Most importantly, the critical residues in the NEAT1IsdBregion between 164 and 168 did not contain mutations in any of the 3277 strains. required for hemoglobin binding. Further, we show that these residues are necessary for heme extraction from hemoglobin and growth on hemoglobin as a single iron source. These processes are found to contribute to the pathogenicity ofS. aureusin a murine model of infection. Together these results build on the model for Isd-mediated hemoglobin binding and heme-iron acquisition during the pathogenesis ofS. aureusinfection. Keywords:Staphylococcus aureus, heme, hemoglobin, iron-regulated surface determinant system, contamination, iron, pathogenesis To colonize the host and cause diseaseStaphylococcus aureusrequires iron. The most abundant iron source within vertebrates is usually hemoglobin. Staphylococci capture hemoglobin and subsequently import and degrade heme Ziyuglycoside II using a series of proteins known collectively as the iron-regulated surface determinant (Isd) system [1]. The Isd system consists Ziyuglycoside II of 10 proteins, which take action in concert to acquire iron from hemoglobin (Physique1A) [1,2]. The current model for Isd-mediated heme capture proposes that IsdA and IsdB are anchored to the cell wall by sortase A and are localized to the bacterial surface [1,3]. IsdC is embedded within the cell wall and is covalently anchored to peptidoglycan by a dedicated sortase B [1,2]. IsdB binds hemoglobin and removes heme from the protein backbone of hemoglobin [1,47]. IsdB then rapidly passes heme to IsdA, which transfers it to IsdC [5,6,810]. Multiple IsdC molecules are suggested to pass heme across the cell wall and deliver heme to the membrane transport component IsdE [5,6,11,12]. The kinetics of heme binding and unidirectional transfer have been extensively studied using mass spectrometry, ultraviolet-visible spectrometry, magnetic circular dichroism spectrometry, nuclear magnetic resonance, and X-ray crystallography [57,9,1318]. IsdH is another protein that binds hemoglobin and heme in vitro [18,19]. Despite significant amino acid similarity to IsdB, IsdH does not appear to be required for hemoglobin binding and hemoglobin-derived iron acquisition in vivo [4]. It is possible that IsdH contributes to hemoglobin binding at low concentrations or to binding of hemoglobin-haptoglobin complexes. Membrane-localized IsdE allows for heme passage into the cytoplasm, where heme is degraded by the heme oxygenases IsdG and IsdI to liberate iron and form staphylobilin [15,2026]. Consistent with the importance of heme-iron acquisition to the pathogenesis of staphylococcal Rabbit Polyclonal to OR1E2 infections, inactivation ofisdA,isdB,isdC,isdG,orisdIreduces bacterial proliferation within the host [3,4,21,2729]. == Figure 1. == S. aureusiron regulated surface determinant system (Isd). (A) Genomic arrangement of the genes encoding the Isd system. Arrows denote promoter regions and the direction of transcription. (B) IsdB and IsdH hemoglobin-binding (white) and heme-binding (grey) NEAT domains. (C) Alignment of the hemoglobin binding motifs from IsdH and IsdB. In contrast to the significant amount of information regarding heme relay between the Isd proteins, hemoglobin capture has not been studied in detail. Binding of hemoglobin and removal of heme are carried out in vitro by distinct domains within IsdB called NEAT1IsdBand NEAT2IsdB, respectively (Figure1B) [5,7,18,28,30,31]. NEAT1IsdBis required and sufficient to bind hemoglobin, whereas NEAT2IsdBis required and sufficient for heme binding [7,28]. Biochemical and crystallographic analyses of NEAT1 from IsdH (NEAT1IsdH) have identified a sequence of consecutive aromatic residues within IsdH that is required for hemoglobin binding in vitro [18,31]. However, differences exist between the residues that are predicted to bind hemoglobin within NEAT1IsdHand those found within NEAT1IsdB(Figure1C). We have previously found that IsdB binds human hemoglobin with a higher affinity than hemoglobin from other species and that mice expressing human hemoglobin (HA) are more susceptible toS. aureusinfection [29,32]. These results suggest that specific hemoglobin binding by IsdB is required for iron acquisition. To directly test this hypothesis, we first interrogated the role of NEAT1IsdBin hemoglobin binding toS. aureusin vivo and identified the residues that are required for hemoglobin capture. Using a strain expressing an IsdB point mutant that is deficient in hemoglobin capture, we demonstrate that hemoglobin binding activity of IsdB is required for heme extraction, iron acquisition, and virulence in HAmice. Together, these results establish the Isd system as a physiologically relevant heme-iron acquisition system withinS. aureusand elucidate the role of Ziyuglycoside II hemoglobin binding by IsdB in the function of the Isd system. == MATERIALS AND METHODS == == Bacterial Strains.