Streptococcus pyogenes and human neutrophils: a paradigm for evasion of innate host defense by bacterial pathogens

Microbes and Infection 6 (2004) 1117–1123 www.elsevier.com/locate/micinf

Review

Streptococcus pyogenes and human neutrophils: a paradigm for evasion of innate host defense by bacterial pathogens Jovanka M. Voyich a, James M. Musser a,b, Frank R. DeLeo a,* a

Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4th Street, Hamilton, MT 59840, USA b Center for Human Bacterial Pathogenesis Research, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 70030, USA Available online 03 August 2004

Abstract Human polymorphonuclear leukocytes (PMNs) are the first line of defense against invading microorganisms. Although most invading bacteria are eliminated by PMNs, some have evolved complex strategies to prevent normal PMN function. This review focuses on the interaction of human PMNs with Streptococcus pyogenes as a paradigm for successful pathogen evasion mechanisms. © 2004 Published by Elsevier SAS. Keywords: Polymorphonuclear leukocytes; Neutrophils; Streptococcus pyogenes; Phagocytosis; Apoptosis; Innate immunity

1. Introduction Human polymorphonuclear leukocytes (PMNs or neutrophils) are essential to the host response against invading microorganisms such as bacteria and fungi. The ability of PMNs to eradicate microorganisms is innate and thus not dependent on previous pathogen exposure. This is a critical component of human host defense against infection because there is immediate activation of effector responses. PMNs kill invading bacteria with potent oxygen-dependent and oxygen-independent microbicidal systems (reviewed in [1]). Phagocytosis of bacteria activates an NADPH-dependent oxidase that produces superoxide [1]. Superoxide is rapidly converted to other potent reactive oxygen species (ROS), such as hydrogen peroxide and hypochlorous acid within forming phagosomes. In addition, cytoplasmic PMN granules fuse with phagosomes (a process called degranulation), enriching phagocytic vacuoles with microbicidal enzymes

Abbreviations: GAS, Group A Streptococcus; PMNs, polymorphonuclear leukocytes; ROS, reactive oxygen species; Sic, streptococcal inhibitor of complement; SpeB, streptococcal pyrogenic exotoxin B. * Corresponding author. Tel.: +1-406-363-9448; fax: +1-406-363-9394. E-mail address: [email protected] (F.R. DeLeo). 1286-4579/$ - see front matter © 2004 Published by Elsevier SAS. doi:10.1016/j.micinf.2004.05.022

and peptides, such as elastase, cathepsin G, hCAP18, and human alpha defensin-1 [1]. Taken together, these neutrophil microbicidal systems are very efficient at killing ingested bacteria. Although PMN microbicidal activity is highly effective at preventing the spread of bacterial and fungal infections, non-specific or misdirected release of cytotoxic agents is often the underlying cause of inflammatory disorders. Therefore, regulation of PMN turnover at sites of infection is a mechanism to moderate inflammation. Recent studies indicate that phagocytosis of bacteria accelerates neutrophil apoptosis, and this process is important for normal resolution of infection and the inflammatory response [2,3]. Notably, some pathogens alter PMN apoptosis to survive and cause disease [2,3]. To establish infections in humans, successful pathogens must avoid ingestion and subsequent killing by PMNs. Streptococcus pyogenes is a Gram-positive bacterium that has evolved sophisticated mechanisms to disrupt many critical aspects of PMN function, including apoptosis [2]. This review highlights some of the newly discovered molecular strategies used by this bacterium to avert destruction by human PMNs.

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2. S. pyogenes (group A Streptococcus, GAS) Group A Streptococcus (GAS) successfully evades PMN phagocytosis and killing, to cause human infections including pharyngitis, impetigo, cellulitis and necrotizing fasciitis (flesh-eating syndrome). These infections and post-infection sequelae, such as acute rheumatic fever, are responsible for high morbidity and mortality worldwide. To avoid phagocytosis, GAS disrupts classical and alternate complement cascades, blocks deposition of opsonins, and employs molecular mimicry to inhibit host cell receptors. During GAS–PMN interaction, GAS engages a global survival response to moderate neutrophil killing mechanisms, and pathogen survival ultimately alters normal PMN apoptosis. These effective strategies are discussed in detail below. 2.1. GAS inhibits PMN recruitment Circulating PMNs are recruited to sites of infection by chemokines, cytokines, matrix metalloproteinases and products produced by the invading microorganisms. Host-derived chemotactic factors are critical in the regulation of inflammatory responses. The anaphylotoxin C5a is a potent neutrophil chemoattractant important in the host response to infection [1]. Disrupting PMN recruitment is one clever strategy used by GAS to evade innate immunity. GAS produces a 130-kDa serine endopeptidase referred to as C5a peptidase (ScpA) that specifically cleaves C5a to inhibit recruitment of PMNs to sites of infection [4,5]. This mechanism of evasion is significant because C5a-activated PMNs have increased capacity to kill strains of phagocytosis-resistant GAS [6]. Importantly, monoclonal antibody specific for CD11b/CD18 abrogates this enhanced PMN phagocytosis and killing [6]. These findings suggest that C5a peptidase may also block activation of CR3 receptors on the PMN surface, thereby inhibiting complement receptor-mediated phagocytosis. 2.2. GAS blocks opsonization by host proteins Although PMNs ingest non-opsonized particles, uptake is greatly enhanced upon opsonization with host-derived components. Phagocytosis is optimally mediated by receptors that recognize antibody (antibody Fc-receptors, FcRs) and serum complement (complement receptors, CRs) [1]. In immune and non-immune hosts the principle serum complement opsonins, C3b and iC3b, are deposited on the surface of microorganisms. PMNs recognize C3b and iC3b with complement receptors 1 (CR1/CD35) and 3 (CR3, CD11b/CD18, Mac-1), respectively, and efficiently phagocytose and kill most complement-coated microbes. However, GAS has evolved multifarious strategies to disrupt opsonization with antibody and complement. As a result, PMN phagocytosis and killing of GAS is far less effective than that of other bacteria. 2.2.1. GAS M protein The well-characterized surface M protein of GAS plays an important role in resistance to PMN phagocytosis. M protein

is a dimeric coiled-coil containing a NH2-terminal hypervariable region with extensive sequence variability. To date, there are over 120 GAS M serotypes [7]. M protein facilitates resistance to phagocytosis in part by binding to the complement regulatory proteins, such as C4b-binding protein, factor H, and factor H-like protein, thereby impeding the binding of opsonic fragment C3b to the GAS surface [8]. The binding of fibrinogen to M protein inhibits deposition of C3b on the surface of GAS. Furthermore, M protein of serotype M22 sequesters IgA [9]. This additional anti-opsonic mechanism may undermine the effects of immune sera comprised of GAS-specific IgA [9]. Thus, M protein contributes to phagocytosis resistance in the immune and non-immune host. Recently, Herwald and colleagues investigated the role of M protein–fibrinogen binding in PMN activation [10]. GAS M protein–fibrinogen complexes activate PMNs through a mechanism that likely involves cross-linking of b2-integrins. The cross-linking promotes release of PMN inflammatory mediators, including heparin-binding protein, a molecule responsible for excessive vascular leakage during shock. This newly described role for M protein in the modulation of PMN function provides important insight into our understanding of streptococcal toxic shock syndrome. 2.2.2. Streptococcal inhibitor of complement (Sic) Streptococcal inhibitor of complement (Sic) is a 31-kDa protein secreted mostly by serotype M1 GAS [11]. The protein is named aptly for its ability to inhibit the formation of the membrane attack complex (MAC) of complement via binding to C5b-C7 [11]. Sic interferes not only with the major complement effector MAC, but also with processes essential for PMN phagocytosis. This was demonstrated by a decrease in the ability of a sic isogenic mutant GAS strain to resist PMN phagocytosis and subsequent killing compared to the wild-type parental strain [12]. Sic was shown to colocalize with ezrin within human PMNs and bind directly to ezrin in vitro [12]. These data suggest that Sic blocks phagocytosis with a mechanism directed at altering normal PMN cytoskeleton function [12]. In addition to its anti-phagocytic properties, Sic inactivates antibacterial peptides such as lysozyme, LL-37, and defensins, including human neutrophil a-defensin-1 [13,14]. This is an important observation in the context of phagosome–granule fusion, at which time GAS is exposed to numerous antibacterial peptides (Fig. 1). Taken together, these studies indicate that Sic protects GAS from innate host defense by interfering with efficiency of PMN phagocytosis and inactivating antibacterial peptides within phagosomes or in the inflammatory milieu. 2.2.3. Streptococcal pyrogenic exotoxin B (SpeB) Several additional proteins secreted by GAS promote pathogen survival. These proteins include the wellcharacterized streptococcal pyrogenic exotoxin B (SpeB), a highly conserved cysteine protease produced by virtually all strains of GAS [15]. SpeB cleaves human fibronectin, degrades vitronectin, activates a human metalloprotease and

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specific IgG. This mechanism is quite effective at blocking antibody-mediated phagocytosis and killing of GAS in blood [20]. Collectively, impairing opsonization is an important mechanism used by GAS to block phagocytosis by PMNs. 2.3. Physical barriers to neutrophil phagocytosis

Fig. 1. Fusion of neutrophil granules with a GAS phagosome. Thirty minutes after initiation of GAS–PMN interaction, samples were fixed and processed for transmission electron microscopy. The sample was examined at 60 kV with a model CM10 transmission electron microscope (Philips Electronics, Eindhoven, NL). Transmission electron microscopy was performed by D.W. Dorward, Rocky Mountain Laboratories, NIAID.

cleaves human interleukin-1b (IL-1b) precursor to its active inflammatory mediator form [15,16]. Using a mouse model of GAS infection, Lukomski et al. [17] found that a speB mutant strain was readily phagocytosed by PMNs and the infection was effectively cleared. In contrast, the wild-type strain lysed PMNs and disseminated, eventually causing host death. The exact mechanism by which SpeB inhibits PMN phagocytosis has yet to be clearly defined. Perhaps the inability of the GAS speB mutant strain to release C5a peptidase from the surface of GAS [18] explains in part PMN-mediated clearance of the mutant strain in infected mice [17]. Alternatively, SpeB might block neutrophil opsonophagocytosis through a mechanism that targets antibody. SpeB cleaves antigen-specific IgG bound to GAS surface molecules at the antigen-binding site, but not at the Fc region [19]. This is a notable finding, because cleavage of IgG in this manner fails to inhibit binding of M-like proteins to the Fc region of IgG [19]. The association of IgG Fc to GAS M-like proteins creates a “host-like coat” that may physically disrupt complement deposition on the pathogen surface. Therefore, this attribute of SpeB function is perhaps an additional antiphagocytic property [19]. 2.2.4. Endoglycosidase (EndoS) Endoglycosidase (EndoS) is an enzyme secreted by GAS that has recently gained attention for its ability to inhibit immunoglobulin-mediated opsonophagocytosis [20]. EndoS hydrolyzes the conserved N-linked oligosaccharides on heavy chains of IgG, thereby decreasing the binding of GAS-

2.3.1. GAS hyaluronic acid capsule Highly mucoid strains of GAS produce an extensive hyaluronic acid capsule composed of N-acetylglucosamine and glucuronic acid repeats. These mucoid GAS serotypes, such as M18, have been linked epidemiologically to rheumatic fever and invasive GAS disease, implying an important role for capsule in immune evasion [8]. Capsular mutant strains demonstrate altered virulence and loss of phagocytosis resistance, and treatment with hyaluronidase increases susceptibility of encapsulated GAS to PMN phagocytosis [21,22]. Unlike M protein, the anti-phagocytic hyaluronic acid capsule of GAS does not seem to block C3 deposition on the surface of GAS. Rather, it acts as a physical barrier to prohibit direct interaction of PMNs with opsonins on the bacterial surface [22]. 2.3.2. A role for collagen and fibronectin in GAS survival The recruitment of collagen by GAS-bound fibronectin is yet another mechanism used by many strains of GAS to block neutrophil phagocytosis [23]. Collagen-recruiting strains are protected from opsonophagocytosis because deposition of a fibronectin-collagen matrix forms large bacterial aggregates. Bacterial aggregates block phagocytosis by masking opsonic epitopes and increasing overall targetparticle size. Notably, this phenomenon does not occur in the presence of human serum, suggesting that this evasion strategy may be important in GAS tissue infections rather than those that occur in the mucosa or blood [23]. 2.4. Molecular mimicry The versatile leukocyte b2-integrin Mac-1 is involved in regulation of critical PMN functions, including adhesion, migration, phagocytosis, cell signaling and NADPHoxidase-mediated killing [1]. GAS Mac is a secreted protein with homology to the a-subunit of human Mac-1 (CD11b) that inhibits phagocytosis, thereby blocking subsequent ROS production and killing of GAS by human PMNs [24]. Sera from patients with GAS infections confirm that Mac is produced during the course of infection [24]. GAS Mac protein interacts with CD16/CD11b at the neutrophil plasma membrane to inhibit antibody and complement receptor-mediated phagocytosis [24]. The protein was later demonstrated to have proteinase activity against human IgG [25], although the role of the proteinase activity in phagocytosis resistance is unclear [26]. Thus, GAS Mac protein is a host–receptor mimetic that significantly impairs our innate immune response to promote GAS survival.

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Table 1 GAS factors that alter PMN function SPy #a Gene Inhibition of chemotaxis 0165 nga 2010 scpA 2039 speB Inhibition of phagocytosis 0861 mac 1361 slr 1813 ndoS 2016 sic 2018 emm1 2039 speB 2200 hasA 2201 hasB 2202 hasC

Encoded protein

Reference for neutrophil interaction

Streptococcal NAD glycohydrolase C5a peptidase Streptococcal pyrogenic exotoxin B

Stevens et al., J. Infect. Dis. 182 (2000) 1117–1128 O’Connor et al., J. Infect. Dis. 156 (1987) 495–504 Ref. [17]

Streptococcal CD11b homolog Mac Streptococcal leucine-rich protein Endo-beta-N-acetylglucosaminidase (EndoS) Streptococcal inhibitor of complement M protein Streptococcal pyrogenic exotoxin B Hyaluronan synthase (capsule synthesis) UDP-glucose 6-dehydrogenase (capsule synthesis) UTP-glucose-1-P uridylyltransferase (capsule synthesis)

Ref. [24] Reid et al., Infect. Immun. 71 (2003) 7043–7052 Ref. [20] Ref. [12] Jacks-Weis et al., J. Immunol. 128 (1982) 1897–1902 Ref. [17] Ref. [21] Ref. [21] Ref. [21]

Detoxification of ROS 0605 bsaA Glutathione peroxidase 1406 sodA Superoxide dismutase 2079 ahpC Alkylhydroperoxidase 2080 nox1 Alkylhydroperoxidase reductase Resistance to components of neutrophil granules 2016 sic Streptococcal inhibitor of complement Neutrophil lysis 0167 slo Streptolysin O 0738 sagA Streptolysin S Gene regulatory systems involved in modulating neutrophil function 2026 ihk Two-component response regulator histidine kinase 2027 irr Two-component response regulator a

Ref. [27] Ref. [27] Ref. [27] Ref. [27] Ref. [14] Andersen et al., J. Infect. Dis. 141 (1980) 680–685 Bernheimer et al., J. Bacteriol. 87 (1964) 1100–1104 Ref. [27,30] Ref. [27,30]

SPy numbers are derived from GAS serotype M1 strain SF370, Ferretti et al., Proc. Natl. Acad. Sci. USA 98 (2001) 4658–4663.

3. Neutrophil phagocytosis triggers a GAS survival response 3.1. Global changes in GAS gene expression during phagocytosis GAS occupies several niches in the human host, depending on the stage or type of disease. Single serotypes of GAS, such as M1, can cause a variety of diseases ranging in severity from mild infections (pharyngitis) to life-threatening disease (necrotizing fasciitis). These observations suggest that GAS actively responds to changes in the host environment. As we have described, several extracellular molecules contribute to the ability of GAS to resist PMN phagocytosis (Table 1 and Fig. 2). However, only recently has a comprehensive analysis of changes in GAS gene expression during PMN phagocytosis revealed the complexity of the GAS–host cell interaction. Microarray analysis of GAS during phagocytic interaction with human PMNs demonstrated that GAS differentially regulates approximately 20% of its entire genome [27]. Several key observations support the idea that global changes in GAS gene expression are essential for survival in the human host. First, the rate of PMN phagocytosis and killing of GAS is maximal during initial host cell– pathogen interaction (within 30 min) and fails to increase thereafter [2,27]. Second, this increased resistance to PMN phagocytosis is accompanied by an increase in the number of differentially expressed GAS genes [27]. During GAS–PMN

Fig. 2. Topology of GAS factors that alter neutrophil function. GAS (orange) produces several proteins (ovals) that are secreted to the extracellular matrix. Most of these proteins inhibit PMN phagocytosis. GAS also generates surface-bound molecules (white squares) that block phagocytosis. It is not clear how much capsule is shed from the GAS cell surface and therefore not surface-associated. Ihk/Irr (yellow box) is a gene-regulatory system directly linked to inhibition of human PMN function. Ihk/Irr expression is triggered by interaction with human PMNs (gray) and/or associated PMN microbicidal components.

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interaction, GAS up-regulates numerous virulence factors that block phagocytosis, including sic, mac, endoS and speB [27]. These genes likely account for the observed increase in resistance to neutrophil phagocytosis described above. GAS is exposed to neutrophil ROS and antimicrobial granule proteins following phagocytosis. To survive these cytotoxic systems, the pathogen up-regulates genes that moderate oxidative stress, such as alkylhydroperoxidase (ahpC), alkylhydroperoxidase reductase (nox1) and glutathione peroxidase (bsaA) [27]. GAS cell envelope components are also up-regulated during PMN phagocytosis. These findings suggest that a gene-regulated program in GAS is triggered to repair damage incurred by PMN ROS and granule components. The idea that global changes in GAS gene expression patterns are critical for resistance to phagocytosis is also supported by the observation that GAS cultured with human PMNs are significantly more virulent in a murine bacteremia model than in broth-grown organisms [28]. Collectively, these data suggest that the stress imparted on GAS by the hostile environment within PMN phagosomes drives a transcription-regulated GAS survival response.

3.2. GAS Ihk/Irr two-component gene regulatory system facilitates evasion of innate host defense Many pathogenic microorganisms use two-component gene regulatory systems to detect and respond to extracellular signals. A transmembrane sensor protein, usually a histidine kinase, senses extracellular stimuli such as oxidative stress or increased temperature [29]. The sensor autophoshorylates and transfers a phosphoryl group to an aspartyl residue of a response regulator. The response regulator acts as a transcription factor for the target gene(s). Genome-scale analysis of GAS revealed 13 potential two-component systems, yet only three of these systems, CsrR/S, FasBCAX and Ihk/Irr, have been linked to GAS–host cell interactions (reviewed in [29]). Importantly, we discovered that genes encoding a GAS two-component gene regulatory system named Ihk/Irr are up-regulated during PMN phagocytosis [27]. Consistent with the notion that ihk and irr are important for immune evasion, an isogenic irr mutant GAS strain is more susceptible to killing by human PMNs [27], and has significantly attenuated virulence in mouse models of GAS soft tissue infection and bacteremia [30]. The increased PMN killing was not attributed to a decrease in phagocytosis nor altered PMN activation. Rather, the irr mutant strain is destroyed more rapidly than the wild-type strain following ingestion [27]. Subsequent work demonstrated that the mutant strain is more susceptible to killing by neutrophil a-granule components and hydrogen peroxide compared with the parental wild-type strain [30]. Collectively, these observations suggest that the mutant strain is in some way unable to moderate the effects of innate immune microbicidal systems.

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3.3. Neutrophil microbicidal components trigger a GAS survival response Consistent with the findings during GAS–PMN interaction, we discovered that neutrophil a-granule components and hydrogen peroxide up-regulate expression of ihk and irr in several GAS M serotypes [30]. Inasmuch as antibacterial proteins and ROS trigger expression of ihk and irr (either directly or indirectly), it might be predicted that genes influenced by Ihk/Irr moderate oxidative stress and regulate cell envelope synthesis. Using comparative microarray analysis of the irr mutant and wild-type GAS strains, we found that expression of genes involved in ROS detoxification as well as cell envelope synthesis are down-regulated [30]. Thus, Ihk/Irr protects GAS from critical neutrophil-derived effector mechanisms, including cationic granule components that target cell envelope, and ROS. These data suggest that Ihk/Irr engages a GAS survival response during PMN phagocytosis.

4. GAS exploits neutrophil apoptosis Modulation of PMN apoptosis or cell fate by bacteria has emerged as a mechanism of pathogenesis [3]. Apoptosis represents the final phase of transcription-regulated neutrophil maturation and superintends multiple processes in these host cells, including resolution of proinflammatory processes [2,3]. Detailed analyses of global changes in PMN gene expression during phagocytosis of bacterial pathogens revealed that GAS induces changes in host cell transcript levels not seen with other pathogens tested [2]. GAS–PMN interaction altered expression of more than 25 neutrophil genes involved in an interferon-response pathway, and over 70 apoptosis and/or cell fate-related genes [2]. Consistent with those observations, GAS greatly accelerates apoptosis in human PMNs compared with other bacterial pathogens studied. The current model of host–pathogen interaction indicates that PMN apoptosis is critical for the resolution of bacterial infections and inflammation [3]. Exploitation of PMN apoptosis by GAS would result in reduced exposure (of the pathogen) to antimicrobial peptides and ROS due to premature PMN lysis, thereby facilitating GAS survival and dissemination.

5. Conclusions GAS is well-equipped to modulate PMN function. To avoid phagocytosis, GAS produces molecules that target critical processes in nearly every stage of the innate host response to infection, including PMN recruitment (Table 1 and Fig. 2). The pathogen produces factors that destroy important host opsonins, such as antibody and complement, critical for efficient ingestion by neutrophils. GAS secretes protein components that interact directly with neutrophils to inhibit phagocytosis. The GAS surface is a barrier to PMN

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uptake, and binds multiple host factors, such as fibrinogen and collagen, to form phagocytosis-resistant bacterial aggregates. Although GAS employs numerous mechanisms to block phagocytosis, the pathogen is only partially successful and is ingested by adherent PMNs or in the presence of specific antibody. Following phagocytosis, PMNs produce highly cytotoxic ROS within phagocytic vacuoles and enrich phagosomes with high concentrations of potent antibacterial peptides that normally kill most bacteria. Nonetheless, several groups report that a percentage of GAS phagocytosed by PMNs survive the harsh intraphagosomal environment [2,27,28,31]. This phenomenon can be explained in part by our recent work describing a GAS survival response following PMN phagocytosis [27,30]. Interaction with human PMNs induces global changes in GAS gene expression, including upregulation of genes involved in cell envelope biosynthesis and oxidative stress [27]. Notably, the survival response is triggered by neutrophil microbicidal systems and regulated by GAS Ihk/Irr [30]. Lackluster phagocytosis combined with prolonged GAS survival during PMN interaction is presumably crucial to altering normal neutrophil turnover at sites of infection. GAS rapidly accelerates neutrophil apoptosis in vitro to cause premature host cell lysis, an event that would facilitate dissemination of the pathogen in vivo. Taken together, the overwhelming evidence indicates that modulation of neutrophil function is a key element of GAS pathogenesis. Genome-wide analyses of host–pathogen interactions have provided new insight into human disease. One of the most important components of these types of analyses is the identification of novel candidates for therapeutic interventions. Many of the GAS molecules identified in recent wholegenome sequencing efforts, global gene expression analyses, or proteomics are potential vaccine antigens and/or targets for therapeutic maneuvers designed to control infections. The GAS–host relationship is indeed complex, but with recent advances in genomics and genome-scale methodologies, it is an auspicious time to further our understanding of host–pathogen interactions.

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