Interaction of human hemoglobin and semi-hemoglobins with the Staphylococcus aureus hemophore IsdB: a kinetic and mechanistic insight

Abstract

Among multidrug-resistant bacteria, methicillin-resistant Staphylococcus aureus is emerging as one of the most threatening pathogens. S. aureus exploits different mechanisms for its iron supply, but the preferred one is acquisition of organic iron through the expression of hemoglobin (Hb) receptors. One of these, IsdB, belonging to the Isd (Iron-Regulated Surface Determinant) system, was shown to be essential for bacterial growth and virulence. Therefore, interaction of IsdB with Hb represents a promising target for the rational design of a new class of antibacterial molecules. However, despite recent investigations, many structural and mechanistic details of complex formation and heme extraction process are still elusive. By combining site-directed mutagenesis, absorption spectroscopy, surface plasmon resonance and molecular dynamics simulations, we tackled most of the so far unanswered questions: (i) the exact complex stoichiometry, (ii) the microscopic kinetic rates of complex formation, (iii) the IsdB selectivity for binding to, and extracting heme from, α and β subunits of Hb, iv) the role of specific amino acid residues and structural regions in driving complex formation and heme transfer, and (v) the structural/dynamic effect played by the hemophore on Hb. The ability of bacterial pathogens to establish infections relies on the adaptation of bacterial metabolism to the environment found within the host, which is often severely nutrient-restricted and a site for competition with commensal microorganisms and other pathogens 1,2. A well-characterized mechanism of adaptation to the nutritional environment of the host is represented by the expression of redundant iron-acquisition systems, both in Gram + and Grambacteria, to provide iron supply to support invasion and proliferation 3. Iron is an essential nutrient for both the pathogen and the host, but also a toxic element due to its involvement in Fenton chemistry and Haber-Weiss reactions that generate reactive oxygen species (ROS) 4. For this reason, vertebrates have put in place strategies to maintain very low concentrations of free iron in body fluids (down to 10 −18-10 −24 M) 4,5 achieving two goals, the first being limiting toxicity and the second creating an iron-restricted environment for pathogens 6,7. In S. aureus, iron scavenging is achieved by at least three different mechanisms: i) acquisition of inorganic iron through production of siderophores, ii) expression of hemoglobin (Hb) receptors, and iii) acquisition of inorganic iron through transporters 4. Heme is the preferred iron source during the initial phase of infection 8

The iron-regulated surface determinant B (IsdB) protein from Staphylococcus aureus acts as a receptor for the host protein vitronectin

“…These CWA proteins share similar mechanisms of binding (5). In parallel with this, the structural basis of Hb binding to IsdB NEAT motifs has been elucidated (72,73). From this perspective, X-ray crystal structure analysis of the recombinant IsdB-binding domain of Vn in complex with NEAT motifs could provide clues about the mechanism and function of this important CWA protein in bacterial colonization of and survival within the host.…”

Staphylococcus Aureus Iron-Regulated Surface Determinant B (IsdB) Protein Interacts with von Willebrand Factor and Promotes Adherence to Endothelial Cells.

“…1A, lane 2) suggesting that binding depends on a protein that is induced by iron starvation. Additional low molecular weight molecules (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) were observed in the material released from bacteria grown in both media.…”

“…Regarding IsdB, no structural data at atomic resolution are available, although the crystallographic structure of IsdB-hemoglobin (Hb) complex 16 indicates that IsdB has a dumbbell-like shape, with the two NEAT domains laying almost orthogonal to each other and joined by a highly exible triple helix linker region 16,17 . Each NEAT domain adopts the characteristic eight-strand immunoglobulin-like β-sandwich fold with a central 3 10 -helix that forms a hydrophobic cavity.…”

“…Notably, the two NEAT domains share low sequence identity (about 12%), in line with their known different molecular recognition properties. In fact, NEAT1 preferentially binds Hb, while NEAT2 is involved in heme extraction from the chains of Hb 16,17 .…”

The Role of Gram-Positive Surface Proteins in Bacterial Niche- and Host-Specialization

“…Heme is then transferred to NEAT domain 2 of IsdB and along a chain of Isd proteins concluding in heme binding to the IsdE membrane-associated lipoprotein. This allows IsdF-mediated translocation of the heme iron into the bacterial cytoplasm for use in metabolism (Muryoi et al, 2008;Zhu et al, 2008;Bowden et al, 2014;Gianquinto et al, 2019). Notably, IsdB is a host-specialized CWA protein with enhanced binding affinity for human hemoglobin, in comparison to hemoglobin from other mammalian sources (Pishchany et al, 2010).…”