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CD163 is a macrophage scavenger receptor central to the clearance of haptoglobin-hemoglobin (HpHb) complexes and the resolution of inflammation. In a remarkable example of near-simultaneous scientific progress, three complementary studies published in Nature Communications 1 , 2 – 3 have resolved the near-atomic resolution structure of the full extracellular domain of human CD163 complexed with HpHb, primarily utilizing cryo-electron microscopy (cryo-EM). The most significant and unified conclusion emerging from this trio of papers is that CD163’s scavenging activity is fundamentally dependent on its ability to form functional multimers.

In the pharmaceutical field, lipid-based nanoparticles are extensively used for drug or vaccine delivery, particularly for treating respiratory disorders. However, their physico-chemical instability, particularly associated with lipid degradation through hydrolysis or oxidation, can affect their encapsulation properties. To monitor the stability of lipid-based formulations over time, we prepared acrylodan-labeled bovine serum albumin (here called albuminodan), and showed it is a fluorescent biosensor capable of concomitantly detect phospholipids as well as their degradation products, i.e., fatty acids and lysophospholipids. We demonstrated that this tool can be used to follow the distribution of lipids in an aqueous phase and hence could be suitable to characterize the hydrolysis of phospholipids in a lipid-based formulation to monitor the stability of nanoparticles.

Nutritional immunity is a form of innate immunity widespread in both vertebrates and invertebrates. The term refers to a rich repertoire of mechanisms set up by the host to inhibit bacterial proliferation by sequestering trace minerals (mainly iron, but also zinc and manganese). This strategy, selected by evolution, represents an effective front-line defense against pathogens and has thus inspired the exploitation of iron restriction in the development of innovative antimicrobials or enhancers of antimicrobial therapy. This review focuses on the mechanisms of nutritional immunity, the strategies adopted by opportunistic human pathogen Staphylococcus aureus to circumvent it, and the impact of deletion mutants on the fitness, infectivity, and persistence inside the host. This information finally converges in an overview of the current development of inhibitors targeting the different stages of iron uptake, an as-yet unexploited target in the field of antistaphylococcal drug discovery.

Infections caused by Staphylococcus aureus depend on its ability to acquire nutrients. One essential nutrient is iron, which is obtained from the heme of the human host hemoglobin (Hb) through a protein machinery called Iron-regulated surface determinant (Isd) system. IsdB is the protein in charge of heme extraction from Hb, which is the first step of the chain of events leading to iron transfer to the bacterium cell interior. In order to elucidate the molecular events leading from the formation of the initial IsdB:Hb complex to heme extraction, we use time-resolved X-ray solution scattering (TR-XSS) in combination with rapid mixing triggering. We succeed in defining the stoichiometry of IsdB:Hb binding and in describing the kinetics of the subsequent structural changes. The presented approach is potentially applicable to unveil the complex kinetic pathways generated by protein-protein interaction in different biological systems. Iron-regulated surface determinant B (IsdB) from Staphylococcus aureus extracts heme from human hemoglobin, which is the first iron transfer step to the bacterial cell. Here, IsdB functional dynamics are explored with X-ray solution scattering techniques including time-resolved WAXS.

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.

Cysteine biosynthesis is increasingly recognized as a critical determinant of bacterial virulence, highlighting this pathway as a promising reservoir of novel antimicrobial targets. In , however, the molecular basis of cysteine production has only recently begun to emerge. Here, we identify PA3816 as the major serine acetyltransferase (PaCysE), the enzyme responsible for generating the activated serine intermediate that feeds -acetylserine sulfhydrylase-mediated cysteine synthesis. Through a combination of biochemical and genetic approaches, we demonstrate that PaCysE efficiently catalyzes L-serine acetylation , and in turn, deletion mutants exhibit cysteine auxotrophy, underscoring its essential contribution to -acetylserine production. Notably, PaCysE is less sensitive to feedback inhibition by cysteine and does not appear to form the canonical cysteine synthase complex, suggesting a regulatory architecture that diverges from well-characterized orthologs. Loss of PaCysE function has broad physiological consequences, including enhanced biofilm formation, reduced pyocyanin production, and attenuated infectivity in an animal model, linking cysteine biosynthesis directly to pathogen fitness. Finally, we identify a thiazole derivative that inhibits PaCysE activity (IC ≈ 30 µM) and suppresses bacterial growth in a cysteine-dependent manner, providing a proof-of-concept for therapeutically targeting this pathway.

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus . This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein–protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico / in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.

Iron surface determinant B (IsdB), a Staphylococcus aureus (SA) surface protein involved in both heme iron acquisition from host hemoglobin (Hb) and bacterial adhesion, is a proven virulence factor that can be targeted for the design of antibacterial molecules or vaccines. Recent single-molecule experiments on IsdB interaction with cell adhesion factors revealed an increase of the complex lifetime upon applying a stronger force (catch bond); this was suggested to favor host invasion under shear stress. An increased bond strength under mechanical stress was also detected by Atomic Force Spectroscopy (AFS) for the interaction between IsdB and Hb. Structural information on the underlying molecular mechanisms at the basis of this behaviour in IsdB-based complexes is missing. Here, we show that the single point mutation of Pro173 in the IsdB domain responsible for Hb binding, which weakens the IsdB:Hb interaction without hampering heme extraction, totally abolishes the previously observed behavior. Remarkably, Pro173 does not directly interact with Hb, but undergoes cis–trans isomerization upon IsdB:Hb complex formation, coupled to folding-upon binding of the corresponding protein loop. Our results suggest that these events might represent the molecular basis for the stress-dependence of bond strength observed for wild type IsdB, shedding light on the mechanisms that govern the capability of SA to infect host cells.

Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but inefficient from Hb either bound to oxygen (oxyHb) or bound to carbon monoxide (HbCO), and encompasses a sequence of structural events that are currently poorly understood. By single-particle cryo-electron microscopy, we determined the structure of two IsdB:Hb complexes, representing key species along the heme extraction pathway. The IsdB:HbCO structure, at 2.9-Å resolution, provides a snapshot of the preextraction complex. In this early stage of IsdB:Hb interaction, the hemophore binds to the β-subunits of the Hb tetramer, exploiting a folding-upon-binding mechanism that is likely triggered by a cis/trans isomerization of Pro173. Binding of IsdB to α-subunits occurs upon dissociation of the Hb tetramer into α/β dimers. The structure of the IsdB:metHb complex reveals the final step of the extraction process, where heme transfer to IsdB is completed. The stability of the complex, both before and after heme transfer from Hb to IsdB, is influenced by isomerization of Pro173. These results greatly enhance current understanding of structural and dynamic aspects of the heme extraction mechanism by IsdB and provide insight into the interactions that stabilize the complex before the heme transfer event. This information will support future efforts to identify inhibitors of heme acquisition by S. aureus by interfering with IsdB:Hb complex formation.

Infections caused by Staphylococcus aureus are closely linked to its ability to secure essential nutrients, including iron, which is extracted from the heme of human hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The compound 4-[[2-[[5- (1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]sulfanyl]acetyl]amino]benzoate (C35) was recently identified as a new potential antimicrobial agent for its ability to bind Hb and hamper its interaction with the staphylococcal hemophore IsdB in vitro. Here, we show that C35 inhibits S. aureus growth by specifically targeting the hemophore-driven iron acquisition system. Our findings confirm both the potential of C35 as a first-in-class protein-protein interaction inhibitor with antimicrobial activity, and the effectiveness of targeting hemophores as a strategy to inhibit S. aureus growth. To gain information for drug discovery purposes, the X-ray structure of Hb in the presence of the compound was solved. Unexpectedly, we discovered that, rather than the predicted binding pose, the molecule binds to tetrameric Hb in a cleft between the alpha subunits, stabilizing an R2 relaxed Hb conformation. This triggered further investigation of the effect of C35 on Hb functional properties, which showed a pronounced left-shift activity on oxygen binding curve (i.e., it strongly increases the Hb oxygen affinity). These results highlight C35 as a promising dual-acting compound with both antimicrobial activity and the ability to modulate Hb function through non-covalent stabilization of a high-affinity state. Staphylococcus aureus is a dangerous bacterium that can cause severe infections in humans. To grow and survive it needs iron, which it steals from our red blood cells by taking it from hemoglobin, the protein that carries oxygen in the blood. In this study, we focused on a small molecule, called C35, that blocks the interaction between hemoglobin and a key bacterial protein involved in heme acquisition. We found that C35 strongly inhibits the growth of S. aureus when hemoglobin is the only available source of iron, showing a potential new method to starve the pathogen and consequently fight the infection. Surprisingly, we also found that C35 increases the affinity of hemoglobin for oxygen. This dual action makes C35 a unique molecule for future therapeutic development, with potential applications both as a new antimicrobial agent and in the treatment of diseases related to hemoglobin function.