysis of the mass spectral data.