Abstract
Iron is an essential nutrient for all living organisms. To acquire iron, many pathogens have developed elaborate systems to steal it from their hosts. The iron acquisition system in the opportunistic pathogen Staphylococcus aureus comprises nine proteins, called iron-regulated surface determinants (Isds). The Isd components enable S. aureus to extract heme from hemoglobin (Hb), transport it into the bacterial cytoplasm, and ultimately release iron from the porphyrin ring. IsdB and IsdH act as hemoglobin receptors and are known to actively extract heme from extracellular Hb. To limit microbial pathogenicity during infection, host organisms attempt to restrict the availability of nutrient metals at the host-pathogen interface. The human acute phase protein haptoglobin (Hp) protects the host from oxidative damage by clearing hemoglobin that has leaked from red blood cells and also restricts the availability of extracellular Hb-bound iron to invading pathogens. To investigate whether Hp serves an additional role in nutritional immunity through a direct inhibition of IsdH-mediated iron acquisition, here we measured heme extraction from the Hp-Hb complex by UV-visible spectroscopy and determined the crystal structure of the Hp-Hb-IsdH complex at 2.9 Å resolution. We found that Hp strongly inhibits IsdH-mediated heme extraction and that Hp binding prevents local unfolding of the Hb heme pocket, leaving IsdH unable to wrest the heme from Hb. Furthermore, we noted that the Hp-Hb binding appears to trap IsdH in an initial state before heme transfer. Our findings provide insights into Hp-mediated IsdH inhibition and the dynamics of IsdH-mediated heme extraction.
Highlights
In order to establish a successful infection, pathogens must be able to obtain nutritional iron from their host as it is essential for a variety of metabolic processes [1]
Heme transfer to IsdH can be determined by measuring decrease in absorbance at a wavelength of 406 nm [19], which is accompanied by a concomitant increase around 370 nm
This is achieved by tightly binding Hb and preventing the S. aureus Hb receptors IsdH and IsdB from extracting the iron-containing heme
Summary
In order to establish a successful infection, pathogens must be able to obtain nutritional iron from their host as it is essential for a variety of metabolic processes [1]. One strategy is to secrete various siderophores that chelate free iron and are subsequently reimported by the bacteria [2] Another strategy is to obtain iron via the extraction and uptake of heme from host hemoproteins e.g. Hb. The S. aureus heme acquisition system is an elaborate system of nine proteins, called the iron-regulated surface determinants (IsdA-I) [3]. The four peptidoglycan-anchored proteins IsdA, IsdB, IsdC and IsdH are structurally related and contain a variable number of conserved NEAr iron Transporter (NEAT) domains [16]. The additional N-terminal NEAT domain of IsdH (IsdHN1) is capable of binding Hb [6], but its significance in heme extraction is not yet fully understood. The HpSP-abHb-IsdHN2-N3 structure, may represent an initial state of the Hb-IsdH complex prior to heme-transfer and provides new insights into the mechanism of IsdH-mediated Hb heme-extraction
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