Abstract
The iron-regulated surface proteins IsdA, IsdB, and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC, and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-containing donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared with that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: 1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC, and apo-IsdE; 2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; 3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and 4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter.
Highlights
Which is a cofactor of hemoglobin and has been shown to be preferred as an iron source in vitro for some bacterial pathogens such as Gram-positive Staphylococcus aureus and Streptococcus pyogenes [1,2,3]
The heme acquisition machinery in S. aureus consists of the iron-regulated surface determinants (Isd), including the surface proteins IsdA, IsdB, and IsdC and the ATP-binding cassette (ABC) transporter IsdDEF [7]
Heme Acquisition in S. aureus apo-IsdB, but not to the other apo-Isd proteins; 2) holo-IsdB can directly transfer its hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; and 3) holo-IsdC does directly transfer its hemin to apo-IsdE, allowing transport into the bacterial cytoplasm
Summary
Gene Cloning—The isdB and isdE genes were cloned from S. aureus MW2 with paired primers 5Ј-TACCATGGAAGCAGCAGCTGAAGAAACA-3Ј/5Ј-TGGATCCTTAAGTTTGTGGTAATGATTTTGC-3Ј and 5Ј-TACCATGGGTCAATCTTCCAGTTCTCAA-3Ј/5Ј-AGGATCCGCAGTTGCGATTAAAATGACT-3Ј, respectively. Preparation of Apo- and Holo-Isd Proteins—Purified IsdB containing apo- and holo-IsdB in 10 mM Tris-HCl, pH 8.0, was loaded onto a Q Sepharose column (1 ϫ 4 cm). The column was extensively washed with 20 mM Tris-HCl to remove free hemin and eluted with 100 mM NaCl to recover holo-IsdB. The IsdB sample obtained contained 95% holo-form according to the measurements of protein and hemin contents. In the separation of Hb from IsdB, IsdA, or IsdE in the reaction mixture, the column was washed with 20 mM Tris-HCl, pH 8, to recover unbound Hb and eluted with 0.2 M NaCl in Tris-HCl to release bound protein(s). The rates of slower hemin transfer from metHb to apoIsdA, apo-IsdC, or apo-IsdE and from holo-IsdA, holo-IsdB, or holo-IsdC to apo-IsdE were measured by monitoring the absorbance changes using a conventional spectrophotometer (SPECTRAmax 384 Plus; Molecular Devices). The time courses of the absorbance changes obtained from these reactions were fitted to a single or double phase exponential expression using GraphPad Prism software to obtain the apparent rate constants for the transfer reactions
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