Photoimmuno-antimicrobial therapy for Staphylococcus aureus implant infection.

INTRODUCTION: Implant infections caused by Staphylococcus aureus are responsible for high mortality and morbidity worldwide. Treatment of these infections can be difficult especially when bacterial biofilms are involved. In this study we investigate the potential of infrared photoimmunotherapy to eradicate staphylococcal infection in a mouse model. METHODS: A monoclonal antibody that targets Wall Teichoic Acid surface components of both S. aureus and its biofilm (4497-IgG1) was conjugated to a photosensitizer (IRDye700DX) and used as photoimmunotherapy in vitro and in vivo in mice with a subcutaneous implant pre-colonized with biofilm of Staphylococcus aureus. A dose of 400 µg and 200 µg of antibody-photosensitizer conjugate 4497-IgG-IRDye700DXwas administered intravenously to two groups of 5 mice. In addition, multiple control groups (vancomycin treated, unconjugated IRDye700DX and IRDye700DX conjugated to a non-specific antibody) were used to verify anti-microbial effects. RESULTS: In vitro results of 4497-IgG-IRDye700DX on pre-colonized (biofilm) implants showed significant (p<0.01) colony-forming units (CFU) reduction at a concentration of 5 µg of the antibody-photosensitizer conjugate. In vivo, treatment with 4497-IgG-IRDye700DX showed no significant CFU reduction at the implant infection. However, tissue around the implant did show a significant CFU reduction with 400 µg 4497-IgG-IRDye700DX compared to control groups (p = 0.037). CONCLUSION: This study demonstrated the antimicrobial potential of photoimmunotherapy for selectively eliminating S. aureus in vivo. However, using a solid implant instead of a catheter could result in an increased bactericidal effect of 4497-IgG-IRDye700DX and administration locally around an implant (per operative) could become valuable applications in patients that are difficult to treat with conventional methods. We conclude that photoimmunotherapy could be a potential additional therapy in the treatment of implant related infections, but requires further improvement.

CD5L is a canonical component of circulatory IgM.

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing.

Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen-antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM's enhanced capacity over IgG to induce complement-mediated lysis of E. coli.

Promoting Fc-Fc interactions between anti-capsular antibodies provides strong immune protection against Streptococcus pneumoniae.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and an important cause of childhood mortality. Despite the introduction of successful vaccines, the global spread of both non-vaccine serotypes and antibiotic-resistant strains reinforces the development of alternative therapies against this pathogen. One possible route is the development of monoclonal antibodies (mAbs) that induce killing of bacteria via the immune system. Here, we investigate whether mAbs can be used to induce killing of pneumococcal serotypes for which the current vaccines show unsuccessful protection. Our study demonstrates that when human mAbs against pneumococcal capsule polysaccharides (CPS) have a poor capacity to induce complement activation, a critical process for immune protection against pneumococci, their activity can be strongly improved by hexamerization-enhancing mutations. Our data indicate that anti-capsular antibodies may have a low capacity to form higher-order oligomers (IgG hexamers) that are needed to recruit complement component C1. Indeed, specific point mutations in the IgG-Fc domain that strengthen hexamerization strongly enhance C1 recruitment and downstream complement activation on encapsulated pneumococci. Specifically, hexamerization-enhancing mutations E430G or E345K in CPS6-IgG strongly potentiate complement activation on S. pneumoniae strains that express capsular serotype 6 (CPS6), and the highly invasive serotype 19A strain. Furthermore, these mutations improve complement activation via mAbs recognizing CPS3 and CPS8 strains. Importantly, hexamer-enhancing mutations enable mAbs to induce strong opsonophagocytic killing by human neutrophils. Finally, passive immunization with CPS6-IgG1-E345K protected mice from developing severe pneumonia. Altogether, this work provides an important proof of concept for future optimization of antibody therapies against encapsulated bacteria.

Monoclonal antibodies effectively potentiate complement activation and phagocytosis of Staphylococcus epidermidis in neonatal human plasma.

Central line associated bloodstream infections (CLABSI) with Staphylococcus epidermidis are a major cause of morbidity in neonates, who have an increased risk of infection because of their immature immune system. As especially preterm neonates suffer from antibody deficiency, clinical studies into preventive therapies have thus far focused on antibody supplementation with pooled intravenous immunoglobulins from healthy donors (IVIG) but with little success. Here we study the potential of monoclonal antibodies (mAbs) against S. epidermidis to induce phagocytic killing by human neutrophils. Nine different mAbs recognizing Staphylococcal surface components were cloned and expressed as human IgG1s. In binding assays, clones rF1, CR5133 and CR6453 showed the strongest binding to S. epidermidis ATCC14990 and CR5133 and CR6453 bound the majority of clinical isolates from neonatal sepsis (19 out of 20). To study the immune-activating potential of rF1, CR5133 and CR6453, bacteria were opsonized with mAbs in the presence or absence of complement. We observed that activation of the complement system is essential to induce efficient phagocytosis of S. epidermidis. Complement activation and phagocytic killing could be enhanced by Fc-mutations that improve IgG1 hexamerization on cellular surfaces. Finally, we studied the ability of the mAbs to activate complement in r-Hirudin neonatal plasma conditions. We show that classical pathway complement activity in plasma isolated from neonatal cord blood is comparable to adult levels. Furthermore, mAbs could greatly enhance phagocytosis of S. epidermidis in neonatal plasma. Altogether, our findings provide insights that are crucial for optimizing anti-S. epidermidis mAbs as prophylactic agents for neonatal CLABSI.

Toward Understanding How Staphylococcal Protein A Inhibits IgG-Mediated Phagocytosis.

IgG molecules are crucial for the human immune response against bacterial infections. IgGs can trigger phagocytosis by innate immune cells, like neutrophils. To do so, IgGs should bind to the bacterial surface via their variable Fab regions and interact with Fcγ receptors and complement C1 via the constant Fc domain. C1 binding to IgG-labeled bacteria activates the complement cascade, which results in bacterial decoration with C3-derived molecules that are recognized by complement receptors on neutrophils. Next to FcγRs and complement receptors on the membrane, neutrophils also express the intracellular neonatal Fc receptor (FcRn). We previously reported that staphylococcal protein A (SpA), a key immune-evasion protein of Staphylococcus aureus, potently blocks IgG-mediated complement activation and killing of S. aureus by interfering with IgG hexamer formation. SpA is also known to block IgG-mediated phagocytosis in absence of complement, but the mechanism behind it remains unclear. In this study, we demonstrate that SpA blocks IgG-mediated phagocytosis and killing of S. aureus and that it inhibits the interaction of IgGs with FcγRs (FcγRIIa and FcγRIIIb, but not FcγRI) and FcRn. Furthermore, our data show that multiple SpA domains are needed to effectively block IgG1-mediated phagocytosis. This provides a rationale for the fact that SpA from S. aureus contains four to five repeats. Taken together, our study elucidates the molecular mechanism by which SpA blocks IgG-mediated phagocytosis and supports the idea that in addition to FcγRs, the intracellular FcRn is also prevented from binding IgG by SpA.

Correction: Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores.

[This corrects the article DOI: 10.1371/journal.ppat.1010051.].

Human monoclonal antibodies against Staphylococcus aureus surface antigens recognize in vitro and in vivo biofilm.

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and potential treatment of biofilm-related infections. Here, we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. The mAbs were also shown to bind a collection of clinical isolates derived from different biofilm-associated infections (endocarditis, prosthetic joint, catheter). We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. Furthermore, we show that a mAb recognizing wall teichoic acid (clone 4497) specifically localizes to a subcutaneously implanted pre-colonized catheter in mice. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo.

Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores.

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and 'locked' C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

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 antibody's 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-Fc 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 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes 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 the 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.

Method for Depletion of IgG and IgM from Human Serum as Naive Complement Source.

Understanding how human complement proteins interact with human antibodies is important for the development of antibody therapies and understanding autoimmune diseases. At present, many groups use baby rabbit serum as a source of complement because, in contrast to human serum, it lacks preexisting antibodies. However, for characterization of human (monoclonal) antibodies, human serum would be a preferred source of complement. To prevent complement activation via naturally occurring antibodies, this human serum ideally lacks IgG and IgM. Here we describe how to deplete human serum of naturally occurring IgG and IgM using fast protein liquid affinity chromatography (FPLC) while minimizing the loss of serum complement activity. We also describe assays that can be used to validate depletion of IgG and IgM (IgG, IgM, and C1q sandwich ELISAs) and functionally assess remaining serum complement activity (hemolytic assays CH50 and AH50). Finally, we demonstrate how captured IgG and IgM can be purified.

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

Immunoglobulin (Ig) G 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 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. Finally, we demonstrate that IgG3 antibodies against S. aureus can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.

Impact of Glycan Linkage to Staphylococcus aureus Wall Teichoic Acid 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 invading S. aureus 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 proinflammatory response. Langerin recognizes the ß-1,4-linked N-acetylglucosamine (ß1,4-GlcNAc) but not α-1,4-linked GlcNAc (α1,4-GlcNAc) modifications, which are added by dedicated glycosyltransferases TarS and TarM, respectively, on the cell wall glycopolymer wall teichoic acid (WTA). Recently, an alternative WTA glycosyltransferase, TarP, was identified, which also modifies WTA with ß-GlcNAc but at the C-3 position (ß1,3-GlcNAc) of the WTA ribitol phosphate (RboP) subunit. Here, we aimed to unravel the impact of ß-GlcNAc linkage position for langerin binding and LC activation. Using genetically modified S. aureus strains, we observed that langerin similarly recognized bacteria that produce either TarS- or TarP-modified WTA, yet tarP-expressing S. aureus induced increased cytokine production and maturation of in vitro-generated LCs compared to tarS-expressing S. aureus. Chemically synthesized WTA molecules, representative of the different S. aureus WTA glycosylation patterns, were used to identify langerin-WTA binding requirements. We established that ß-GlcNAc is sufficient to confer langerin binding, thereby presenting synthetic WTA molecules as a novel glycobiology tool for structure-binding studies and for elucidating S. aureus molecular pathogenesis. Overall, our data suggest that LCs are able to sense all ß-GlcNAc-WTA producing S. aureus strains, likely performing an important role as first responders upon S. aureus skin invasion.

The Orphan Immune Receptor LILRB3 Modulates Fc Receptor-Mediated Functions of Neutrophils.

Neutrophils are critical to the generation of effective immune responses and for killing invading microbes. Paired immune receptors provide important mechanisms to modulate neutrophil activation thresholds and effector functions. Expression of the leukocyte Ig-like receptor (LILR)A6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) paired-receptor system on human neutrophils has remained unclear because of the lack of specific molecular tools. Additionally, there is little known of their possible functions in neutrophil biology. The objective of this study was to characterize expression of LILRA6/LILRB3 receptors during human neutrophil differentiation and activation, and to assess their roles in modulating Fc receptor-mediated effector functions. LILRB3, but not LILRA6, was detected in human neutrophil lysates following immunoprecipitation by mass spectrometry. We demonstrate high LILRB3 expression on the surface of resting neutrophils and release from the surface following neutrophil activation. Surface expression was recapitulated in a human PLB-985 cell model of neutrophil-like differentiation. Continuous ligation of LILRB3 inhibited key IgA-mediated effector functions, including production of reactive oxygen species, phagocytic uptake, and microbial killing. This suggests that LILRB3 provides an important checkpoint to control human neutrophil activation and their antimicrobial effector functions during resting and early-activation stages of the neutrophil life cycle.

Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models.

Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative pathway (AP) C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by AP convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases [C3bBb(C3b)n] is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a new classical pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases but also inhibit formation of a functional classical pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.

Identification of a staphylococcal complement inhibitor with broad host specificity in equid Staphylococcus aureus strains.

Staphylococcus aureus is a versatile pathogen capable of causing a broad range of diseases in many different hosts. S. aureus can adapt to its host through modification of its genome (e.g. by acquisition and exchange of mobile genetic elements that encode host-specific virulence factors). Recently, the prophage φSaeq1 was discovered in S. aureus strains from six different clonal lineages almost exclusively isolated from equids. Within this phage, we discovered a novel variant of staphylococcal complement inhibitor (SCIN), a secreted protein that interferes with activation of the human complement system, an important line of host defense. We here show that this equine variant of SCIN, eqSCIN, is a potent blocker of equine complement system activation and subsequent phagocytosis of bacteria by phagocytes. Mechanistic studies indicate that eqSCIN blocks equine complement activation by specific inhibition of the C3 convertase enzyme (C3bBb). Whereas SCIN-A from human S. aureus isolates exclusively inhibits human complement, eqSCIN represents the first animal-adapted SCIN variant that functions in a broader range of hosts (horses, humans, and pigs). Binding analyses suggest that the human-specific activity of SCIN-A is related to amino acid differences on both sides of the SCIN-C3b interface. These data suggest that modification of this phage-encoded complement inhibitor plays a role in the host adaptation of S. aureus and are important to understand how this pathogen transfers between different hosts.

LukMF' is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis.

Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal leukocidins in the pathogenesis of bovine S. aureus disease. We show that LukAB, in contrast to the γ-hemolysins, LukED, and LukMF', was unable to kill bovine neutrophils, and identified CXCR2 as a bovine receptor for HlgAB and LukED. Furthermore, we assessed functional leukocidin secretion by bovine mastitis isolates and observed that, although leukocidin production was strain dependent, LukMF' was most abundantly secreted and the major toxin killing bovine neutrophils. To determine the role of LukMF' in bovine mastitis, cattle were challenged with high (S1444) or intermediate (S1449, S1463) LukMF'-producing isolates. Only animals infected with S1444 developed severe clinical symptoms. Importantly, LukM was produced in vivo during the course of infection and levels in milk were associated with the severity of mastitis. Altogether, these findings underline the importance of LukMF' as a virulence factor and support the development of therapeutic approaches targeting LukMF' to control S. aureus mastitis in cattle.

Molecular insights into the surface-specific arrangement of complement C5 convertase enzymes.

BACKGROUND: Complement is a large protein network in plasma that is crucial for human immune defenses and a major cause of aberrant inflammatory reactions. The C5 convertase is a multi-molecular protease complex that catalyses the cleavage of native C5 into its biologically important products. So far, it has been difficult to study the exact molecular arrangement of C5 convertases, because their non-catalytic subunits (C3b) are covalently linked to biological surfaces through a reactive thioester. Through development of a highly purified model system for C5 convertases, we here aim to provide insights into the surface-specific nature of these important protease complexes. RESULTS: Alternative pathway (AP) C5 convertases were generated on small streptavidin beads that were coated with purified C3b molecules. Site-specific biotinylation of C3b via the thioester allowed binding of C3b in the natural orientation on the surface. In the presence of factor B and factor D, these C3b beads could effectively convert C5. Conversion rates of surface-bound C3b were more than 100-fold higher than fluid-phase C3b, confirming the requirement of a surface. We determine that high surface densities of C3b, and its attachment via the thioester, are essential for C5 convertase formation. Combining our results with molecular modeling explains how high C3b densities may facilitate intermolecular interactions that only occur on target surfaces. Finally, we define two interfaces on C5 important for its recognition by surface-bound C5 convertases. CONCLUSIONS: We establish a highly purified model that mimics the natural arrangement of C5 convertases on a surface. The developed model and molecular insights are essential to understand the molecular basis of deregulated complement activity in human disease and will facilitate future design of therapeutic interventions against these critical enzymes in inflammation.

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3).

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.

The staphylococcal toxin Panton-Valentine Leukocidin targets human C5a receptors.

Panton-Valentine Leukocidin (PVL) is a staphylococcal bicomponent pore-forming toxin linked to severe invasive infections. Target-cell and species specificity of PVL are poorly understood, and the mechanism of action of this toxin in Staphylococcus aureus virulence is controversial. Here, we identify the human complement receptors C5aR and C5L2 as host targets of PVL, mediating both toxin binding and cytotoxicity. Expression and interspecies variations of the C5aR determine cell and species specificity of PVL. The C5aR binding PVL component, LukS-PV, is a potent inhibitor of C5a-induced immune cell activation. These findings provide insight into leukocidin function and staphylococcal virulence and offer directions for future investigations into individual susceptibility to severe staphylococcal disease.