Demonstration of the Iron-regulated Surface Determinant (Isd) Heme Transfer Pathway in Staphylococcus aureus

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. 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. Staphylococcus aureus is widely known as an important human pathogen that causes a range of infections and diseases from minor, such as impetigo and abscesses, to life-threatening, such as pneumonia, meningitis, endocarditis, and septicemia (1Gordon R.J. Lowy F.D. Clin. Infect. Dis. 2008; 46: S350-S359Crossref PubMed Scopus (665) Google Scholar). The emergence of resistant bacteria has taken place over several decades and methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) are particularly clinically challenging, often accompanied by limited treatment options (2McGowan Jr., J.E. Tenover F.C. Nat. Rev. Microbiol. 2004; 2: 251-258Crossref PubMed Scopus (39) Google Scholar). Furthermore, MRSA, initially limited to hospitals, has since spread to the community (CA-MRSA). Adding to the complexity is our incomplete understanding of mechanisms of drug resistance. Iron is an essential nutrient for the majority of living organisms. Although the human body contains abundant iron, its low solubility in the ferric state of aerobic systems and the fact that the majority is intracellular means that very little soluble iron (estimated as ∼10–9 m) is directly available to bacteria. As such, bacteria have developed multiple iron acquisition systems to facilitate their survival in such low iron environments. The most abundant potential iron source for bacteria is heme, or iron-protoporphyrin IX, which is typically bound in proteins such as hemoglobin and myoglobin. Pathogenic bacteria have evolved specialized mechanisms for acquiring heme-iron from the host (for recent reviews, see Refs. 3Genco C.A. Dixon D.W. Mol. Microbiol. 2001; 39: 1-11Crossref PubMed Scopus (187) Google Scholar, 4Wandersman C. Stojiljkovic I. Curr. Opin. Microbiol. 2000; 3: 215-220Crossref PubMed Scopus (252) Google Scholar, 5Skaar E.P. Schneewind O. Microbes Infect. 2004; 6: 390-397Crossref PubMed Scopus (166) Google Scholar, 6Wandersman C. Delepelaire P. Annu. Rev. Microbiol. 2004; 58: 611-647Crossref PubMed Scopus (750) Google Scholar). The iron-regulated surface determinant (isd) 3The abbreviations used are: isd, iron-regulated surface determinant; MCD, magnetic CD; ESI-MS, electrospray ionization mass spectrometry; NEAT, NEAr iron Transporter; rIsdA, recombinant IsdA. gene cluster was first identified in 2002 (7Mazmanian S.K. Ton-That H. Su K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2293-2298Crossref PubMed Scopus (309) Google Scholar, 8Morrissey J.A. Cockayne A. Hammacott J. Bishop K. Denman-Johnson A. Hill P.J. Williams P. Infect. Immun. 2002; 70: 2399-2407Crossref PubMed Scopus (38) Google Scholar, 9Taylor J.M. Heinrichs D.E. Mol. Microbiol. 2002; 43: 1603-1614Crossref PubMed Scopus (103) Google Scholar), and then in 2003, Mazmanian et al. (10Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar) were the first to propose that the Isd series of proteins formulate a heme transfer pathway across the cell wall and through the membrane. In recent years, there has been a significant amount of research into defining the Isd-mediated heme acquisition system, especially in S. aureus. Briefly, the Isd system in S. aureus consists of nine iron-regulated proteins: IsdA, IsdB, IsdC, and IsdH/HarA, which are cell wall-anchored surface proteins, and IsdDEF, which constitute a membrane-localized transporter, and, finally, IsdG and IsdI, which encode heme-degrading enzymes in the cytoplasm (12Skaar E.P. Gaspar A.H. Schneewind O. J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). IsdA is highly expressed on the cell wall of iron-limited S. aureus and, in addition to several other reported functions (13Clarke S.R. Foster S.J. Infect. Immun. 2008; 76: 1518-1526Crossref PubMed Scopus (55) Google Scholar, 14Clarke S.R. Mohamed R. Bian L. Routh A.F. Kokai-Kun J.F. Mond J.J. Tarkowski A. Foster S.J. Cell Host Microbe. 2007; 1: 199-212Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15Clarke S.R. Wiltshire M.D. Foster S.J. Mol. Microbiol. 2004; 51: 1509-1519Crossref PubMed Scopus (124) Google Scholar), is effective at scavenging heme (16Vermeiren C.L. M. J. Heinrichs D.E. PubMed Scopus Google Scholar, C.L. Heinrichs D.E. Mol. Microbiol. 2007; PubMed Scopus Google Scholar). reported functions of Isd proteins the by IsdB of hemoglobin Humayun M. Schneewind O. Skaar E.P. J. PubMed Scopus Google Scholar), of and A. D. A. E. Mol. Microbiol. 2003; PubMed Scopus Google Scholar), and heme by IsdC and IsdE J. C. Heinrichs D.E. 2004; PubMed Scopus Google Scholar, S. Cockayne A. M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, C.L. Heinrichs D.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, M. C.L. J. Heinrichs D.E. J. 2007; Google Scholar). a and a of the of proteins, heme into a the of the protein, and to to the heme-iron C.L. Heinrichs D.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins IsdA, IsdB, IsdC, and each at that a to heme into a with heme-iron via C.L. Heinrichs D.E. Mol. Microbiol. 2007; PubMed Scopus Google Scholar, S. Cockayne A. M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the series of Isd proteins in S. aureus are to with heme proteins, the heme and transport it across the cell wall through to the membrane it is then into the cytoplasm (for a recent to Skaar E.P. 2007; PubMed Scopus Google Scholar). As by has been as has the heme-degrading of IsdG and (12Skaar E.P. Gaspar A.H. Schneewind O. J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). et al. M. H. D.M. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar) used to that heme transfers from IsdA to In this we the of magnetic circular dichroism spectral data together with electrospray ionization mass spectrometry to unambiguously the or of a heme and heme are has been in and by in the heme of the complete proteins IsdA, IsdB, IsdC, and as the NEAT domains of IsdA, IsdB (domain 2), (domain 3), and IsdC to to the heme-binding and state (16Vermeiren C.L. M. J. Heinrichs D.E. PubMed Scopus Google Scholar, J. C. Heinrichs D.E. 2004; PubMed Scopus Google Scholar, M. C.L. J. Heinrichs D.E. J. 2007; Google Scholar, J. of Heme Iron The of and K. M., K. M., and Scholar, S. S. A. A. PubMed Scopus Google Scholar, M. Heinrichs D.E. J. 2008; PubMed Scopus Google Scholar, M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). D. E. and M. J. of the mass spectral data have that the readily from the and the (16Vermeiren C.L. M. J. Heinrichs D.E. PubMed Scopus Google Scholar, M. Heinrichs D.E. J. 2008; PubMed Scopus Google Scholar, M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar). the of we for the first for an heme transfer system the surface Isd to is that the is a of the and state of the heme-iron and that the in as a of heme the data show in their in in heme heme the very Isd proteins and the IsdE on the heme of IsdA, of IsdB, of IsdC, of IsdH/HarA, and of IsdE were from S. aureus and into in such a as to was of and into D. J. J. PubMed Scopus Google Scholar) into a J.M. H. R. J. PubMed Scopus Google Scholar). the of the study, were used to each of and in a in in the of recombinant The of in of each of the proteins was used in and and was used in and was in heme the spectral data for of proteins heme transfer from the Isd domains and to state and mass for a of Isd and was in and are identified in the state with the abbreviations for with a mass of with a mass of and with a mass of state and mass for a of Isd and was in and are identified in the state with the abbreviations for with a mass of with a mass of and with a mass of state and mass for a of Isd and was in and are identified in the state with the abbreviations for with a mass of with a mass of and with a mass of In each heme transfer to Isd NEAT-C was in a of the of and and NEAT-B2 or NEAT-H3 was in their mass with The in of the proteins on the of the proteins the in with the other of were used in each of the spectral data In each of the was that there was transfer from and to state and mass and for of with apo-IsdE as a heme and with apo-IsdE as a heme In the data show that transfers heme to In each mass are that to the that to apo-IsdE to at that to to at and that to to at The the the addition of IsdE by the from the and with IsdE for by the from the with The was by heme to 2), which was a that used to NEAT-B2 which was used for the data in and in the the The experimental results were by the of the In the data show that transfers heme to In each mass are that to the that to apo-IsdE to at that to to at and that to to at The of the the addition of IsdE by from the and with apo-IsdE for by from the with The to that of The was a that used for the NEAT-H3 which was used to the data in and in the were the The experimental results were by the of the transfer from to and to and state and mass and for of with as a heme with as a heme and with as heme In the data show that transfers heme to In each mass are that to the in the mass that to and at that to and at and that to to at In the data show that transfers heme to In each mass are that to the that to and at that to and at and that to to at In the data show that transfers heme to In each mass are that to the and that to and at that to and at and that to at and to to at and recombinant were at in and were and in were then in a and cell were to at for to were purified a and system with an were then a The were by with at for and through a for was in for of in were the at and at These were the as a and for PubMed Scopus Google Scholar). were the at of for for NEAT-A and for and for of the of in mass spectrometry was used to a NEAT as in were with in were then to and purified of were to that heme was were in a at at were in at in an in a was Absorption were the were in the and in the were was used with through a The mass source were at of was for several was used to the in were to to was Heme heme transfer the NEAT domains and and the were with an of the heme The were from to In the the as a to from Isd heme-binding NEAT domains from IsdA IsdB IsdC and or as as IsdE were overexpressed in and were by of with The proteins were by mass spectrometry to that each was or and to the of heme bound to each The mass spectral data are as the in with the in in provide an of the of the or to the M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar). In the of in state the and for and in addition to the data for IsdE M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar), that significant take place with heme in of The data in see that there is a very in of NEAT-A heme the state to from of the heme we the in the of the and in for and the is that there are the heme in in this a of with that take place for IsdE M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar), the in of NEAT-A and NEAT-H3 with heme are and for NEAT-A in agreement with the of the apo of the C.L. Heinrichs D.E. Mol. Microbiol. 2007; PubMed Scopus Google Scholar). heme mass spectrometry was used to that the were The mass spectral data provide important that the were the only source of heme in the transfer of the of a mass not is an important as we have that is not a to the complete and of heme from a The of heme as a on the of and with its in heme transfer each of the heme-binding Isd NEAT domains heme-binding that in heme transfer very the for each of the heme-binding NEAT domains and IsdE. that the of the bound in IsdA, IsdB NEAT IsdC, and NEAT are very NEAT heme transfer via and to as the spectral of the and for the NEAT domains of IsdA, IsdC, and and M. Heinrichs D.E. J. 2008; PubMed Scopus Google M. Heinrichs D.E. J. 2008; PubMed Scopus Google M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar) in a circular dichroism that provide from proteins, and we have demonstrated this with the proteins and the NEAT domains (16Vermeiren C.L. M. J. Heinrichs D.E. PubMed Scopus Google Scholar, M. Heinrichs D.E. J. 2008; PubMed Scopus Google Scholar, M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar). the data from are with mass data, heme transfer in and identified together with their The of this was the of heme to transfer Isd proteins to directly the that the proteins in as a heme system in the cell is the transfer are and of the or, on the other highly specific to the heme as would the specific protein-protein interactions were each NEAT contains a and, a mass mass spectral demonstrated in 2), heme transfer could readily the it was important to results with other that have been used to heme proteins, and magnetic circular dichroism Heme from NEAT-A through NEAT-C to the spectral of the NEAT domains are very to the in of the IsdE contains a heme-binding which is from the NEAT domains and, as a a very and IsdE a heme a and a the NEAT domains a heme a for heme-iron (16Vermeiren C.L. M. J. Heinrichs D.E. PubMed Scopus Google Scholar, C.L. Heinrichs D.E. Mol. Microbiol. 2007; PubMed Scopus Google Scholar, S. Cockayne A. M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, C.L. Heinrichs D.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar). The is to for from the of the NEAT Heme transfer for transfer the in spectral NEAT-A and the spectral are the heme-iron in NEAT-C is in the the sequence of for in the presence of of there is a in the on data it was as to heme transfer place. data heme transfer in this was from the data that the heme in NEAT-A to In other the data for this transfer are very and the data provide data, the data provide data to the that heme transfer place. that the of apo-IsdE to is in the is significant in the The data show that the heme not transfer from NEAT-A to IsdE. an was that the data show that spectral take place at the heme in the fact that heme is not which with a in the of the ferric a a is by a The data that heme transfer place NEAT-A and the data from MCD, not the fact that an the proteins is of the in this study, the the spectral from heme transfer as of apo-IsdE are to a of and as that used to the spectral data in in as that to and the spectral of the heme in are the heme is bound in or holo-NEAT-C, there are and that are in the and These data that transfers NEAT domains are to by that heme transfer to IsdE is readily and the provide with to the at the of the In the we report the of the to unambiguously that specific heme transfer occur the NEAT domains of (domain 3), IsdB (domain 2), IsdA, and IsdC, and the IsdE Heme from and we show mass spectral data that heme is and from each of and to The heme proteins were with a of the heme are in each of the in and in which are the of the the of the and the of the of the is for the of the that the of the used to proteins the by the mass is for each the of the and of the In data the and there is and the of the only the reverse in the presence of protein, then would of the would from was for only for the heme transfer NEAT-B2 and Our from data are the data from and the data unambiguously show that heme was to in the and the data by IsdE to Heme from and in we show the mass spectral data of and apo-IsdE and, in we show the for the proteins, and heme transfer from the the mass spectral data that heme was to at apo-IsdE at and at are The mass is with to the mass of the and we a from to is only of for the NEAT-C as there is The state with the of IsdE. of the heme not the of IsdE or NEAT-C see the state for IsdE and for NEAT-C heme The in show the for apo-IsdE is this the are the of IsdE to the Heme from data in show that there is heme transfer apo-IsdE is with holo-NEAT-A. is the only with data not we transfer to the Isd at in from in the experimental of the and from the presence of there is in the of the and the of the of transfer is in the the of with apo-IsdE is the as the in the other that the in that there is proteins, transfer of the Heme from and data in show unambiguously that heme, it was initially in NEAT-B2 or NEAT-H3 was and to The in and that IsdE is the heme there are significant in the The of and apo-IsdE and of and apo-IsdE are Heme from and data in and show that the heme in and transfers to The transfer from to is the data in show the presence of The Isd series of proteins are proposed to a complete heme acquisition and transport system, together to heme from proteins such as then to heme across the cell wall through to membrane which the heme into the cytoplasm it is by proteins IsdG and significant for the proteins (10Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar, E.P. Gaspar A.H. Schneewind O. J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, C.L. M. J. Heinrichs D.E. PubMed Scopus Google Scholar, C.L. Heinrichs D.E. Mol. Microbiol. 2007; PubMed Scopus Google Scholar, Humayun M. Schneewind O. Skaar E.P. J. PubMed Scopus Google Scholar, J. C. Heinrichs D.E. 2004; PubMed Scopus Google Scholar, S. Cockayne A. M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, C.L. Heinrichs D.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. D.M. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Heinrichs D.E. J. 2008; PubMed Scopus Google Scholar, M. C.L. J. Heinrichs D.E. 2007; 46: PubMed Scopus Google Scholar, A. D. C. M. A. R. A. E. J. 2007; PubMed Scopus Google Scholar), the first for heme transfer Isd proteins was only very for the IsdA to IsdC transfer M. H. D.M. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). In the study, a complete of heme transfer of the heme-binding Isd proteins that on the of the cell has been for the first heme-binding Isd NEAT domains heme has been to a ferric heme through a (16Vermeiren C.L. M. J. Heinrichs D.E. PubMed Scopus Google Scholar, C.L. Heinrichs D.E. Mol. Microbiol. 2007; PubMed Scopus Google Scholar, S. Cockayne A. M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Heinrichs D.E. J. 2008; PubMed Scopus Google Scholar) and, as such, the of the bound in IsdA, IsdB, IsdC, and are very The and data reported to not provide on the or that would the Isd proteins are to and transfer heme Although the heme of the NEAT domains very the data show that the NEAT domains in The majority of heme transfer and in a unidirectional fashion for the transfer NEAT-B2 and and, in the of IsdA, very via IsdE. is important to that of the Isd proteins used in this were of heme from conditions not The state of the heme in not the as ferric could not not the results that specific protein-protein interactions must occur to the of heme from Isd proteins, since of heme were low and were not to occur, would in very transfer this is not was in the of protein-protein interactions we would to in data the and the presence of see for significant of and is that or of the NEAT that or in heme from or In of it is that hemoglobin Humayun M. Schneewind O. Skaar E.P. J. PubMed Scopus Google Scholar) A. D. C. M. A. R. A. E. J. 2007; PubMed Scopus Google Scholar). In the proposed Isd-mediated heme transport system, the Isd proteins with proteins such as hemoglobin or which are in of the heme then by heme transfer to other Isd proteins in the cell wall of the data the in for transfer the NEAT the provide there is only a transfer from to The is the transfer place to IsdE. The transfer to IsdE was by since a of the from to was the other the mass data show that heme to other NEAT domains as as to IsdE. heme to with was the only of heme transfer that was in this and that IsdB and are in agreement with on of this is that the IsdA NEAT NEAT-B2 or NEAT-A accepted heme from NEAT-B2 and NEAT-H3 in a unidirectional and only heme to NEAT-C not to mass MCD, and that IsdE was to accept heme from and show the and of and the addition of in the or in the was spectrometry that of of with of heme transfer was not IsdC has been proposed to as the central conduit in the cell wall for heme to the cell membrane. The of NEAT-C to accept heme from and NEAT-H3 and to only transfer it to apo-IsdE and for this in was with a significant in was of apo-IsdE with see In the data a model for heme transport as in in agreement with the proposal first by Mazmanian et al. (10Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar). in challenge experiments, is that the of heme is from the and IsdB through to the IsdE to the is that heme not transfer from NEAT-A directly to is the taken together with the demonstrated of the transfer that specific protein-protein interactions must a of the to the heme transfer the membrane. the of this a to this available on in H. M. M. D.M. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus (103) Google Scholar).

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