bioRxiv Science⌕ Search

Biology subjects

van Strijp, J.

Publications and source records attributed to van Strijp, J..

9 recordsLinked to original sources

LaeA-dependent production of small molecules of Aspergillus niger that compete with specific antibodies that bind to human immune receptors

Microorganisms secrete a variety of compounds into their environment such as proteins, carbohydrates, and secondary metabolites. These molecules play diverse roles in the interaction of microbes with their abiotic and biotic environment. Little is known about secreted fungal molecules mediating immune evasion. Here we screened culture media of three Aspergilli to assess whether these fungi secrete molecules that can compete with specific antibodies that bind to human immune receptors. Culture media of Aspergillus fumigatus Af293, Aspergillus tubingensis CBS 133792 and the non-acidifying mutant strain Aspergillus niger D15#26 contained components that showed competition for binding to a total of 13 receptors, of which PSGL-1, CXCR1, and CXCR2, were shared between the three species. Filtration experiments showed that most, if not all, interacting components were [≤] 3 kDa. Production of the components that competed with antibodies to bind to CD88 and CXCR2 was shown to be regulated by LaeA. The component(s) that competed for binding to CXCR1 was not only produced in the non-acidifying strain Aspergillus niger D15#26 but also in the non-acidifying oahA deletion strain of Aspergillus niger. Together, these data show that Aspergillus species might produce small molecules that interact with human immune receptors.

microbiology↗

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibodys constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fcs are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various DNP-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contribute to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in absence and presence of C1r2s2. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

immunology↗

The impact of Staphylococcus aureus cell wall glycosylation on langerin recognition and Langerhans cell activation

Staphylococcus aureus is the leading cause of skin and soft tissue infections. It remains incompletely understood how skin-resident immune cells respond to S. aureus invasion and contribute to an effective immune response. Langerhans cells (LCs), the only professional antigen-presenting cell type in the epidermis, sense S. aureus through their pattern-recognition receptor langerin, triggering a pro-inflammatory response. Langerin specifically recognizes the {beta}-1,4-linked N-acetylglucosamine ({beta}-GlcNAc) modification, which requires the glycosyltransferase TarS, on the cell wall glycopolymer Wall Teichoic Acid (WTA). Recently, an alternative WTA glycosyltransferase, TarP, was identified in methicillin-resistant S. aureus strains belonging to clonal complexes (CC) 5 and CC398. TarP also modifies WTA with {beta}-GlcNAc but at the C-3 position of the WTA ribitol phosphate (RboP) subunit. Here, we aimed to unravel the impact of {beta}-GlcNAc linkage position for langerin binding and LC activation. In addition, we performed structure-binding studies using a small panel of unique chemically-synthesized WTA molecules to assess langerin-WTA binding requirements. Using FITC-labeled recombinant human langerin and genetically-modified S. aureus strains, we observed that langerin similarly recognized bacteria that produce either TarS- or TarP-modified WTA. Furthermore, using chemically-synthesized WTA, representative of the different S. aureus WTA glycosylation patterns, established that {beta}-GlcNAc is sufficient to confer langerin binding. Functionally, tarP-expressing S. aureus induce increased cytokine production and maturation of in vitro-generated LCs compared to tarSexpressing S. aureus. Overall, our data suggest that LCs are able to sense all {beta}-GlcNAc-WTA producing S. aureus strains, likely performing an important role as first responders upon S. aureus skin invasion.

microbiology↗

Staphylococcal protein A inhibits complement activation by interfering with IgG hexamer formation

IgG molecules are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, a reaction that triggers a variety of responses that help to kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that Staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of Staphylococcus aureus, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)6:C1q complexes and prevents downstream complement activation on the surface of S. aureus. Lastly, we demonstrate that IgG3 antibodies against S. aureus can potently induce complement activation even in the presence of SpA. Altogether, this study identifies SpA as an immune evasion protein that specifically blocks IgG hexamerization.

microbiology↗

Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria at mucosal surfaces. Though several GBS adhesins have been identified, the host receptor targets of these adhesins remain unknown. We report here that surface-expressed {beta} protein from GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that the IgSF domain in {beta} represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessments revealed that this newly identified IgI3 fold is not exclusively present in GBS. Instead, the IgI3 fold is predicted to be present in adhesins from other clinically important human pathogens. We confirmed the interaction between CEACAM1 and the predicted IgI3-containing adhesin in two different streptococcal pathogens. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.

microbiology↗

Bacterial immune evasion proteins: The therapeutic potential of CHIPS

Bacterial pathogens have evolved to secrete strong anti-inflammatory proteins that target the immune system. It was long speculated whether these virulence factors could serve as therapeutics in diseases in which abnormal immune activation plays a role. We adopted the secreted Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) as a model virulence factor-based therapeutic agent for diseases in which C5aR1 stimulation plays an important role. We show that administration of CHIPS in human C5aR1 knock-in mice successfully dampens C5a mediated neutrophil migration during immune complex-initiated inflammation. Subsequent CHIPS toxicology studies in animal models were promising. However, during a small phase-I trial, healthy human volunteers showed adverse effects directly after CHIPS administration. Subjects showed clinical signs of anaphylaxis with mild leukocytopenia and increased C-reactive protein concentrations, suggesting an inflammatory response, which are believed to be related to the presence of relatively high circulating anti-CHIPS antibodies. Even though our data in mice shows CHIPS as a potential anti-inflammatory agent, safety issues in human subjects temper the use of CHIPS in its current form as a therapeutic candidate. The use of staphylococcal proteins, or other bacterial proteins, as therapeutics or immune-modulators in humans is severely hampered by pre-existing circulating antibodies.

microbiology↗

Staphylococcal superantigen-like protein 13 activates neutrophils via Formyl Peptide Receptor 2

Staphylococcal Superantigen-Like (SSL) proteins, one of major virulence factor families produced by Staphylococcus aureus, were previously demonstrated to be immune evasion molecules that interfere with a variety of innate immune defenses. However, in contrast to these characterized SSLs, that inhibit immune functions, we show that SSL13 is a strong activator of neutrophils via the formyl-peptide receptor 2 (FPR2). Moreover, our data show that SSL13 acts as a chemoattractant, induces degranulation and oxidative burst in neutrophils. As with many other staphylococcal immune evasion proteins, SSL13 shows a high degree of human specificity. SSL13 is not able to efficiently activate mouse neutrophils, hampering in vivo experiments.\n\nIn conclusion, SSL13 is a neutrophil chemoattractant and activator that acts via the FPR2. Therefore, SSL13 is a unique SSL member that does not belong to the immune evasion class, but is a pathogen alarming molecule.

microbiology↗

Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration

backgroundStaphylococcus aureus Panton Valentine Leukocidin (PVL) is a pore-forming toxin targeting the human C5a receptor (hC5aR), enabling this pathogen to battle the immune response by destroying phagocytes through targeted lysis. The mechanisms that contribute to rapid cell lysis are largely unexplored.\n\nResultsHere we show that cell lysis may be enabled by a process of toxins targeting receptor clusters and receptor recycling which allows multiple toxin pores to be formed close together. Using live cell single-molecule super-resolution imaging, Forster resonance energy transfer (FRET) and nanoscale total internal reflection fluorescence (TIRF) colocalization microscopy we visualized toxin pore formation in the presence of its natural docking ligand.\n\nConclusionsWe demonstrate disassociation of hC5aR from toxin complexes and simultaneous binding of new ligands. This effect may free mobile receptors to amplify hyper inflammatory reactions in early stages of microbial infections and have implications for several other similar bi-component toxins and the design of new antibiotics.

microbiology↗

Characterization Of The Stoichiometry Of The Complex Formed By Staphylococcal LukSF And Human C5aR Receptor In Living Cells

Staphylococcus aureus Panton Valentine Leukocidin (PVL) is a pore-forming toxin comprising protein subunits LukS and LukF. Binding of LukS to human C5a receptor (hC5aR) on leukocytes induces secondary binding of LukF and assembly of lytic complexes. Previous analysis suggests that PVL consists of 4-plus-4 LukS/LukF subunits but the exact stoichiometry between LukS, LukF and hC5aR is not yet known. In this study we determine the stoichiometry and spatiotemporal dynamics of functional LukS/LukF-hC5aR complexes in living eukaryotic cells. By using rapid total internal reflection fluorescence (TIRF) and single-molecule photobleaching analysis we found that tetrameric LukS-hC5aR complexes are formed within a cluster of receptors. Upon binding to hC5aR each LukS subunit binds LukF leading to lytic pore formation and simultaneous dissociation of receptors from the complex. Our findings corroborate a hetero-octamer model but provide a new view on the kinetics of crucial virulence factor assembly on integrated host cell membrane receptors.

biophysics↗