IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes

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IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes Ulrich von Pawel-Rammingen and Lars BjoÈrcky The Gram-positive bacterium Streptococcus pyogenes is a major human pathogen causing substantial morbidity and mortality in society. S. pyogenes has evolved numerous molecular mechanisms to avoid the various actions of the human immune system and has established means to modulate both adaptive and innate immune responses. S. pyogenes produces and secretes proteolytic enzymes, which have an important impact on the ability of the bacteria to survive in the human host. Prominent among these are two immunoglobulin-degrading enzymes: the newly discovered streptococcal cysteine proteinase, IdeS, and the classical cysteine proteinase of S. pyogenes, SpeB. Addresses Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, Biomedical Research Centre, B14, SE-221 84 Lund, Sweden  e-mail: [email protected] y e-mail: [email protected]

Current Opinion in Microbiology 2003, 6:50±55 This review comes from a themed issue on Host±microbe interactions: bacteria Edited by Hans Wolf-Watz and Virginia Miller 1369-5274/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1369-5274(03)00003-1 Abbreviations FcR IgFc receptor IdeS IgG-degrading enzyme of Streptococcus pyogenes Ig immunoglobulin PMN polymorphonuclear leukocyte PSGN post-streptococcal glomerulonephritis RGD arginine-glycine-aspartic acid SpeB streptococcal pyrogenic exotoxin B

Introduction

The Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is one of the most important human bacterial pathogens and the causative agent for a variety of relatively mild diseases such as impetigo, scarlet fever and pharyngitis. It can also cause severe, life-threatening conditions such as septicemia, necrotizing fascitis and streptococcal toxic-shock syndrome [1]. Sequelae to initially mild infections include serious clinical conditions such as acute rheumatic fever and post-streptococcal glomerulonephritis (PSGN). Acute rheumatic fever is the most common cause of pediatric heart disease worldwide [2], and PSGN is an important cause of renal failure [3]. Although streptococcal Current Opinion in Microbiology 2003, 6:50±55

infections have shown decreased severity during the past century, invasive S. pyogenes infections have reoccurred worldwide since the late 1980s, which has attracted considerable attention and concern [4,5]. Streptococcal infections are usually treated with b-lactam antibiotics, but despite the fact that S. pyogenes remains susceptible to these antibiotics, hyperacute and toxic infections are still connected with high mortality. The survival of S. pyogenes depends on its ability to avoid the various actions of the innate and adaptive immune systems and to modulate the in¯ammatory responses induced by the pathogen. Thus, intracellular proteinases from neutrophils and damaged host tissue will be released at the site of infection and cause the intense in¯ammatory state, which is a typical feature of S. pyogenes infections [6]. However, the pathogen has also evolved several mechanisms to utilize and manipulate host proteolytic cascades, and it produces and secretes proteolytic enzymes that in¯uence its ability to avoid host defenses. In this review, we will focus primarily on two cysteine proteinases of S. pyogenes that have multiple effects on host immune responses: the newly discovered IdeS, and the classical SpeB enzyme.

S. pyogenes and immunoglobulins

Immunoglobulins (Igs) play a key role in the human immune defense system by speci®cally recognizing invading microorganisms and mediating their killing by professional phagocytes or the complement system, or both. Igs consist of antigen-recognizing Fab regions, which are linked through a ¯exible hinge region with the constant Fc effector region. The Fc effector region triggers the activation of the classical pathway of complement and mediates contact with Fc receptors (FcRs) on phagocytic cells. The classical pathway of complement is initiated by the interaction of complement factor C1q with speci®c IgG or IgM. Binding of C1q triggers the activation of a proteolytic cascade that subsequently results in the attachment of complement factor C3b to the surface of the immune complex. C3b-opsonized particles are ef®ciently recognized by complement receptors on phagocytes, which mediate the phagocytosis of these complexes. Thus, to persist and proliferate, pathogenic bacteria have to ®nd ways to avoid identi®cation by Igs and to interfere with IgFc-mediated phagocytosis. S. pyogenes expresses cell-wall-anchored surface proteins of the so-called M protein family. These proteins have the ability to interact with several abundant extracellular www.current-opinion.com

Streptococcal cysteine proteinases von Pawel-Rammingen and BjoÈrck 51

Figure 1

(a)

IgG bound via Fc interactions

Opsonizing IgG Unbound Fc fragment

Unbound Fab fragment

IdeS Fc Fc M surface protein

Fab Bacterial surface protein

S. pyogenes Cell wall

(b)

Cleavage and activation of multiple host proteins

Fab

SpeB Fc

Fc

Fab Bacterial surface protein

S. pyogenes Cell wall Current Opinion in Microbiology

Effects of IdeS and SpeB on the interactions between IgG antibodies and S. pyogenes. (a) Early phase of infection. Secretion of IdeS starts at early exponential growth phase. The enzyme cleaves IgG antibodies in the hinge region and will degrade opsonizing IgG bound to bacterial surface structures, but also IgG bound to M and M-like surface proteins through Fc interactions. As a result, Fab fragments bound to specific antigens and Fc fragments bound by M and M-like proteins remain at the surface, whereas unbound Fab and Fc fragments are released. (b) Late phase of infection. Secretion of SpeB starts at late exponential growth phase. SpeB degrades bacterial surface proteins. Thus, Fc and Fab fragments in complexes with bacterial surface proteins are released during late phase of infection. Cleavage sites for the enzymes are indicated by small arrows; red arrows, IdeS; green arrows, SpeB.

human proteins, including albumin, ®brinogen IgG, IgA and others [7]. Nonimmune binding of IgG by M proteins occurs through Fc interactions (Figure 1a). When bound to M proteins, the Fc region is blocked and cannot bind C1q of complement, resulting in a reduced surface deposition of opsonic complement factor C3b [8]. In contrast to IgG bound via the Fc region, IgG antibodies directed against surface antigens expose their Fc region to Fc receptors present on phagocytes (Figure 1a). Thus, bacteria, recognized by speci®c IgG antibodies, are rapidly eliminated from human blood. To avoid the detrimental actions of speci®c IgG, S. pyogenes has developed different strategies to interfere www.current-opinion.com

with IgG function. Fc glycosylation is critical for IgG recognition by FcR [9,10]. The secreted endoglycosidase of S. pyogenes EndoS [11] hydrolyzes conserved N-linked oligosaccharides on IgG. The hydrolyzed IgG has a reduced ability to bind FcR and is therefore impaired in mediating opsonophagocytosis [12]. Another strategy involves the two cysteine proteinases of S. pyogenes, SpeB and IdeS, which are discussed below.

SpeB, immunoglobulins and the immune response

The streptococcal cysteine proteinase SpeB was isolated and characterized in 1945 and was the ®rst cysteine proteinase to be isolated from a prokaryote [13]. SpeB Current Opinion in Microbiology 2003, 6:50±55

52 Host±microbe interactions: bacteria

is secreted as a 40 kDa pro-enzyme that is converted into a mature 28 kDa active proteinase by autocatalytic cleavage [13]. Mature SpeB has broad proteolytic activity and degrades or activates several different human proteins [6]. Besides its ability to cleave host proteins, SpeB also exerts proteolytic activity on streptococcal surface proteins, including IgG-binding proteins [6,14]. Released IgGbinding proteins in complex with IgG have the ability to activate and consume complement at a distance from the pathogen [8], thereby avoiding complement activation at the bacterial surface. SpeB expressed by the clinically important M1 serotype contains an arginineglycine-aspartic acid (RGD) motif, which mediates binding of SpeB to eukaryotic cell surfaces [15] and suggests that SpeB might have additional functions not yet elucidated. The crystal structure of the 40 kDa precursor of SpeB reveals a structural homology to enzymes of the papain superfamily [16]. Like papain, SpeB cleaves IgG in the ¯exible hinge region of the IgG heavy chain, generating two Fab fragments and one Fc fragment. SpeB cleaves the heavy chain at a de®ned site between glycine residues 236 and 237 [11], which differs from the reported cleavage site of papain at position 224 [17]. The proteolytic activity of SpeB towards immunoglobulins is not restricted to IgG; SpeB also degrades the carboxy-terminal parts of the heavy chains of IgA, IgD, IgE and IgM [18]. Overnight incubation of opsonizing IgG with SpeB impairs the ability of these antibodies to promote phagocytic killing of S. pyogenes in human blood, indicating that the IgG-cleaving activity of SpeB could be important for the bacteria to evade the adaptive immune system [12]. However, under laboratory conditions SpeB expression is repressed until S. pyogenes reaches stationary phase [19,20], which questions whether SpeB has a role in cleaving opsonizing IgG antibodies. Given the detrimental effect of speci®c IgG, it should be important for an IgG-cleaving proteinase to be continuously present during infection. Furthermore, a proteinase potentially able to remove opsonizing antibodies has to be highly effective and should be speci®c enough to avoid the other redundant substrates, occupy the enzyme, and affect the cleavage of speci®c Igs. Thus, although IgG might be a substrate for SpeB under certain environmental conditions, it seems unlikely that SpeB is part of the ®rst line of defense against speci®c antibodies. Even though SpeB might not be directly involved in the attenuation of the adaptive immune response, its proteolytic activity towards streptococcal surface proteins, including IgGbinding proteins and the streptococcal C5a peptidase, could certainly be important for the modulation of the complement system [8]. Furthermore, SpeB-induced release of dermatan sulphate from the extracellular matrix results in the inactivation of antimicrobial peptides [21], Current Opinion in Microbiology 2003, 6:50±55

suggesting another mechanism whereby SpeB activity affects the innate immune response.

IdeS and immunoglobulins

Recently, the unexpected observation that S. pyogenes expresses proteolytic activity towards IgG, even under conditions repressing the production of SpeB, led to the identi®cation of IdeS (IgG-degrading enzyme of S. pyogenes), a previously unknown cysteine proteinase from S. pyogenes [22]. IdeS is a secreted 35 kDa protein that, in contrast to SpeB, does not require processing or activation to exert its enzymatic activity. IdeS cleaves the heavy chain of IgG at glycine residue 237 (i.e. at the same site as SpeB), generating two stable Fab fragments and one Fc fragment. IdeS was identi®ed by its cleavage of IgG in human plasma, and subsequent experiments demonstrated that IdeS is also active in whole human blood. Although active in these protein-rich environments, the only degradation products identi®ed were the IgG-derived fragments, and to date no additional substrate of IdeS is known. The other classes of human Ig (IgM, IgA, IgD and IgE) were also found to be resistant to IdeS cleavage [22]. Although IdeS might have other unidenti®ed substrates, it has the degree of speci®city required to protect the bacteria from opsonizing IgG antibodies. In addition, IdeS expression has already started during early logarithmic growth, and proteolytic activity is detected into late stationary phase. This continued presence makes the enzyme suitable for cleaving opsonizing IgG antibodies as soon as they appear at the bacterial surface [22]. The ability of IdeS to interfere with Fc-mediated phagocytic killing has been demonstrated in a variety of bactericidal assays [22,23,24]. Opsonized bacteria were incubated with IdeS and mixed with macrophage-like cells, puri®ed polymorphonuclear leukocytes (PMNs), or human immune blood, containing antibodies against S. pyogenes. The presence of IdeS signi®cantly increased survival of the bacteria compared with buffer controls [22,23,24]. Thus, because of its unique and highly speci®c proteolytic activity and early and sustained expression, IdeS enables S. pyogenes to evade the adaptive immune response. Compared with SpeB, IdeS represents a tailor-made and more effective defense against opsonizing IgG antibodies.

IdeS and innate immunity

S. pyogenes has evolved several mechanisms to interfere with the complement system and thereby disarm this branch of the innate immune system. These mechanisms include binding of regulatory components of the complement system to the streptococcal surface [25±27], proteolytic degradation of chemotactic substances [28], inhibition of membrane attack complex formation, and the inhibition of antibacterial proteins and peptides ([29±31], IM Frick, www.current-opinion.com

Streptococcal cysteine proteinases von Pawel-Rammingen and BjoÈrck 53

P AÊkesson, M Rasmussen, A Schmidtchen and L BjoÈrck, unpublished data). Recently, a streptococcal protein (Mac) was suggested to mimic parts of the CD11b/CD18 complement receptor and to inhibit phagocyte function by blocking the interaction of the FcgIIIb receptor CD16 with complement factors and/or speci®c antibodies [24]. Interestingly, the streptococcal protein Mac is identical to IdeS. This ®nding raised the question of whether the enzymatic activity of IdeS/Mac towards IgG is a prerequisite for the reported interference with phagocyte function [24]. Thus, if IdeS/ Mac inhibits CD16 function by mimicking the host-cell receptor CD11b/CD18, then a similar, but enzymatically inactive, protein should also be able to prevent phagocytic killing. This approach was used in two independent studies that resulted in controversial results. One study suggested that an enzymatically inactive protein retains the ability to interfere with phagocytic killing [32], whereas another study demonstrated that the enzymatically inactive mutant protein IdeSCys!Gly does not interfere with IgG-mediated phagocytic killing [23]. The latter study was challenged by the speculation that the mutant protein might be structurally altered [32] (see also Update), but ¯ow cytometry analyses demonstrated that IdeSCys!Gly is not affected in its ability to interact with PMNs and that the binding of IdeSCys!Gly was indistinguishable from the binding of IdeS, indicating that the mutant protein retained its binding properties [23]. Further results are required to elucidate the precise role of IdeS in virulence, although it appears that the enzymatic activity of IdeS/Mac is essential for its ability to interfere with phagocytic killing and that steric hindrance of the CD16 receptor per se is not suf®cient to mediate protection. However, IdeS interacts with puri®ed PMNs, and the enzyme could hypothetically bind to the neutrophil surface to cleave IgG associated with the bacterial surface that is presented to the PMNs.

Other effects of IdeS

Most S. pyogenes strains express surface proteins that bind IgG through the Fc region with high af®nity. Consequently, bacteria surrounded by plasma or in¯ammatory exudates are covered with IgG bound in a non-immune fashion. This IgG population, present in vast amounts compared with antigen-speci®c antibodies, is also cleaved by IdeS. Furthermore, a cooperative action of IdeS and SpeB might also release bacterial surface proteins in complex with either Fab or Fc fragments (Figure 1). Thus, IdeS cleavage will generate substantial amounts of Fc and Fab fragments, which might have secondary biological effects (e.g. by affecting PMN functions). The FcgIIIb receptor CD16 is, in addition to its membrane-bound form, also present as a soluble receptor in www.current-opinion.com

body ¯uids, and it is likely, at least during in¯ammation, to exceed the concentration of secreted IdeS [33,22]. CD16 binds preferentially to immune complexes and only weakly to monomeric IgG [34]. In addition, IdeS competes with the binding of IgG and immune complexes to CD16 [24]. Soluble CD16 affects PMN function and lifespan, an effect that might perpetuate local in¯ammatory processes [35]. A potential binding of IdeS to soluble CD16 might therefore interfere with these functions and modulate the local in¯ammatory response in favor of the pathogen. Analogous to SpeB, IdeS also contains an RGD motif, which is important for ligand recognition by integrins. Several bacterial and viral pathogens have the ability to bind to host-cell integrins and to trigger signal transduction pathways [36]. The RGD motif is not involved in the proposed interaction with CD16 [24], indicating that IdeS could have additional functions through the binding to other eukaryotic cell receptors. Finally, IdeS was recently shown to bind human IgA and IgM in vitro [37], although these Ig classes are not substrates for IdeS (see above). Whether these interactions have biological functions in vivo remains to be demonstrated.

Role of IdeS in streptococcal virulence

IdeS is produced by strains of clinically relevant serotypes of S. pyogenes, including the M1 serotype, associated with severe invasive infections [22,24]. Moreover, strains connected with PSGN, M12 and M55 [38,39], produced proteolytically active IdeS in vitro [22], suggesting a role for IdeS both during acute infections and in aseptic sequelae following acute S. pyogenes infections. This notion is further supported by the ®nding that antibodies reacting with IdeS are present in serum from patients suffering from diverse streptococcal infections, such as pharyngitis, acute rheumatic fever and invasive infections [24,40]. To de®ne the role of IdeS in streptococcal pathogenicity in vivo requires further investigation. However, such analyses might turn out to be complicated, because IdeS activity towards IgG is species-speci®c (U von PawelRammingen and L BjoÈrck, unpublished data). For instance, mouse IgG is not cleaved by IdeS, which precludes established mouse models of S. pyogenes infections.

Conclusions

S. pyogenes is an important human bacterial pathogen and the cause of substantial morbidity and mortality worldwide. The pathogen has evolved a series of complex virulence mechanisms designed to avoid host defenses, enabling it to establish itself in human hosts. Previous studies have indicated that SpeB and IdeS play important roles in S. pyogenes pathogenicity. Both are cysteine proteinases that cleave IgG in the hinge region; both contain an RGD integrin-binding motif. Although Current Opinion in Microbiology 2003, 6:50±55

54 Host±microbe interactions: bacteria

the degradation of IgG by IdeS and SpeB could be complementary under certain conditions (Figure 1), most available information suggests that IdeS is more signi®cant in bacterial defense against opsonizing IgG antibodies. Whereas SpeB cleaves numerous substrates, IdeS has a uniquely high degree of speci®city; IgG is the sole substrate identi®ed so far. Cleavage of IgG by IdeS is also much more ef®cient than by SpeB. Moreover, unlike SpeB, IdeS is already expressed at early exponential phase and does not require proteolytic processing and reduction to become activated. Thus, as soon as IdeS is secreted, it will be able to cleave IgG antibodies bound to the surface of S. pyogenes. A role of SpeB in virulence is suggested by its other proteolytic activities, such as the release of bradykinin [41], activation of interleukin-1b [42], and degradation of extracellular matrix proteins [21,43]. During the course of infection, removal of opsonizing IgG antibodies should represent a crucial line of defense at an early growth phase, while the induction of proin¯ammatory responses leading to exudation and tissue breakdown could provide starving bacteria with nutrients and facilitate the spread of the infection to new locations. Thus, IdeS protects the pathogen during colonization and growth, whereas SpeB activity is induced at a later phase, when the infection is losing momentum. Further studies on the activities of these cysteine proteinases should add to our understanding of temporal and spatial aspects of S. pyogenes infections and might help to identify novel therapeutical possibilities.

Finally, as suggested above, it is also known that the RGD amino acid motif of Mac/IdeS is involved in the interaction of the enzyme with human integrins avb3 and aIIb3.

Acknowledgements

The authors are supported by grants from the Swedish Research Council (projects 1055 (U von Pawel-Rammingen), 14379 and 7480 (L Bjorck); the foundations of Crafoord, Wiberg, Royal Physiogra®c Society (U von PawelRammingen), the Medical Faculty, Lunds University, the foundations of È sterlund; and Hansa Medical AB. Kock, Lundberg, and O

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Update

An extensive analysis of the mac/ideS gene in 67 S. pyogenes strains representing 36 different M protein serotypes has recently been published [44]. Sequence comparison revealed the existence of two families of protein variants of Mac/IdeS, which differ from each other in around 15% of the amino acids. Interestingly, members of the protein family designated as complex II showed considerably less homology to the human Mac-1 receptor, but retained the enzymatic activity towards IgG, emphasizing the importance of the enzymatic activity for the biological role of IdeS. Furthermore, this study presents data to support the earlier notion [32] that an enzymatically inactive mutant protein retains the ability to inhibit phagocytosis and to promote streptococcal survival. Unfortunately, data are only presented on the ability of the enzymatically inactive mutant Mac/IdeS to interfere with the phagocytosis of IgG-coated latex beads; the experiments did not address the survival of S. pyogenes under conditions similar to those in vivo. Thus, experimental evidence demonstrating that the enzymatic activity against IgG is not necessary for the critical biological function of IdeS has not yet been presented. Current Opinion in Microbiology 2003, 6:50±55

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Current Opinion in Microbiology 2003, 6:50±55