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Approximately 30% of the human population is contin- uously colonized with Staphylococcus aureus, whereas some individuals are hosts for intermittent colonization^1. S. aureus typically resides in the nares but is also found on the skin and in the gastrointestinal tract. Although colonization is not a prerequisite for staphylococcal disease, colonized individuals more frequently acquire infections^1. Skin and soft-tissue infections (SSTIs) are the most frequent disease form of S. aureus, and these infections can progress to bacteraemia and invasive disease (that is, bloodstream infection, endocarditis or sepsis^2 ). In addition, S. aureus can cause pneumonia, osteomyelitis, infectious arthritis, abscesses in many organ tissues and infections of surgical wounds or prosthetic materials^2. Annual attack rates for S. aureus disease range between 1–3% and vary with age, ethnic- ity and geographical location of human populations^2. At elevated risk for staphylococcal infection are low- birth-weight infants, children, the elderly and patients with indwelling catheters, endotracheal intubation, medical implantation of foreign bodies, trauma, surgical procedures, haemodialysis, diabetes or immunosuppres- sive or cancer therapy^2. A key feature of S. aureus disease is its recurrence, which occurs for 8–33% of cases of SSTI and bloodstream infections^3. Prior disease does not elicit protection against subsequent S. aureus infection^2. Neutrophils play a central part in protecting humans against S. aureus infection. Staphylococcal entry and replication in host tissues leads to the release of bacterial products (formyl-peptides, lipoproteins or peptidogly- can) and to damaged tissues that produce inflammatory signals (that is, chemoattractants and cytokines^4 ). Staphylococcal products are detected by immune cells via Toll-like receptors (TLRs) and G protein-coupled receptors, whereas cytokines activate cognate immune receptors. Neutrophils answer this call, extravasate from blood vessels, and migrate towards the site of infection to phagocytose and kill bacteria or to immobilize and damage the pathogen through NETosis — the release of neutrophil extracellular traps (NETs) comprising DNA and antimicrobial peptides^4. The importance of neutro- phils in controlling S. aureus infection has been docu- mented through the study of immune defects. Mutations in genes encoding NADPH oxidase, the enzyme generat- ing bactericidal superoxide in phagocytes, cause chronic granulomatous disease (CGD), which is associated with defects in phagocytic killing of S. aureus and frequent infection^5. Individuals with inborn errors of signal trans- ducer and activator of transcription 1 (STAT1) or STAT signalling of immune cells have perturbed interleukin- 17 (IL-17) cytokine pathways, which diminishes mucocu- taneous immunity and promotes S. aureus infection^6. IL- 17 - dependent T cell signalling is a key activator of neutrophils and of anti-staphylococcal defences^7. Finally, cancer patients with diminished blood neutrophil counts are highly susceptible to S. aureus infection^8. Nevertheless, the vast majority of S. aureus disease occurs in immune-competent individuals without defects in phagocyte function. To achieve this, S. aureus deploys an arsenal of immune-evasive strategies that together prevent phagocytosis and killing by neutro- phils. Furthermore, the ability of the pathogen to cause Abscesses The pathological product of Staphylococcus aureus infection: the harbouring of a staphylococcal abscess community within a pseudocapsule of fibrin deposits that is surrounded by layers of infiltrating immune cells destroying physiological organ tissue. Recurrence The propensity of S. aureus infections to reoccur when surgery and/or antibiotic therapy are initially effective.
Staphylococcal manipulation of host
immune responses
Vilasack Thammavongsa1,2, Hwan Keun Kim^1 , Dominique Missiakas^1 and
Olaf Schneewind^1
Abstract | Staphylococcus aureus , a bacterial commensal of the human nares and skin, is a
frequent cause of soft tissue and bloodstream infections. A hallmark of staphylococcal
infections is their frequent recurrence, even when treated with antibiotics and surgical
intervention, which demonstrates the bacterium’s ability to manipulate innate and adaptive
immune responses. In this Review, we highlight how S. aureus virulence factors inhibit
complement activation, block and destroy phagocytic cells and modify host B cell and
T cell responses, and we discuss how these insights might be useful for the development
of novel therapies against infections with antibiotic resistant strains such as
methicillin-resistant S. aureus.
(^1) Department of Microbiology, University of Chicago, 920 East 58th^ Street, Chicago, Illinois 60637, USA. (^2) Regeneron Pharmaceuticals, 755 Old Saw Mill River Road, Tarrytown, New York 10591, USA. Correspondence to O.S. e‑mail: oschnee@bsd.uchicago.edu doi:10.1038/nrmicro NATURE REVIEWS | MICROBIOLOGY VOLUME 13 | SEPTEMBER 2015 | 529
Superantigen Molecules that crosslink B cell receptors (that is, IgM) or T cell receptors and major histocompatibility complexes to trigger lymphocyte proliferation, thereby diverting adaptive immune responses. Anaphylatoxin Protein fragments generated during complement activation of C3a and C5a that trigger immune responses via C3a and C5a receptors on immune cells. recurrent disease implies the presence of mechanisms that effectively block the development of adaptive immune responses. Here, we review recent work on the immune evasive attributes of S. aureus, including the subversion of the innate and adaptive immune systems and the killing of immune cells, along with epidemiologi- cal features of the corresponding genes. We also discuss how the characterization of bacterial immune evasive factors can have translational effects in the therapy of autoimmune diseases or the development of vaccines and immunotherapeutics against S. aureus infection. Subversion of innate immune responses Neutrophil extravasation and chemotaxis. Pro- inflammatory signals promote neutrophil adhesion and extravasation across capillary endothelia, relying on reciprocal interactions between endothelial recep- tors (such as P-selectin, E selectin, intercellular adhe- sion molecule 1 (ICAM1) and hyaluronan) and ligands on neutrophil surfaces (such as P-selectin glycoprotein ligand 1 (PSGL1), lymphocyte function-associated antigen 1 (LFA1; also known as αLβ2 integrin), αMβ 2 integrin and CD44)^9. Although neutrophils function to migrate towards bacterial invaders, S. aureus can interfere with neutrophil extravasation and chemotaxis through the secretion of staphylococcal superantigen-like proteins (SSLs), phenol-soluble modulins (PSMs), chemotaxis inhibitory protein of S. aureus (CHIPS), formyl pep- tide receptor-like 1 inhibitor (FLIPr) and its homologue FLIPr-like (FLIPrL). SSLs are a family of secreted proteins with structural homology to staphylococcal superantigens^10 –^12. The ssl genes are arranged as tandem repeats in genomic island-α (GIα; for ssl1–ssl11) and in the immune evasion cluster 2 (IEC2; for ssl12–ssl14) on the bacterial chromosome^13. GIα-encoded ssl genes vary between lineages as does the coding sequence of individual ssl genes; the number of different alleles ranges from 1 to 13 and most alleles are uniquely associated with specific S. aureus lineages^13. ssl1, ssl2, ssl3, ssl11, ssl12, ssl13 and ssl14 are found in all S. aureus isolates^13 (BOX 1). Purified, recombinant SSL and SSL11 bind PSGL1 on leukocytes and, when assayed in vitro, interfere with the binding of neutrophils to P-selectin and neutrophil adhesion and rolling^14 ,^15 (FIG. 1a). SSL5 also interferes with chemokine- and anaphylatoxin- mediated activation of neutrophils by binding to the glyco- sylated amino termini of G protein-coupled receptors^14 ,^16. Moreover, SSL5 has been shown to activate platelets and support their adhesion involving platelet surface receptors GPIbα and GPVI^17 ,^18. Intravenous administration of SSL caused intravascular platelet-rich thrombi and increased bleeding of C57Bl/6 mice^19. Other work demonstrated SSL5-mediated inactivation of leukocyte matrix metallo- proteinase 9 (REF. 20 ). The affinity of SSL5 for different host factors is mediated via its glycan binding pocket, an attribute that is shared by other members of the SSL fam- ily^21. SSL3 binds to TLR2 and blocks immune cell recogni- tion of staphylococcal lipoproteins and peptidoglycan via TLR1–TLR2 and TLR2–TLR6 heterodimers^22 , and SSL blocks C-X-C chemokine receptor 4 (CXCR4)-mediated Box 1 | Variability of Staphylococcus aureus immune evasion determinants Genome sequencing of Staphylococcus aureus isolates from humans and animals has provided insights into the origin, diversification and spread of the pathogen. Over the past 10,000 years, S. aureus evolved as a colonizer and pathogen of humans and their lifestock^142 , generating lineages with unique genetic traits and discrete host ranges^143. Staphylococcal evolution was accompanied by the loss of genes encoding the CRISPR–Cas system, which protect the genome against phages and mobile genetic elements. S. aureus relies on horizontal gene transfer mediated by these elements for adaptation, and it preserves its identity through restriction modification systems and satellite phage-encoded pathogenicity islands that block phage replication^143. When placed under selection in different hosts, S. aureus acquires mobile genetic elements that contain genes for antibiotic resistance, immune evasion and adhesion to specific anatomic niches. Analysis of large genome datasets described the core genome, which is common to all S. aureus isolates, and found that these core genes contribute to colonization, tissue invasion, establishment of abscess lesions, dissemination, immune evasion and pathogenesis of reiterative disease^36. Variable genes are associated with S. aureus colonization or invasion of specific host species or may be present in subsets of strains associated with increased virulence or specific disease (for example, enterotoxin-mediated gastroenteritis)^93. Generally, capsular polysaccharide and cell wall-anchored surface proteins are components of the core genome and contribute to immune evasion by synthesizing adenosine (by adenosine synthase A), binding fibrinogen or fibrin (by clumping factors A and B and fibronectin binding proteins A and B) and binding immunoglobulin (by staphylococcal protein A (SpA)). Several secreted proteins are components of the core genome: proteases that cleave host factors (such as aureolysin and staphopain), coagulases that activate prothrombin (such as coagulase and von-Willebrand factor binding protein), toxins that lyse immune cells (such asγ-haemolysin AB, γ-haemolysin CB and leukocidin AB), inhibitors of host proteases (staphylococcal extracellular adherence protein and its homologues) and phenol-soluble modulins, peptides that perturb host cell membranes and trigger neutrophil chemotaxis. Genetic determinants that interfere with neutrophil chemotaxis, phagocytosis, complement activation, promote lysis of immune cells or activate T cells often represent constituents of the variable genome.TABLE 1 summarizes core genome and variable immune evasion factors contributing to staphylococcal disease pathogenesis.Isolates of the CC75 lineage are predominantly found in the South-West Pacific and were originally isolated from superficial skin lesions of individuals from the indigenous communities of Australia^144. CC75 strains lack the staphyloxanthin gene cluster, retain the CRISPR–Cas system and lack S. aureus pathogenicity islands (SaPIs), but they are endowed with genomic island-α and genomic island-β, a unique coa gene and a unique spa sequence type, which are elements important for staphylococcal evasion of innate and adaptive immune responses^144. These strains, with the species designation Staphylococcus argenteus , may represent an early and terminal branch in the development of S. aureus in which mobile genetic elements were not incorporated into the genome because the retained CRISPR–Cas system prevented horizontal gene transfer^144. 530 | SEPTEMBER 2015 | VOLUME 13 www.nature.com/reviews/micro
Staphopain a Inhibition of neutrophil extravasation and chemotaxis b Inhibition of complement and phagocytosis c Inhibition of neutrophil-mediated killing d Staphyococcal killing of host cells FLIPr CHIPS C5aR FPR FPR TLR1–2 CXCR TLR2– PSGL Neutrophil Neutrophil cytoplasm Neutrophil T cell Macrophage Red blood cells Phagosome CD S. aureus S. aureus S. aureus CHIPS SSL3 SSL5 (^) SelX SSL C3b (^) C4b C3b C C C1q C1r Zn 2+ fH fI C3a + SCIN SpA Efb Ecb Eap Aur C3d C3d (^) fH C3 convertase Peptidoglycan Immunoglobulin Capsule FLIPr-L Lysozyme Nitric oxide synthase NADPH oxidase ROS
- ++ +^ NO • C3b C3b C C C SSL Sak Sbi SSL Cna AdsA OatA KatG AhpC SodA SodM Ldh Hmp DltA-D MprF Aur Eap EapH1 or EapH LukED LukED LukAB HlgAB Hla HlgAB PVL PSMα HlgCB LukED HlgAB PVL HlgCB Degranulation Antimicrobial peptides Serine protease H 2 O (^2) Adenosine AdoR A (^) A A PLG Nature Reviews | Microbiology A PAMP^ A P^ ADP ATP P A P P P 532 | SEPTEMBER 2015 | VOLUME 13 www.nature.com/reviews/micro
Fibrinogen An abundant glycoprotein of vertebrates that, when cleaved by thrombin or staphylothombin, self-assembles into fibrin clots. FcαRI The IgA Fc receptor, which regulates mucosal immune responses in humans. Fcγ domain The portion of antibodies dedicated to C1q complement and Fc-receptor activation. Core genome The portion of the genome shared by all members of a bacterial species. homologues of SCIN, designated SCIN-B and SCIN-C, are encoded by genes in the IEC2 locus. scn, scnB and scnC are found in many, but not all, human clinical isolates (BOX 1); SCIN, SCIN-B and SCIN-C associate with C3 convertase from humans but not with that of other vertebrates^43. The genes encoding extracellular fibrinogen- binding protein (Efb) and its homologue, extracellular complement-binding protein (Ecb), are also located on IEC2. Both Efb and Ecb bind to C3d (a cleavage prod- uct of C3b that activates innate and adaptive responses by binding to complement receptor 2 (CR2)) and inhibit C3bBb and the C5 convertases^43 ,^44 (FIG. 1b). Ecb associ- ates with both fH and C3b to facilitate the complement inhibitory attributes of fH^45. Efb also binds fibrinogen and prevents fibrinogen interaction with αMβ2, an integrin on neutrophils that activates pro-inflammatory responses, as well as fibrinogen-mediated platelet activation^46 ,^47. Efb and Ecb inhibitory activities have been observed for human and mouse convertases and fibrinogen. In the mouse intravenous challenge model, the S. aureus ΔefbΔecb mutant displayed reduced time-to-death and increased survival, as well as diminished abscess formation in organ tissues^48. The ecb gene is found in all S. aureus genomes sequenced to date, whereas efb is found in many, but not all, human clinical isolates^13. SSLs also interfere with complement activation and phagocytosis. For example, SSL7 binds to human IgA and complement C5, interfering with IgA binding to FcαRI, the production of C5a and the oxidative burst of phagocytes in vitro; the in vivo contributions of SSL towards S. aureus pathogenesis are not known^49. SSL binds to human and non-human primate IgG1, but not to immunoglobulins of lower vertebrates, and inhibits IgG1 binding to Fcγ receptors and the in vitro phago- cytosis of IgG1-opsonized bacteria by immune cells^50 ,^51. Staphylococcal binder of immunoglobulin (Sbi) is a secreted protein with two immunoglobulin binding domains (IgBDs; designated Sbi-I and Sbi-II), which are triple-helical bundles that associate with the Fcγ domain of human and vertebrate immunoglobulin (BOX 2). Sbi-I and Sbi-II interfere with C1q binding to immunoglobulin and block the classical complement pathway^52 ,^53 (FIG. 1b). The Sbi-III and Sbi-IV domains associate with C3 and fH to form tripartite complexes that inhibit the alternative pathway^54 ,^55 (FIG. 1b). The sbi gene is located in the sbi–hlg locus of the core genome of all isolates^13. Staphylokinase forms enzymatically active complexes with plasminogen, cleaving fibrin, defensins, human IgG, C3b and its prote- olytically inactivated product iC3b on bacterial surfaces, thereby blocking complement activation56–58^ (FIG. 1b). Collagen adhesin (Cna), a surface protein expressed by some S. aureus isolates, binds C1q and interferes with classical pathway activation, blocking the association between C1q and C1r^59. Neutrophil-mediated killing. Once phagocytosed, staphy- lococci are exposed to a variety of toxic products that kill and degrade the engulfed bacteria: antimicrobial pep- tides, nitric oxide (NO), ROS (that is, hydrogen peroxide, superoxide and hydroxyl radicals), cell wall hydrolases and proteolytic enzymes^4. However, S. aureus has evolved a number of strategies to survive in this environment (FIG. 1c). Peptidoglycan acetylation (by the protein OatA), d-alanylation of teichoic acids (by the DltABCD com- plex), and lysyl- or alanyl-phosphatidylglycerol synthesis (by the protein MprF) provide staphylococcal resistance against lysozyme- and antimicrobial peptide-mediated killing by blocking enzymes (such as lysozyme) or pep- tides binding to the envelope target60–62. Staphyloxanthin, a carotenoid pigment synthesized by all S. aureus iso- lates^63 , provides resistance against hydrogen peroxide and/or hydroxyl radicals, the bactericidal compounds of neutrophils^64 (this is not the case for CC75 isolates but we consider these to belong to a separate species, Staphylococcus argenteus (BOX 1)). Similarly, two super- oxide dismutases (SodA and SodM), fulfill overlapping functions in eliminating neutrophil superoxide^65 , whereas catalase (KatG) and alkylhydroperoxide reductase (AhpC) protect staphylococci against hydrogen peroxide^66. Figure 1 | Staphylococcus aureus interference with chemotaxis, complement and killing by phagocytes. a | Neutrophil extravasation and chemotaxis is inhibited by Staphylococcus aureus through the secretion of staphylococcal superantigen-like (SSL) molecules. SSL3 inhibits Toll-like receptor (TLR) heterodimers, SSL5, SSL11 and SelX inhibit PSGL1 signalling (SSL11 is not shown), and SSL6 inhibits the G protein-coupled receptor CD47. Other secreted proteins include chemotaxis inhibitory protein of S. aureus (CHIPS), which inhibits the complement receptor C5aR and formyl-peptide receptor 1 (FPR1) and FPR2, formyl peptide receptor-like 1 inhibitor (FLIPr) and FLIPr-like (FLIPrL), which inhibit FPR1 and FPR2, and staphopain, which inhibits signalling from the chemokine receptor C-X-C chemokine receptor (CXCR2). b | Complement activation and phagocytosis of staphylococci are blocked through the secretion of inhibitory factors that interfere with opsonization. Collagen adhesin (Cna) blocks the association of complement factor C1q bound to immunoglobulin with complement receptor C1r. Staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) binding to immunoglobulin blocks its association with C1q. Sbi, SpA, SSL7 and SSL sequester immunoglobulins to block their ability to promote complement activation. Sbi (when associated with the host factors C3d and factor H (fH)) and SSL7 also inactivate the complement factors C3 and C5, respectively. Sak associates with plasminogen (PLG) and activates the zymogen to cleave complement factor C3b and immunoglobulin. Extracellular complement-binding protein (Ecb), extracellular fibrinogen-binding protein (Efb), staphylococcal complement inhibitor (SCIN) and extracellular adherence protein (Eap) inhibit C3 convertases, and aureolysin (Aur) cleaves the complement factor C3, which compromises opsonization because the cleavage product C3b is degraded by a complex of the host proteins fI and fH. c | S. aureus inhibits neutrophil- mediated killing of phagocytosed bacteria by expressing several enzymes and inhibitors. The adenosine-synthesizing enzyme AdsA enables the inhibition of granulation via adenosine receptor (AdoR) signalling. Staphyloxanthin, superoxide dismutase A (SodA) and SodM, the catalase KatG and alkylhydroperoxide reductase (AhpC) are antioxidants that reduce oxidative stress caused by phagosomal reactive oxygen species (ROS) and H 2 O 2 generation. Aureolysin (Aur) cleaves antimicrobial peptides and DltA–DltD promote d-alanyl esterification of teichoic acids to protect staphylococci from antimicrobial peptides. MprF modifies phosphatidylglycerol with alanine or lysine, another mechanism to protect staphylococci against antimicrobial peptides. l‑lactate dehydrogenase (Ldh) and flavohaemoglobin (Hmp) inhibit nitrosative stress, Eap and its homologues EapH1 and EapH2 inhibit neutrophil serine proteases, and OatA O - acetylates peptidoglycan, which prevents its lysozymal degradation. d | Secreted β-barrel pore forming toxins (β-PFTs) bind specific receptors on immune cells to impair immune cell functions or promote cell lysis. These β-PFTs include leukocidin ED (LukED) (which binds to neutrophils, T cells and macrophages), γ-haemolysin AB (HlgAB) (which binds to neutrophils, macrophages and red blood cells), HlgCB and Panton–Valentine leukocidin (PVL) (which bind to neutrophils and macrophages), and LukAB and α-haemolysin (Hla) (which bind to neutrophils). Phenol-soluble modulin-α (PSMα), which is another factor secreted by S. aureus (but not a β-PFT), can also lyse leukocytes.
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Leukocidins Bacterial secreted toxins targeting white blood cells (leukocytes) for destruction. and with the expression of virulence factors, including α-haemolysin^25. S. aureus Δpsma1–psma4 mutants are attenuated in the mouse bloodstream infection model^69 , a phenotype that may be due to defects in biofilm for- mation, virulence gene expression and/or contribu- tions of PSMα1–PSMα4 towards lysis of immune cells, presumably via membrane insertion and pore formation^70. β-barrel pore-forming toxins (β-PFTs) are secreted by the bacterium as soluble monomers and, on associa- tion with receptors on cell surfaces, assemble into multi- meric pore structures, penetrating the lipid bilayer to invoke alterations in the physiology of injured cells or their outright lysis^71. α-haemolysin (Hla), the prototype β-PFT of S. aureus, is encoded by the hla gene, which is located within IEC2. Although conserved among all S. aureus isolates, some lineages of S. aureus carry a non- sense mutation that blocks hla expression^72. Hla binds to its receptor on host cells, ADAM10, and assembles into a heptameric pore; through the metalloproteinase activ- ity of ADAM10, Hla modulates the function of immune cells, including neutrophils, or triggers lysis of epithelial cells^73 ,^74. S. aureus hla mutants display defects in disease severity in mouse models for lethal pneumonia, bacte- raemia and SSTI; however, hla is not required for the establishment of S. aureus abscess lesions75–77. Based on ADAM10 expression on the surface of myeloid cells, organ epithelia and the vascular endothelium, Hla causes global, as well as organ-specific, changes to host physiology during S. aureus infection^74. Leukocidins are other β-PFTs secreted by S. aureus (FIG. 1d). Following leukocidin association with receptors on myeloid cells and erythrocytes, these toxins assemble from two different subunits (F and S) into an octameric pore structure^78. All S. aureus strains produce at least three leukocidins, γ-haemolysin AB (HlgAB), HlgCB and leukocidin AB (LukAB; also known as LukGH), whereas other strains may also secrete Panton–Valentine leucocidin (PVL; which is encoded by lukPV) and LukED or LukMF^79 (BOX 1). The operon encoding LukAB is located immediately adjacent to hlb, whereas the operon encoding γ-haemolysin (hlgABC) is part of the sbi–hlg locus. LukAB binds to the I domain of human, but not mouse, αM integrin on myeloid cells^80. Purified LukAB can trigger human neutrophils to release NETs that, at least temporarily, ensnare staphylococci^81. LukAB has also been reported to promote S. aureus escape from the phagosome of neutrophils^82. Purified HlgAB γ-haemolysin, but not purified HlgCB γ-haemolysin, is able to lyse human and rabbit red blood cells^83. HlgAB binds to chemokine receptors CXCR1, CXCR2 and CC-chemokine receptor 2 (CCR2), whereas HlgCB uses complement receptors C5aR and C5L2 to associate with target cells^84. Following staphylococcal inoculation into human blood, hlgABC is upregulated 34–145-fold^85 , and the S. aureus ΔhlgABC mutant displays reduced sur- vival, presumably because HlgAB and HlgCB promote release of iron-compounds from erythrocytes, thereby enabling bacterial acquisition of this essential nutrient^83. Name Gene Genome Proposed Function Target Alleles^13 Staphylokinase sak IEC1 (var) Phagocytosis inhibition (^) Plasminogen, fibronectin, C3 and IgG 1 Sbi sbi sbi – hlg (con) Phagocytosis inhibition (^) IgG Fcγ, C3 and factor H 4 SCIN scn IEC1 (var) Complement inhibition C3bBb None SCIN-B scnB IEC2 (var) Complement inhibition C3bBb 7 SCIN-C scnC IEC2 (var) Complement inhibition C3bBb 7 SpA spa core Phagocytosis inhibition and B cell superantigen Ig Fcγ and Ig Fab (VH3) Xr (SpA typing) SSL3 ssl3 (^) GIα (var) TLR signalling inhibition TLR2 13 SSL5 ssl5 (^) GIα (var) Chemotaxis and platelet inhibition (^) PSGL1, GPCRs, GPIbα and GPVI 5 SSL6 ssl6 GIα (var) Chemotaxis inhibition PSGL1 2 SSL7 ssl7 GIα (var) Phagocytosis inhibition IgA and C5 4 SSL10 ssl10 GIα (var) Phagocytosis inhibition IgG, fibrinogen, fibronectin, thrombin and factor Xa
SSL11 ssl11 GIα (con) Chemotaxis inhibition PSGL1 10 Staphyopain scpA core Chemotaxis inhibition CXCR2 1 TSST1 tst SaPI1 T cell superantigen Vβ2 TCR and MHC class II α-chain 2 vWbp vwb core Phagocytosis inhibition Thrombin, fibrinogen, factor XIII and fibronectin
C5aR, C5a receptor; CCR, CC-chemokine receptor; CHIPS, chemotaxis inhibitory protein of S. aureus; Clf, clumping factor; con, conserved; Cna, Collagen adhesin; dAdo, deoxyadenosine; CXCR, C-X-C chemokine receptor; Eap, extracellular adherence protein; Ecb, extracellular complement-binding protein; Efb, extracellular fibrinogen-binding protein; FLIPr, formyl peptide receptor-like 1 inhibitor; FLIPrL, FLIPr-like; FPR, formyl-peptide receptor; FnBP, fibronectin-binding protein; GI, genomic island; GPCR, G protein-coupled receptor; Hlg, γ-haemolysin; ICAM1, intercellular adhesion molecule 1; IEC, immune evasion cluster; Ig, immunoglobulin; Luk, leukocidin; MHC, major histocompatibility complex; PMN, polymorphonuclear leukocyte; PSGL1, P-selectin glycoprotein ligand 1; PSM, phenol-soluble modulin; PVL, Panton–Valentine leukocidin; SaPI, S. aureus pathogenicity island; Sbi, staphylococcal binder of immunoglobulin; SCIN, staphylococcal complement inhibitor; SpA, staphylococcal protein A; SSL, S. aureus superantigen-like; TCR, T cell receptor; TLR, Toll-like receptor; TSST1, toxic shock syndrome toxin 1; var, variable; vWbp, von Willebrand factor-binding protein. Table 1 (cont.) | Staphylococcus aureus immune evasion determinants, their function and epidemiology NATURE REVIEWS | MICROBIOLOGY VOLUME 13 | SEPTEMBER 2015 | 535
Dabigatran A small molecule that directly binds and inhibits thrombin as well as staphylothrombin, the complex formed between coagulase or von Willebrand Factor-binding protein and prothrombin. Sortase The bacterial transpeptidase responsible for anchoring surface proteins to the cell wall envelope. Both purified HlgAB and HlgCB promote lysis of neu- trophils, monocytes and macrophages from humans, as well as non-human primates, and to a lesser degree rabbits and mice^83. In a mouse intravenous challenge model, animals infected with a S. aureus ΔlukAB mutant displayed increased time-to-death and survival. Using subcutaneous inoculation in mice or rabbits, the S. aureus lukAB mutant did not display defects in skin abscess formation^86. The ΔhlgAB mutant displayed a virulence defect in the intraperitoneal challenge model in mice^84. lukED is present in the GIβ locus of ~70% of clinical S. aureus isolates^13 (BOX 1). Purified LukED triggers lysis of macrophages, dendritic cells and T cells from many different vertebrates, as the toxin binds to the chemokine receptors CCR5, CXCR1 and CXCR2 (REFS 87 , 88 ). For S. aureus strain Newman, which harbours GIβ, the ΔlukED mutation increased the time-to-death and survival of mice following intravenous challenge with mutant staphylococci^89. PVL is secreted by S. aureus lysogenized with PVL phage^90. PVL binds to the C5aR on neutrophils, monocytes and macrophages, but its activity is restricted towards human and rabbit cells^9. By virtue of binding C5aR, PVL not only exerts its lytic activity on target host cells but can also facilitate the priming of human polymorphonuclear leukocytes by pro-inflammatory stimuli (for example, formyl pep- tides). Injection of purified recombinant PVL leads to increased immune cell recruitment and increased archi- tectural destruction of the lung, owing to toxin-mediated recruitment and subsequent lysis of immune cells^9. Only ~2% of S. aureus isolates secrete PVL; however, community-acquired MRSA isolates frequently harbour PVL phages, and PVL expression is also associated with necrotizing pneumonia^91. S. aureus ΔlukPV variants display defects in the pathogenesis of SSTIs and lung infections in rabbits, but not in mice, which seems to be due to neutrophil-mediated inflammatory responses and tissue distruction^76 ,^92. lukMF, genes for another phage- encoded leukocidin, are found in S. aureus isolates associate d with bovine mastitis^13. Staphylococcal agglutination. Coagulation, the conver- sion of fibrinogen to a crosslinked fibrin meshwork by activated thrombin, is an innate defence of all vertebrates that immobilizes microbial invaders and attracts immune cells for phagocytic clearance of bacteria. Therefore, every successful bacterial pathogen must evolve mechanisms for escape from fibrin entrapment and subsequent phago- cytosis by infiltrating immune cells. A hallmark of all S. aureus isolates is the secretion of two coagulases: coag- ulase (Coa) and von Willebrand factor-binding protein (vWbp)^93. Coa and vWbp associate with prothrombin, a zymogen, to generate enzymatically active staphylothrom- bin, which cleaves the A and B peptides of fibrinogen to generate fibrin fibrils^94 (FIG. 2). As staphylothrombin does not cleave other substrates of thrombin, it avoids the acti- vation of clotting and inflammatory factors that ordinar- ily accompany fibrin polymerization^95. Staphylothrombin activity is not subject to feedback inhibition through host antithrombin. However, staphylothrombin is blocked by dabigatran and other direct thrombin inhibitors of the same family^96. The staphylothrombin-generated fibrin meshwork protects S. aureus from phagocytes and contributes to the formation of staphylococcal abscess lesions and lethal bacteraemia in mice^97. Activation of prothrombin is mediated by the D1 and D2 domains in the N-terminal region of Coa and is blocked by spe- cific antibodies, which provide protection from S. aureus bloodstream infection in the mouse model^98. Perhaps owing to purifying selection, coa is one of the most vari- able genes in the core genome of S. aureus, with >50% sequence variation in the coding sequence for its D1–D domains and 14 distinct isoforms^93 (TABLE 1). vWbp also has conserved D1–D2 domains for association with pro- thrombin, but this complex generates fibrin at a reduced rate and contributes to abscess formation without affect- ing staphylococcal escape from phagocytosis^99. The gene encoding vWbp, vwb, has limited sequence variability^98. S. aureus agglutinates with Coa- or vWbp-derived fibrin fibrils, which requires clumping factor A (ClfA), a glycosylated, sortase-anchored surface protein, the immunoglobulin-like domains of which bind to Box 2 | Structural features of immune evasion factors Crystallographic analysis of Staphylococcus aureus immune evasion determinants revealed five discrete structural domains that enable specific interactions with the host’s immune system: oligonucleotide-binding (OB) fold,β-grasp domain, triple-helical bundle (THB), leukocidin domain and immunoglobulin-like fold. Varying the amino acid sequence for these domains has created panoplies of ligands that interact with the defence molecules of infected hosts at the places that matter most^4 ,^145. Thus, the study of S. aureus immune evasion factors laid bare the most intricate workings of the human immune system and identified new avenues for the therapy of autoimmune and inflammatory diseases. Examples for immune-evasion factors with OB fold and β-grasp domains include the staphylococcal T cell superantigens (SEA, SEB, SEC1–3, SED, SEE, SEG, SHE, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, SEU, toxic shock syndrome toxin 1 (TSST1), TSST2 and SelX) and the staphylococcal superantigen-like (SSL) family (SSL1–SSL13)^125. Chemotaxis inhibitory protein of S. aureus (CHIPS), formyl peptide receptor-like 1 inhibitor (FLIPr), FLIPr-like (FLIPrL), extracellular adherence protein (Eap), EapH1 and EapH2 have β-grasp domains but not an OB fold^68 ,^146. Immune evasion factors with triple helical bundles include extracellular complement-binding protein (Ecb), extracellular fibrinogen-binding protein (Efb), staphylococcal protein A (SpA), staphylococcal binder of immunoglobulin (Sbi), staphylococcal complement inhibitor (SCIN), SCIN-B and SCIN-C^147 , whereas members of the β-PFT family (γ-haemolysin proteins, leukocidin proteins, Panton–Valentine leukocidin (PVL) and Hla) have leukocidin domains^79. Surface proteins with IgG-like domains include the immune evasion factor clumping factor A (ClfA) and its relatives ClfB, fibronectin-binding protein A (FnBPA) and FnBPB^102 (TABLE 1). 536 | SEPTEMBER 2015 | VOLUME 13 www.nature.com/reviews/micro
Nature Reviews | Microbiology S. aureus AdsA Nuc AdsA CD CD ADP AMP ATP A A A A A A P dA dA P P A P P A P P P Macrophage AdoR1/2A/2B/ Macrophage apoptosis Neutrophil AdoR1/2A/2B/ B cell AdoR2A T cell AdoR2A/2B Tissue injury Staphylococcal abscess community Infiltrated PMNs NETs NETosis dAMP dAdo a Adenosine signalling b Deoxyadenosine signalling ↓Superoxide burst ↓MHC II ↓IL- Pro-caspase 3 Caspase 3 Fab domains The portions of antibodies dedicated to antigen binding. VH3 clan IgM IgM derived from one of three clans of variable heavy chain (VH) genes, the products of which provide the scaffold for the antigen-binding determinants of antibodies. Plasmablasts Immature B cells in the blood that secrete antibodies. AdsA activity also modulates immune responses fol- lowing the degradation of NETs. During bloodstream infection in mice, S. aureus disseminates to many dif- ferent organ tissues to establish abscess lesions. These lesions are composed of a bacterial nidus, designated as the staphylococcal abscess community (SAC), encased within a pseudocapsule of fibrin deposits, and sur- rounded by layers of immune cells^97. In spite of large numbers of infiltrated neutrophils, mice are unable to eliminate staphylococci from abscess lesions and eventually succumb to persistent infection^36. Although neutrophils use NETosis to entangle staphylococci, NETs are degraded by staphylococcal nuclease (Nuc) and thereby fail to exert bactericidal activities^110 (FIG. 3b). Nucdigestion of NETs releases 5ʹ and 3ʹ monophosphate nucleotides that are converted by AdsA into deoxyaden- osine^111 (FIG. 3b). Deoxyadenosine production triggers caspase 3 induced apoptosis of macrophages and pre- vents phagocyte entry into the SAC, the core of staphy- lococcal abscess lesions, thereby promoting bacterial survival within the lesion^111. Manipulation of adaptive immune responses B cell responses. S. aureus is capable of manipulating B cell survival and function, especially through pro- duction of staphylococcal protein A (SpA), which is a sortase-anchored surface protein with high affinity for vertebrate immunoglobulin, including human IgA, IgD, IgG1–IgG4, IgM and IgE^112. SpA is initially deposited in the staphylococcal envelope and subsequently released by cell wall hydrolases (LytM)^113. spa is expressed by all clinical S. aureus isolates; the immunoglobulin binding domains are conserved in the genomes of these isolates, but region X, the cell wall spanning domain of SpA, is a highly polymorphic sequence^114 ,^115 (BOX 1). The immunosuppressive attributes of SpA have been ascribed to two distinct binding activities: asso- ciation with the Fcγ domain and with the Fab domains of antibodies^116 ,^117. SpA binding to the Fcγ domain of IgG blocks phagocytosis of staphylococci^118 , whereas SpA binding to the Fab domains and crosslinking of VH3 clan IgM promotes B cell superantigen activity^119 (FIG. 4a). Of note, SpA binds specifically to VH3 clan IgM antibodies, which mediate the predominant anti- body responses to infection and immunization, but not to other clan antibodies. In the intravenous chal- lenge model of S. aureus infected mice, spa expression suppresses antibody responses against many different staphylococcal antigens and provides antiphagocytic attributes, promoting staphylococcal survival in blood^120. Infection of mice with S. aureus spa variants that can- not bind immunoglobulin is associated with attenuated disease and with antibody responses against many differ- ent antigens that can protect animals against subsequent lethal challenge with other S. aureus isolates^120. Mice harbour a limited repertoire of VH 3 +^ B cells, whereas humans possess large populations of these cells, yet both species cannot develop SpA-neutralizing anti- bodies during infection^121. S. aureus infection in humans triggers expansions of VH3 idiotypic plasmablasts (>90% of blood plasmablasts), the antibodies of which (that is, the B cell receptors) associate with SpA via their Fab domains but do not display pathogen-specific binding activities^121 (FIG. 4b). When mice are treated with puri- fied SpA, crosslinking of VH3 clonal B cells triggers proliferation and apoptotic collapse of expanded pop- ulations of B cells^122. It is not clear, however, whether apoptotic collapse of expanded lymphocyte populations occurs during S. aureus infection in mice or in humans. Non-toxigenic SpA, designated SpAKKAA, was engi- neered by substituting twenty amino acid residues essen- tial for its association with the Fcγ and Fab regions^123. Although SpAKKAA has twenty amino acid substitutions, this antigen elicits antibodies that neutralize SpA when Figure 3 | Staphylococcus aureus AdsA perturbs adenosine and deoxyadenosine signalling. a | Staphylococcus aureus infection and its associated inflammatory damage promote the release of ATP, which is converted by adenosine synthase A (AdsA) into the immune suppressive signalling molecule adenosine (A). Adenosine inhibits activation of B cells, T cells, macrophages and dendritic cells via adenosine receptor (AdoR) signalling by acting on four different receptors (AdoR 1 , AdoR2A, AdoR2B and AdoR 3 ). Under physiological conditions, CD39 and CD73 generate adenosine signals to limit inflammatory responses; CD39 and CD73 are also responsible for the adenosine halo surrounding immune cells and for immune suppressive states involving regulatory T cells (T cells expressing the FOXP3+^ marker protein (not shown)). b | S. aureus induced NETosis of infiltrating neutrophils leads to nuclease-mediated degradation of the DNA fibres that are the major components of neutrophil extracellular traps (NETs) and AdsA-mediated conversion of 5ʹ-monophosphate-deoxyadenosine (dAMP) into deoxyadenosine (dAdo), which promotes autocleavage of the apoptosis factor pro-capsase 3 to caspase 3. Caspase 3 induces macrophage death, thereby protecting S. aureus against professional phagocytes. IL‑12, interleukin‑12; MHC II, major histocompatibility complex class II; Nuc, staphylococcal nuclease; PMN, polymorphonuclear leukocyte. 538 | SEPTEMBER 2015 | VOLUME 13 www.nature.com/reviews/micro
a S. aureus manipulation of B cells c S. aureus manipulation of T cells b S. aureus manipulation of plasmablasts VH 3 +^ B1, MZ and B2 cells B cell expansion Apoptotic deletion SpA SpA SpA SpA (^) IgM IgM (VH 3 +) SpA crosslinking of B cell receptors (IgM) VH 3 +^ plasmablast Somatic hypermutation and class-switch recombination Non-specific activation and antibody secretion SpA SpA SpA SpA IgM IgG SpA SpA SpA SpA SpA SpA SpA SpA Inability to develop adaptive immunity Inability to develop adaptive immunity Inability to develop adaptive immunity MHC II TCR SAg Staphylococcal antigen Antigen- presenting cell TH cells T cell expansion and anergy Cytokine storm ↑ IL- ↑ IFNγ ↑ IL-1β ↑ TNF Nature Reviews | Microbiology injected into animals^123. The SpAKKAA-derived polyclonal antibodies promote phagocytosis of staphylococci and display adjuvant attributes by suppressing staphylococ- cal B cell superantigen activity and promoting humoral immune responses against a wide range of S. aureus antigens^123. Studies with mouse monoclonal antibodies corroborate this concept^53. T cell responses. Staphylococcal T cell superantigens bind to MHC class II molecules on the surface of anti- gen-presenting cells, providing antigen-independent crosslinking with T cell receptors on T helper cells^124 (FIG. 4c). S. aureus strains have been shown to express 23 different enterotoxins and T cell superantigens^125. Three superantigens are most frequently associated with human disease — toxic shock syndrome toxin 1 (TSST1), staphylococcal enterotoxin B (SEB) and SEC — each providing high-affinity interactions with distinct subsets of Vβ chain T cell receptors^126. In humans with toxic shock syndrome, S. aureus secretion of TSST1 or other enterotoxins trigger expansions of cognate T cell populations, up to 30% of blood lymphocytes and nonspecific release of cytokines, preventing a focused adaptive immune response^127. Depending on the site and severity of S. aureus infection or intoxication, superantigen-mediated activation of T cell responses may be associated with cytokine storms and toxic shock syndrome pathology^128. Staphylococcal superantigens are also thought to interfere with antigen-specific pro- liferation of T cells and with antibody responses against specific subsets of staphylococcal antigens, including staphylococcal superantigens^129. It is not yet known whether superantigens have a crucial role in the sup- pression of T cell responses in mice that are observed during S. aureus bloodstream infections^130. S. aureus can also manipulate T cell responses by promoting T cell lysis. For example, δ-toxin (Hld; also known as δ-haemolysin), a member of the PSMα family, can lyse T cells^131 and has also been reported to trigger mast cell degranulation, which could be a key factor in Figure 4 | Staphylococcus aureus manipulates B cell and T cell responses. a | Staphylococcus aureus releases staphylococcal protein A (SpA) into host tissues, where it binds to and crosslinks VH3 clan B cell receptors. In B1 cells, marginal zone (MZ) B cells and B2 cells, SpA crosslinking is associated with proliferative expansion and apoptotic collapse. The death of these cells impedes the development of adaptive immunity during S. aureus infections. b | In VH 3 + plasmablasts, SpA crosslinking promotes somatic hypermutation and class switching from IgM antibodies to IgG antibodies, followed by the secretion of antibodies that are not specific for the S. aureus antigen. c | S. aureus secretes T cell superantigen (SAg), which crosslinks major histocompatibility complex class II antigens (MHC II) on the surface of antigen-presenting cells and T cell receptors (TCRs) on the surface of T helper (TH) cells, triggering T cell expansion and anergy and causing cytokine storms (including interleukin-2 (IL-2), interferon-γ (IFNγ), IL- 1 β and tumour necrosis factor (TNF)). As a result, a T cell response specific for S. aureus antigens is not produced. NATURE REVIEWS | MICROBIOLOGY VOLUME 13 | SEPTEMBER 2015 | 539
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