Maartje Ruyken

Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases

The complement system is pivotal in host defense but also contributes to tissue injury in several diseases. The assembly of C3 convertases (C4b2a and C3bBb) is a prerequisite for complement activation. The convertases catalyze C3b deposition on activator surfaces. Here we describe the identification of staphylococcal complement inhibitor, an excreted 9.8-kilodalton protein that blocks human complement by specific interaction with C4b2a and C3bBb. Staphylococcal complement inhibitor bound and stabilized C3 convertases, interfering with additional C3b deposition through the classical, lectin and alternative complement pathways. This led to a substantial decrease in phagocytosis and killing of Staphylococcus aureus by human neutrophils. As a highly active and small soluble protein that acts exclusively on surfaces, staphylococcal complement inhibitor may represent a promising anti-inflammatory molecule.

Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor

Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg2+-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.

Staphylococcus aureus Metalloprotease Aureolysin Cleaves Complement C3 To Mediate Immune Evasion

Complement is one of the first host defense barriers against bacteria. Activated complement attracts neutrophils to the site of infection and opsonizes bacteria to facilitate phagocytosis. The human pathogen Staphylococcus aureus has successfully developed ways to evade the complement system, for example by secretion of specific complement inhibitors. However, the influence of S. aureus proteases on the host complement system is still poorly understood. In this study, we identify the metalloprotease aureolysin as a potent complement inhibitor. Aureolysin effectively inhibits phagocytosis and killing of bacteria by neutrophils. Furthermore, we show that aureolysin inhibits the deposition of C3b on bacterial surfaces and the release of the chemoattractant C5a. Cleavage analyses show that aureolysin cleaves the central complement protein C3. Strikingly, there was a clear difference between the cleavages of C3 in serum versus purified conditions. Aureolysin cleaves purified C3 specifically in the α-chain, close to the C3 convertase cleavage site, yielding active C3a and C3b. However, in serum we observe that the aureolysin-generated C3b is further degraded by host factors. We pinpointed these factors to be factor H and factor I. Using an aureolysin mutant in S. aureus USA300, we show that aureolysin is essential and sufficient for C3 cleavage by bacterial supernatant. In short, aureolysin acts in synergy with host regulators to inactivate C3 thereby effectively dampening the host immune response.

Early expression of SCIN and CHIPS drives instant immune evasion by Staphylococcus aureus

Chemotaxis inhibitory protein of staphylococci (CHIPS) and Staphylococcal complement inhibitor (SCIN) are small, excreted molecules that play a crucial role in the staphylococcal defence against the human innate immune system. Here we show that they both counteract crucial acute responses of our immune system such as complement activation, neutrophil chemotaxis and neutrophil activation. By studying gene expression via promoter–green fluorescent protein fusions, Northern blots and protein expression analyses, we show that SCIN and CHIPS are produced during the early (exponential) growth stages. Although the SCIN and CHIPS genes are expressed simultaneously, they are differently regulated by various Staphylococcus aureus regulatory loci. However, the sae locus is crucial for upregulation of both SCIN and CHIPS. This is the first study that presents the expression of two extracellular S. aureus proteins early during growth. Because SCIN and CHIPS are both efficient modulators of neutrophil chemotaxis, phagocytosis and killing, their early expression is necessary for efficient modulation of the early immune response.

Pseudomonas aeruginosa Alkaline Protease Blocks Complement Activation via the Classical and Lectin Pathways

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.

Staphylococcal Complement Inhibitor: Structure and Active Sites

The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 Å resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31—Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

Significance Neutrophils are among the first immune cells to migrate to the site of infection and clear invading bacteria. They store large amounts of neutrophil serine proteases (NSPs) that play key roles in immune defense. Unfortunately, NSPs also contribute to tissue destruction in a variety of inflammatory disorders. In this study we discover that the pathogenic bacterium Staphylococcus aureus secretes a family of highly potent and specific NSP inhibitors that promote the pathogenicity of this bacterium in vivo. From crystallography experiments, we conclude that these proteins constitute a unique class of NSP inhibitors, which can be used to design novel treatment strategies against excessive NSP activity. Furthermore, this study significantly increases our understanding of the complex nature of S. aureus infections. Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity.

Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

The CXC chemokine receptor 2 (CXCR2) on neutrophils, which recognizes chemokines produced at the site of infection, plays an important role in antimicrobial host defenses such as neutrophil activation and chemotaxis. Staphylococcus aureus is a successful human pathogen secreting a number of proteolytic enzymes, but their influence on the host immune system is not well understood. Here, we identify the cysteine protease Staphopain A as a chemokine receptor blocker. Neutrophils treated with Staphopain A are unresponsive to activation by all unique CXCR2 chemokines due to cleavage of the N‐terminal domain, which can be neutralized by specific protease inhibitors. Moreover, Staphopain A inhibits neutrophil migration towards CXCR2 chemokines. By comparing a methicillin‐resistant S. aureus (MRSA) strain with an isogenic Staphopain A mutant, we demonstrate that Staphopain A is the only secreted protease with activity towards CXCR2. Although the inability to cleave murine CXCR2 limits in‐vivo studies, our data indicate that Staphopain A is an important immunomodulatory protein that blocks neutrophil recruitment by specific cleavage of the N‐terminal domain of human CXCR2.

Staphylococcus aureus Virulence Is Enhanced by Secreted Factors That Block Innate Immune Defenses

Staphylococcus aureus is a leading human pathogen that causes a large variety of diseases. In vitro studies have shown that S. aureus secretes several small proteins that block specific elements of the host innate immune system, but their role in bacterial pathogenicity is unknown. For instance, the extracellular complement-binding protein (Ecb) impairs complement activation by binding to the C3d domain of C3. Its homolog, the extracellular fibrinogen-binding protein (Efb), is known to block both complement activation and neutrophil adhesion to fibrinogen. Here, we show that targeted inactivation of the genes encoding Ecb and Efb strongly attenuates S. aureus virulence in a murine infection model: mice experienced significantly higher mortality rates upon intravenous infection with wild-type bacteria (79%) than with an isogenic ΔEcbΔEfb mutant (21%). In addition, Ecb and Efb are both required for staphylococcal persistence in host tissues and abscess formation in the kidneys (27% for wild-type vs. 7% for the ΔEcbΔEfb mutant). During staphylococcal pneumonia, Ecb and Efb together promote bacterial survival in the lungs (p = 0.03) and block neutrophil influx into the lungs. Thus, Ecb and Efb are essential to S. aureus virulence in vivo and could be attractive targets in future vaccine development efforts.

Staphylococcal Complement Inhibitor Modulates Phagocyte Responses by Dimerization of Convertases

The human pathogen Staphylococcus aureus produces several complement-evasion molecules that enable the bacterium to withstand the host immune response. The human-specific staphylococcal complement inhibitor (SCIN) blocks the central C3 convertase enzymes that trigger critical complement functions, such as C3b deposition, phagocytosis, and C5a generation. SCIN effectively blocks the conversion of C3 by alternative pathway C3 convertases (C3bBb), but also induces dimerization of these enzymes. In this study, we show that formation of dimeric convertases by SCIN is important for S. aureus immune evasion because it modulates complement recognition by phagocytic receptors. Dimeric, but not monomeric, SCIN convertases showed an impaired binding to complement receptor 1 and the complement receptor of the Ig superfamily. The dimerization site of SCIN is essential for its strong antiphagocytic properties. These studies provide critical insights into the unique immune-evasion strategies used by S. aureus.