Structural basis for the procoagulant activity of Staphylococcus aureus staphylocoagulase

Plasminogen Recruitment by Staphylococcus aureus SdrC Reveals a Tractable Antivirulence Target.

Staphylococcus aureus exploits host extracellular matrix components to promote tissue invasion and dissemination. Here, we identify the serine-aspartate repeat protein C (SdrC) as a previously unrecognized plasminogen-binding protein on the S. aureus surface. Using recombinant domains and isogenic mutants, we show that SdrC is a major determinant of plasminogen recruitment at the cellular level. Biochemical and biophysical analyses demonstrate that plasminogen recognition is enhanced by the cooperative action of the SdrC N2 and N3 domains, which together bind plasminogen with submicromolar to low-micromolar affinity. This interaction is lysine-dependent and is selectively inhibited by lysine and 6-aminocaproic acid, with measurable IC50 values, and requires plasminogen kringle domain 4. Importantly, SdrC-bound plasminogen remains readily activatable by host plasminogen activators, generating active plasmin capable of degrading fibrinogen. Consistently, heterologous expression of SdrC enhances plasminogen binding and promotes plasmin activity at the bacterial surface. These findings link a defined staphylococcal adhesin to localized engagement of the host fibrinolytic system and suggest that SdrC-mediated plasminogen recruitment may contribute to persistence and tissue dissemination during invasive infection. Overall, our results establish the SdrC-plasminogen axis as a mechanistically characterized and pharmacologically tractable antivirulence target.

Surface-associated phosphoglycerate kinase (PGK) of Staphylococcus aureus binds human plasminogen in a partially lysine-independent manner.

Staphylococcus aureus, a potent opportunistic pathogen causes a variety of infections ranging from mild skin infections to life-threatening diseases. With the rapid increase in antimicrobial resistant pathogens, it is critical to comprehensively understand the virulence strategies employed by the pathogen to invade the host. The glycolytic enzymes of S. aureus have been reported to possess additional roles promoting pathogenesis. In this study, we cloned, overexpressed and purified the S. aureus glycolytic protein, phosphoglycerate kinase (PGK) (EC 2.7.2.3) and investigated its localization and interaction with human plasminogen. To elucidate the localization of PGK, a whole cell ELISA and cell fractionation was performed. We report the localization of the wild type protein in the cytosol as well as the cell surface associated fractions of S. aureus. Whole-cell ELISA with anti-PGK antibodies showed significant antibody binding compared to control with secondary antibody (∼11-fold increase, p = 0.0005), indicating surface exposure of PGK. Purified recombinant PGK bound human plasminogen (p < 0.0001) and enhanced its conversion to its active form in the presence of tissue plasminogen activator (p < 0.0001). Plasminogen binding to PGK was only partially inhibited by ε-aminocaproic acid, indicating that lysine-mediated interactions are not the sole determinants. These results were fully corroborated with the molecular dynamics which revealed stable binding throughout the entire 500ns period and a similar disposition of the lysines. These findings suggest that PGK may function as a moonlighting protein, playing an essential role in the interaction of S. aureus with its host through plasminogen binding.

Helicolysins emerge as a new family of metzincin metallopeptidases in host-pathogen interactions.

The Helicobacter pylori Hom family of outer-membrane proteins (OMPs) has been implicated in virulence-related processes. Here, we present a structural and functional characterization of HomA from H. pylori strain 26695. Comparative in vivo analyses of the wild-type strain and a homA deletion mutant demonstrate a role for HomA in focal adhesion kinase phosphorylation at Y397 and in bacterial adhesion to human leukaemia-derived MEC-1 cells. Recombinant production of several HomA variants identified a soluble ectodomain (ECDHomA) that harbours the conserved zinc-binding motif of metzincin metallopeptidases (MPs) and displays dimerization capacity, acid resistance, and immunomodulatory activity by triggering a pro-inflammatory response in Raji human B-lymphoblastoid cancer cells. Site-directed mutagenesis of the zinc-binding motif confirmed its contribution to zinc coordination and thermal stability. Structural analysis of ECDHomA revealed a two-domain organization comprising an N-terminal accessory β-sandwich domain (ND) and a C-terminal catalytic domain (CD), in which a non-canonical 'Thr-turn' replaces the hallmark metzincin 'Met-turn'. Integrating bioinformatics with AI-based structure predictions uncovered putative HomA homologues across 44 proteomes, predominantly from Gram-negative bacteria. Structural and biochemical characterization of the closest non-Helicobacter homologue from the oral pathogen Capnocytophaga canimorsus, dubbed canilysin, revealed an architecture equivalent to ECDHomA and highly specific MP activity. Together, these findings define the Hom family and canilysin as helicolysins, a previously uncharacterized metzincin subfamily distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host-pathogen interactions, adhesion, and immunomodulation.

A Procoagulant Peptide Analog of the SARS-CoV-2 Nucleocapsid C-terminal Domain

Structural and biochemical insights into the antibiofilm activity of cationic lipoamino acid ester against methicillin-resistant Staphylococcus aureus

Three-dimensional structures, primarily derived from X-ray crystallography, are crucial for understanding the solid-state properties of active pharmaceuticals and for enabling structure-based drug design. Herein, we report the first single crystal X-ray structure of cationic lipoaminoacid ester [(2-((N-(2-methoxy-2-oxoethyl)tetradecanamido)methyl)-1-methylpyridin-1-ium iodide, QPyN14Ge]. QPyN14Ge crystallized in the triclinic space group P1̄. QPyN14Ge has a highly ordered and coherent packing arrangement due to tail-to-tail interdigitation of hydrophobic layers, stabilization by planar trans-amide head groups, controlled flexibility at the distal ends of the hydrocarbon chains, and heavy-atom anchoring by iodine near the polar region. QPyN14Ge exhibits antimicrobial activity against Methicillin-resistant Staphylococcus aureus by compromising membrane integrity. Molecular docking reveals that QPyN14Ge binds to the Staphylococcal protein FemX, which is responsible for peptidoglycan biosynthesis. Molecular dynamics simulations support the formation of a stable, energetically favourable FemX-QPyN14Ge complex, suggesting its potential as a cell wall biosynthesis inhibitor. QPyN14Ge shows strong antibiofilm activity at the MIC and can inhibit biofilm formation (40%) even at sub-MIC levels. Confocal microscopy shows that sub-MIC QPyN14Ge reduces the biofilm thickness from 11.88 ± 1.16 µm to 7.01 ± 0.95 µm. The antibiofilm mechanism is attributed to the inhibition of major virulence factors of MRSA, such as cell surface hydrophobicity, slime synthesis, exopolysaccharide production, and staphyloxanthin production. The findings suggest that ability of QPyN14Ge to form interdigitated molecular packing, in which molecules insert themselves between lipid bilayers, thinning and compromising membrane integrity, resulted in antibacterial and antibiofilm activities.

The Staphylococcus aureus carotenoid staphyloxanthin modifies the structure of phosphoglycerol lipid bilayers

Staphyloxanthin (STX) is a carotenoid synthesized by the human pathogen Staphylococcus aureus. The golden color of this bacterium is due to this carotenoid. STX protects Staphylococcus aureus from oxidative stress by scavenging free radical species. Furthermore, STX has been shown to mechanically strengthen the Staphylococcus aureus membrane and to form microdomains that recruit antibiotic-resistance factors. Thus, inhibition of STX is a promising strategy for intervening against multidrug-resistant strains of this pathogen. However, the molecular mechanisms by which STX regulates the membrane structure and function of Staphylococcus aureus remain unclear. More specifically, the localization of STX within phosphatidylglycerol (PG) bilayers, the primary phospholipid of this bacterium’s membrane, and how this localization drives macroscopic biophysical changes remain unresolved questions. Here, we addressed this issue by integrating molecular dynamics (MD) simulations, X-ray scattering experiments, and fluorescence spectroscopy. We developed an atomistic model of STX, which was validated against X-ray scattering data and which is suitable for all-atom MD simulations. We demonstrate that STX significantly increases lipid packing and acyl chain order of STX–PG bilayer mixtures. In addition, STX self-assembles into clusters, where the long and rigid conjugated triterpenoid chain interdigitates across both leaflets, modifying locally the density of the surrounding PG molecules. These findings provide a molecular explanation to the reduced headgroup spacing and core dynamics observed in fluorescence experiments and are consistent with the formation of structurally-distinct STX-enriched microdomains. Notably, STX reduces the gel-to-liquid crystalline phase transition temperature, indicating a general stabilizing effect for the fluid phase of PG lipids of varying length. Overall, our findings provide molecular insights into how STX enhances membrane mechanical integrity. It will be highly interesting to establish how the membrane remodeling effects observed here connect with STX’s dual roles, acting as an antioxidant and preventing pore formation and other mechanical perturbations induced by antimicrobial molecules.