Molecular mechanisms used by Staphylococcus aureus to access iron from human haemoglobin

Iron is used as a co-factor in a range of biological reactions and is an essential nutrient across all domains of life. During infection, pathogens must acquire iron from their host environment. The tetrameric oxygen transport protein, haemoglobin A (Hb), represents the largest pool of mammalian iron, accounting for ~ 65 % of iron in a healthy individual. Genes encoding for Hb receptors are found in the genomes of Gram-positive and Gramnegative pathogens and their deletion results in attenuated virulence, suggesting that Hb is an important source of iron during host colonization. As yet, the mechanisms by which these receptors interact with Hb and capture the haem co-factor (containing the iron) are largely unknown. Staphylococcus aureus is a Gram-positive human pathogen and is a significant health burden in both community and health care settings. S. aureus expresses an elaborate system of proteins known as the iron regulated surface determinant (Isd) system, which is specialized to strip iron from Hb. Two related Hb receptors have been identified in the staphylococcal genome, IsdB and IsdH. IsdB and IsdH bind to Hb on the bacterial cell surface, actively remove the haem cofactor and relay it to the down stream haem binding proteins IsdA and IsdC in the cell wall. IsdB and IsdH are multi-domain proteins, with separate domains dedicated to Hb binding and haem binding. Recent structural studies have revealed how Hb and haem are bound by the isolated domains, however, as the isolated domains do not liberate the haem cofactor from Hb these studies provide limited insight into the receptor mechanism. The aim of this project was to characterize the structure and function of the intact Hb receptors and reveal the molecular mechanism by which they capture the haem from Hb and relay it through the Isd pathway. Small angle X-ray scattering studies were used to show that a conserved three-domain region of IsdB and IsdH, which includes one Hb-binding and one haem-binding domain, adopts a conserved bi-lobed structure. Two X-ray crystal structures of IsdB and IsdH at 4.2 ‚àövñ and 3.7 ‚àövñ resolution, respectively, indicate that this architecture is maintained upon binding to Hb. Each Hb globin chain is bound independently by one receptor molecule and functional studies confirmed that haem is captured from all four globin haem pockets, suggesting a mechanism where the receptors can access all the globin haem groups regardless of the Hb oligomeric state. The Hb-binding domains of the full-length receptors bind to a surface of Hb that is distant from the globin haem coordination site. The haem-binding domain is positioned via a scaffolding linker domain directly adjacent to the globin haem pocket suggesting that a second set of specific interactions is involved in liberating the haem cofactor. As this interface is not well resolved in the low-resolution structures, rational mutagenesis was used to alter the Hb-binding properties of IsdH. A version of IsdH that bound through only the subunit of Hb yielded crystals that diffracted to 2.5 ‚àövñ resolution. The higher resolution structure revealed specific contacts on the haem transfer interface, and a conformational change in the globin haem pocket, which begin to illustrate how the haem cofactor is extracted from the globin. To investigate the mechanism of haem relay through the Isd pathway, haem transfer assays were designed. IsdH and IsdB captured haem from Hb with similar efficiency. However, IsdB showed superior activity in transferring haem to IsdA and IsdC. Haem capture from Hb required the specific recognition of Hb though the Hb-targeting domain, but, the speed of haem relay to IsdA/C was inversely related Hb-binding affinity suggesting that haem relay activity relies on a weak interaction with the Hb molecule. Using site directed mutagenesis, Hb binding affinity was attenuated or enhanced in a range of IsdB constructs. The activity of these mutants in haem relay experiments was consistent with the hypothesis that Hb binding affinity is fine tuned to allow for a specific interaction while maximizing the transfer speed of the haem cargo. Hb utilization is a common strategy employed by pathogenic bacteria to enhance growth and survival during infection. This work has contributed to our understanding of how the human pathogen, S. aureus, interacts with and utilizes host Hb, and is the first study to characterize the molecular mechanism by which the haem ligand is captured from Hb by a bacterial receptor.