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Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016-.
Group A streptococci produce a wide variety of secreted extracellular products, many of which are considered virulence factors. The number of these potential virulence factors exceeds that of many other pathogens, and probably correlates to this organism’s ability to be a successful pathogen. This chapter reviews those virulence factors that are secreted and released from the streptococcus, including: streptokinase, proteinases, esterase, the hemolysins SLO and SLS, CAMP factor, DNases, hyaluronidases, complement inhibitor, superoxide dismutase, and immunoglobulin degrading enzymes. Many of these presumed virulence factors may also function as digestive enzymes that provide the bacteria with nutrients from the host.
The group A streptococci produce a variety of extracellular products, many of which are secreted proteins that are often considered to be virulence factors. Historically, many of these proteins have been studied, either individually or in isolation, to determine their mechanism of action in evading host defenses, as well as to determine possible candidate antigens for a potential vaccine. The total number of these potential virulence factors exceeds that of many other pathogens—and probably correlates with this organism’s ability to be a successful pathogen. How each or any of these virulence factors functions in the disease process is not fully understood; it is unlikely that all these factors play a role in all diseases. The role of various virulence factors in some group A streptococcal diseases has recently been reviewed (Reglinski & Sriskandan, 2014; Walker, et al., 2014).
Recent genomic studies have provided a great deal of new information about these extracellular products at the cell-system level since many of their genes have previously been individually cloned in order to study the specific proteins and their role in pathogenesis. The number of secreted proteins produced can vary among serotypes and strains, since some genomes either do not contain the specific gene for a particular protein, or the gene has mutations that do not allow for proper transcription and/or translation. Additionally, differences in the functional regulatory networks between different strains will influence the number and types of products produced. Another reason for the variation in secreted proteins among different strains is that some of the extracellular protein genes are chromosomally located, while others are encoded in prophages that are located in the genome.
This chapter reviews those virulence factors that are secreted and released from the streptococcus; that is, the released extracellular products (Figure 1, Table 1). Other virulence factors, those that are cell bound or surface associated, are discussed elsewhere (Hynes, 2004), as well as in various chapters of this book (as in the chapters by Fischetti, Chaussee, Vega, and Cattoir). Additionally, the mechanism of transport and secretion of the various virulence factors are also discussed elsewhere in this work. Most (but not all) of the prophage encoded genes specify superantigens, such as the streptococcal exotoxins SpeA, SpeC, SpeH, SpeI, SpeK, and streptococcus superantigen (SSA) are described further in the chapter on superantigens.
Secreted Extracellular Virulence Factors based on hypothesized effects*
Streptokinase (Ska) is a single-chain 414 amino acid protein, secreted by group A, C, and G streptococci, that has an activity similar to two host proteins (urokinase-type and tissue-type plasminogen activators), in that it non-enzymatically converts inactive plasminogen to proteolytically active plasmin. Plasminogen is a single-chain glycoprotein zymogen of plasmin, a serine protease. Plasminogen is a key component of the fibrinolytic system and is found in plasma and extracellular fluids, with activation to plasmin having a number of effects. Although streptokinase is a plasminogen activator protein, it is not itself a protease. Streptokinase activates plasminogen non-enzymatically; the binding of streptokinase to plasminogen induces a conformational change that results in the formation of an active site in the streptokinase-plasminogen complex. Within this complex, the intermolecular cleavage of plasminogen to plasmin occurs. The enzymatically active complex then proteolytically converts its substrate, plasminogen, to plasmin (Boxrud & Bock, 2004; Boxrud, Fay, & Bock, 2000). Figure 2 presents a hypothesized mechanism of plasminogen (Pg) activation by streptokinase (SK) using a two-cycle catalytic pathway (Nolan, Bouldin, & Bock, 2013).
The streptokinase gene from a group C strain was the first streptococcal gene to be cloned and sequenced (Malke & Ferretti, 1984; Malke, Roe, & Ferretti, 1985) and was shown to be 85% identical in amino acid sequence to that of the group A streptokinase (Huang, Malke, & Ferretti, 1989b), with heterogeneity seen in the genes (Huang, Malke, & Ferretti, 1989a). Since that time, streptokinase has been shown to be composed of three distinct domains: α (aa 1–150), β (aa 151–287) and γ (aa 288–414) (Kalia & Bessen, 2004; Wang, Lin, Loy, Tang, & Zhang, 1998) separated by coiled-coil regions with the N- and C- termini of the protein being disordered flexible structures (Wang, Lin, Loy, Tang, & Zhang, 1998). The β-domains of streptokinases from different strains of group A streptococci show sequence variability (55% identity), while the α- and γ-domains showed higher levels of conservation (77% and 84% identity) (Zhang, Liang, Glinton, Ploplis, & Castellino, 2013). Phylogenetic studies indicate that sequence variation in the β-domains divides the streptokinases into 2 clusters (SK1 and SK2); within the SK2 cluster, the sequences can be further subdivided into SK2a/SK2b (Kalia & Bessen, 2004; McArthur, et al., 2008; Zhang, Liang, Glinton, Ploplis, & Castellino, 2013). Phylogenetic studies also found that the streptokinases form discrete lineages associated with tissue specific emm patterns of the organism (McArthur, et al., 2008). SK2a-expressing strains are nasopharngeal isolates, many of which express fibrinogen binding M protein, while SK2b is secreted by skin-tropic strains that express plasminogen-binding M protein (Kalia & Bessen, 2004; Zhang, Liang, Glinton, Ploplis, & Castellino, 2013). The sequence variation observed among the different streptokinases leads to structural β-domain differences that lead to functional differences in SK1, SK2a, and SK2b plasminogen activation. Variations in the streptokinase sequences of different isolates of S. pyogenes results in distinct plasminogen activation pathways being used by the pathogen, which can directly affect the pathogenesis of this organism (Cook, Skora, Gillen, Walker, & McArthur, 2012). Isogenic chimeric strains (streptokinase and M protein) were shown to influence streptococcal virulence by changes in plasminogen activation and plasmin binding; virulence was partially explained by disparate plasminogen activation, coupled with the M protein of the strain (Chandrahas, et al., 2015). In addition to the different activation pathways, it is also important to note that streptokinase expressed by human isolates is only active against human plasminogen (Marcum & Kline, 1983; Sun, et al., 2004). The importance of activation of (human) plasminogen was shown experimentally by using transgenic mice. Mice expressing human plasminogen were more susceptible to group A streptococci expressing streptokinase than mice expressing their own plasminogen, or those without streptokinase expression (Marcum & Kline, 1983; Sun, et al., 2004).
The interaction of streptokinase with the plasminogen activation systems of the host is considered to be a virulence mechanism for the pathogenesis of this organism (Khil, et al., 2003; Sun, et al., 2004; Sun, et al., 2012; Walker, McArthur, McKay, & Ranson, 2005). Not only does S. pyogenes produce streptokinase for activating plasminogen, but the organism also binds plasminogen and plasmin to the cell surface via numerous cell wall-associated proteins such as M proteins, glyceraldehyde-3-phosphate, and streptococcal enolase (Cole, Barnett, Nizet, & Walker, 2011; Gase, Gase, Schirmer, & Malke, 1996; Lähteenmäki, Kuusela, & Korhonen, 2001; Malke, Mechold, Gase, & Gerlach, 1994). These cell surface-bound proteins, along with streptokinase, result in the availability of both soluble and cell-bound plasmin activity to the invading pathogen. Plasmin also binds and cleaves fibrinogen, the product of which (fibrinogen D domain fragments) can interact with the cell surface fibrinogen receptors, and in doing, so mediate the acquisition of plasmin onto the bacterial surface (Sanderson-Smith, De Oliveira, Ranson, & McArthur, 2012).
The generation of plasmin from plasminogen at the infection site may result in the activation of host matrix metalloproteinases that lead to fibrinolysis and degradation of extracellular matrix and basement membrane components, which allow streptococci to spread from its primary or initial site of infection into surrounding sites (Sun, et al., 2004). The initial host response is to confine the infection, but by degradation of the extracellular matrix, tissue barriers, and degradation of fibrin networks, plasmin plays a role in spreading the bacteria to other sites in the body (Walker, McArthur, McKay, & Ranson, 2005). In addition, streptokinase has been shown to induce inflammation by complement activation, which may play a role in post-infectious sequelae of S. pyogenes; streptokinase has also been suggested to play a role in acute poststreptococcal glomerulonephritis (APSGN) (Nordstrand, Norgren, Ferretti, & Holm, 1998; Ohkuni, et al., 1991). While playing this apparently important role in virulence, streptokinase may also be degraded by another virulence factor, the cysteine proteinase SpeB (discussed below), which is produced by the same organism (Sumby, Whitney, Graviss, Deleo, & Musser, 2006).
Expression of the streptokinase gene, ska, is negatively controlled by the CovRS regulatory system (Walker, et al., 2014), with phosphorylated CovR repressing the expression of ska (Treviño, et al., 2009); ska expression is upregulated in covRS mutants (Sumby, Whitney, Graviss, Deleo, & Musser, 2006). In addition to the CovRS system, streptokinase expression is also controlled by the FasBCA regulatory system in S. pyogenes, and similarly in S. equisimilis (Kreikemeyer, Boyle, Buttaro, Heinemann, & Podbielski, 2001; Malke & Steiner, 2004; Steiner & Malke, 2002). This regulation is through FasA acting indirectly by controlling FasX, a sRNA that post-transcriptionally regulates streptokinase production. It does this by enhancing ska transcript stability, which results in an approximately 10-fold increase in streptokinase activity (Ramirez-Peña, Treviño, Liu, Perez, & Sumby, 2010).
The cysteine proteinase, SpeB, is also known as streptococcal pyrogenic erythrogenic toxin B or streptococcal cysteine proteinase. The proteinase was originally thought to be two distinct proteins, one with pyrogenic activity and the other with proteinase activity. However, when it was cloned, it was found that “both activities” were encoded by the same gene, which is designated speB (Bohach, Hauser, & Schlievert, 1988; Gerlach, Knöll, Köhler, Ozegowski, & Hríbalova, 1983). Cloning allowed for the analysis of the protein in the absence of any contaminating streptococcal proteins, and showed the proteinase precursor to be both pyrogenic and mitogenic (Gerlach, Knöll, Köhler, Ozegowski, & Hríbalova, 1983). Although the SpeB protein is not an exotoxin, the SpeB designation has been retained over the years and is still used in the current literature. The streptococcal cysteine proteinase is produced as an inactive zymogen (40 kDa) that undergoes autocatalytic cleavage and conversion to a mature 28 kDa active enzyme (Chen, et al., 2003; Liu & Elliott, 1965a; Musser, Stockbauer, Kapur, & Rudgers, 1996). In addition to cleavage, the protein also needs to be reduced (cysteine residue) in order to be active (Liu & Elliott, 1965b; Musser, Stockbauer, Kapur, & Rudgers, 1996). In culture, it is produced in the late log to the early stationary phase, in response to starvation (Chaussee, Phillips, & Ferretti, 1997). The crystal structure of the zymogen form of the enzyme revealed it to be a distant homolog of the papain superfamily (Kagawa, et al., 2000). Additionally, the structure revealed an integrin-binding motif located on the protein surface of some isolates (M1 and 20% of other isolates) and suggested a link to the pathogenesis of invasive streptococcal strains (Kagawa, et al., 2000). This proteinase is one of a number of proteolytic enzymes produced by S. pyogenes; others include the C5a peptidase, the cell-associated aspartic apoproteinase or opacity factor (see the chapter on antibiotic resistance), a serine protease, and other cysteine proteinases, such as IdeS (Rasmussen & Björck, 2002).
The cysteine proteinase of S. pyogenes is one of the most studied of this organism’s virulence factors, but its role in pathogenesis is still not fully understood (Nelson, Garbe, & Collin, 2011). The highly conserved gene for the proteinase is found in all strains of group A streptococci (Chaussee, Phillips, & Ferretti, 1997; Yu & Ferretti, 1991) and has been implicated in multiple studies as having a role in virulence, yet other studies suggest that it has no obvious role. The proteinase has broad specificity, as it is able to degrade a number of host proteins (such as cytokines, chemokines, complement components, various immunoglobulins, protease inhibitors found in the serum, and extracellular matrix components), as well as streptococcal proteins (Nelson, Garbe, & Collin, 2011).
One role of proteinases in bacterial virulence is attributed to their abilities to facilitate or enhance bacterial spread by degradation of tissue structure; this degradation allows for either the organism or its products to spread in the host. In the case of the streptococcal cysteine proteinase, different roles in virulence have been suggested, including effects on degradation of host extracellular matrix material, effects on the immune system, and effects on proteins of the producer strain; however, other results contradict some of these findings (Hynes W. , 2004; Nelson, Garbe, & Collin, 2011; Walker, et al., 2014). The proteinase has been shown to be required for growth in saliva (Shelburne, 3rd, et al., 2005), the establishment of skin infections (Cole, et al., 2006; Svensson, et al., 2000), and virulence in a mouse model (Cole, et al., 2006). In the case of S. pyogenes M1T1, survival at the site of local infection requires SpeB (Cole, et al., 2006). However, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system, which results in decreased systemic spread; decreased streptococcal proteinase activity allows for increased surface binding of plasmin, which can result in an increase in the systemic spread of the organism (Cole, et al., 2006; Sumby, Whitney, Graviss, Deleo, & Musser, 2006).
The cysteine proteinase also degrades human cathelicidin LL-37, a cationic peptide component of the innate immune system (Nyberg, Rasmussen, & Björck, 2004); this peptide is also degraded by surface-bound plasmin (Hollands, et al., 2012) that is partially controlled by the level of SpeB. Retention of proteinase activity at the bacterial surface protects against killing by the antibacterial peptide LL-37 (Nyberg, Rasmussen, & Björck, 2004). The enzyme can also be retained on the bacterial surface by being trapped by alpha2-M-binding protein bound to the protein G-related alpha2-M-binding protein (GRAB) (Rasmussen, Müller, & Björck, 1999); GRAB also protects virulence factors from degradation by both host and bacterial proteases. The proteinase also promotes cleavage of H-kininogen, which has a role in kinin activation and inflammation (Herwald, Collin, Müller-Esterl, & Björck, 1996). Another function assigned to the cysteine protease has been as an immunoglobulin-degrading enzyme that is capable of degrading IgG, IgA, IgM, IgD and IgE (Collin & Olsén, 2001a); however, recent results indicate that SpeB shows no immunoglobulin degrading activity under physiological conditions (Persson, Vindebro, & von Pawel-Rammingen, 2013). SpeB has also been suggested to play a role in APSGN, perhaps as the much sought-after nephritis-associated antigen; evidence suggests that SpeB is the major antigen involved in the pathogenesis of most cases of APSGN (Batsford, Mezzano, Mihatsch, Schlitz, & Rodríguez-Iturbe, 2005).
SpeB has many conflicting immune-response–related activities: it induces inflammation but possesses anti-inflammatory properties, it inhibits recruitment of neutrophils to an infection site while preventing degradation of neutrophil traps, it cleaves IgG while inhibiting other antibody-degrading enzymes, and it both activates and inhibits complement activation (Nelson, Garbe, & Collin, 2011). One question that still remains is whether the proteinase would be active under the physiological conditions found during an infection (Nelson, Garbe, & Collin, 2011).
Cysteine proteinase production is positively regulated by the two-component regulatory system CovRS (Walker, et al., 2014), with non-phosphorylated CovR repressing the expression of speB (Treviño, et al., 2009). Mutations in the CovRS system result in lack of production of the proteinase, and such variants have been shown to have an enhanced ability to evade innate immune responses (Li, et al., 2014). Recent results have shown that the divalent metals zinc and copper post-translationally inhibit SpeB activity (Chella Krishnan, Mukundan, Landero Figueroa, Caruso, & Kotb, 2014). The authors suggest that availability of zinc and/or copper in the bacterial microenvironment may modulate the SpeB activity that protects other virulence factors essential for bacterial survival and dissemination within the host. The proteinase has the ability to change the phenotype of the producing strain, not only by indirectly affecting transcription, but also by various post-translational events, such as releasing proteins from the bacterial surface, modifying them to altered forms (active or inactive), or degrading them to terminate activity (Nelson, Garbe, & Collin, 2011).
Streptococcus pyogenes secretes a serine proteinase, SpyCEP, (Streptococcus pyogenes cell envelope protease, prtS) capable of cleaving and inactivating neutrophil chemokines in a specific manner (Lawrenson & Sriskandan, 2013); in particular the chemokine CXCL-8/IL-8, which the host uses to recruit polymorphonucleocytes when confronted with a microbial challenge. SpyCEP is able to cleave all human CXC chemokines that contain the ELR motif (Lawrenson & Sriskandan, 2013; Zingaretti, et al., 2010); this specificity makes it unique among the many proteinases produced by group A streptococci. SpyCEP cleaves CXCL8/IL-8 at the C-terminal α-helix; this results in diminished function of this host-manufactured signal protein (Kurupati, et al., 2010).
SpyCEP has been determined to exist in secreted, as well as cell-associated forms; it is produced as a large immature form, 170kDa, which is composed of two polypeptide fragments (Zingaretti, et al., 2010). The protein undergoes an autocatalytic cleavage, but the two fragments of SpyCEP act in concert to form an active protease (Zingaretti, et al., 2010). SpyCEP is produced throughout exponential growth, as it is found in both the supernatant and in the cell wall (Turner, Kurupati, Jones, Edwards, & Sriskandan, 2009). The way in which the proteinase is released from the cell wall is unknown, but there is no evidence for either an autocatalytic release or cleavage mediated by an alternative protease (Lawrenson & Sriskandan, 2013). SpyCEP transcription appears to be under the regulation of the CovR/CovS two-component regulatory system (Sumby, et al., 2008).
A variety of pathogenic streptococci produce homologs of SpyCEP, including S. equi, S. pneumoniae, S. agalactiae, and S. suis (Lawrenson & Sriskandan, 2013). In addition, the C5a peptidase of S. pyogenes is considered to be a homolog of SpyCEP. The C5a peptidase, ScpA, is a cell-bound peptidase anchored to the cell wall by sortase A (Raz & Fischetti, 2008) that inactivates the complement factor C5a, which is responsible for stimulating polymorphonuclear leukocytes to migrate to the site of infection (Kagawa & Cooney, 2013). As discussed in the chapter on vaccines, C5a peptidase has shown promise as a potential vaccine component.
The widespread distribution of homologs of this proteinase suggests more than a passing involvement in pathogenesis, although studies with mutants that lack SpyCEP have found differing results (Hidalgo-Grass, et al., 2006; Kurupati, et al., 2010; Sjölinder, et al., 2008; Sumby, et al., 2008). Studies have demonstrated that the expression of SpyCEP both impedes bacterial clearance and assists in bacterial spread to the regional lymph node, as well as in systemic circulation. When SpyCEP was introduced into and expressed by L. lactis, hallmark features of a group A streptococcal-induced systemic infection occurred in a mouse model using the strains expressing SpyCEP, but not the wild-type L. lactis strain (Kurupati, et al., 2010).
Streptococcus secreted esterase, SsE, is an extracellular product that appears to have a role in virulence and pathogenesis, being essential for invasive infections and systemic dissemination (Zhu, Liu, Sumby, & Lei, 2009). SsE is a carboxylic acid esterase similar to that found in other organisms that hydrolyzes platelet-activating factor (PAF). PAF is a receptor and serves as a phospholipid mediator manufactured by endothelial cells, neutrophils, macrophages, and granular eosinophiles. PAF mediates IL-12-induced chemotaxis of natural killer cells and neutrophils, and can induce the migration of neutrophils to an exposed endothelium. SsE has been demonstrated to diminish the solicitation and the recruitment of neutrophils, which provides a mechanism for bacterial evasion of the host immune system (Liu, et al., 2012). The esterase has been shown to be important in the virulence of group A streptococci, as it has an important role in subcutaneous infections and dissemination from the skin, though its role has yet to be fully determined (Zhu, Liu, Sumby, & Lei, 2009).
Two distinct variants of SsE have been reported, and are described as either complex I or complex II. Complex I esterases are produced by serotypes M1, M2, M3, M5, M6, M12, and M18, while complex II proteins are produced by serotypes M4, M28, and M49. The two SsE complexes share greater than 98% identity in their amino acid sequence, but can have a sequence variation of up to 37% between the complexes. SsE appears to be under the control of the CovR/CovS two-component regulatory system (Liu, Liu, Xie, & Lei, 2013), as it is negatively controlled by the regulator (Zhu, Liu, Sumby, & Lei, 2009).
Streptococcus pyogenes secretes two well-known hemolysins, streptolysin O and streptolysin S, which have effects on a variety of cell types. Other putative hemolysins, including CAMP factor, may also be encoded in the genomes of group A streptococci (Ferretti, et al., 2001), but their role in bacterial growth and virulence remains to be determined.
Streptolysin O (SLO) is a pore-forming, cholesterol-dependent, oxygen-labile, thiol-activated cytotoxin (Tweten, 2005). Similar types of hemolysins are produced by a variety of other pathogens, and the structure of SLO is similar to these other cholesterol-dependent cytolysins, but there are also some differences (Feil, Ascher, Kuiper, Tweten, & Parker, 2014). One difference is in the binding of the cytolysins to cholesterol-rich membranes, where there is a structural difference in the membrane-binding interface between SLO and perfringolysin O (Farrand, et al., 2015). The SLO hemolysin is 69 kDa in size, which is subject to N-terminal cleavage by the cysteine proteinase (Pinkney, et al., 1995). The hemolysin is produced with a 70-residue N-terminal region that is required for the translocation of another streptococcal product, the NAD-glycohydrolase (nga) into host cells (Madden, Ruiz, & Caparon, 2001); with slo and nga being co-transcribed (Madden, Ruiz, & Caparon, 2001). Streptolysin O pore formation occurs in stages, including cholesterol-dependent binding of monomeric forms to the cell membrane, followed by oligomerization, which results in the development of pores (Bhakdi & Tranum-Jensen, 1985; Bhakdi, Tranum-Jensen, & Sziegoleit, 1985). In addition to cholesterol, the membrane-binding domain of SLO also implicates a glycan (galactose) receptor involvement in binding and pore formation (Mozola & Caparon, 2015; Shewell, et al., 2014). These pores result in disruption of the integrity of host cell membranes and induce apoptosis (Timmer, et al., 2009). An alternate pathway utilized by S. pyogenes adhering to cells does not involve the galactose receptor, but an unknown receptor that associates with the streptococcal NAD-glycohydrolase (NADase, Nga, or SPN); this results in translocation of the NADase and orients the SLO, which allows for pore formation (Mozola & Caparon, 2015). Streptolysin O has also been shown to induce intracellular Ca2+ oscillations that result from the depletion of intracellular stores and activation of store-operated Ca2+ in host cells, the mechanisms of which remain unknown (Usmani, et al., 2012).
An interesting function of SLO is that it is required for the transfer of streptococcal NAD-glycohydrolase into epithelial cells (Madden, Ruiz, & Caparon, 2001). The NADase of group A streptococci is encoded by the nga gene, which is found next to the slo gene, and was originally reported as being an unusual glycohydrolase with three activities. However, on reevaluation, it was shown to possess only the β-NAD hydrolytic activity (Ghosh, Anderson, Chandrasekaran, & Caparon, 2010). Like SLO, NADase is encoded on a 36 kb chromosomal region of M1T1 that was acquired prior to the global dissemination of the invasive clone commonly found in developed countries (Walker, et al., 2014).
The structure of NADase showed two functional domains; the amino terminal domain responsible for translocation via the SLO-mediated system, and the carboxyl terminal domain that contains the NADase activity (Ghosh & Caparon, 2006). However, pore formation, per se, by SLO is not required for the transport of the NADase (Magassa, Chandrasekaran, & Caparon, 2010). The role of NADase is not fully understood, but data suggests its function in pathogenesis is as a NAD-glycohydrolase (Ghosh, Anderson, Chandrasekaran, & Caparon, 2010) when injected across the membrane into the cytoplasm, using the cytolysin mediated translocation pathway. Once in the cytoplasm, the toxin functions to deplete the intracellular pool of NAD (Ghosh, Anderson, Chandrasekaran, & Caparon, 2010; Yoon, et al., 2013). In S. pyogenes, the SLO operon encodes genes for NAD-glycohydrolase (nga) and its inhibitor (ifs) (Kimoto, Fujii, Hirano, Yokota, & Taketo, 2006; Madden, Ruiz, & Caparon, 2001). As bacterial cells are also susceptible to the action of NADase, the immunity factor for the glycohydrolase functions as a competitive inhibitor of enzyme function (Kimoto, Fujii, Hirano, Yokota, & Taketo, 2006). The intracellular enzyme-inhibitor complex is dissociated during transport through the streptococcal membrane and into the host cell (Yoon, et al., 2013). Although all strains appear to possess the nga gene, some isolates produce a protein that lacks NADase activity (Ajdic, McShan, Savic, Gerlach, & Ferretti, 2000). NADase without activity had a toxic effect on E. coli but not on S. pyogenes; the reason for this is currently unknown, and has been attributable to an unknown NADase-independent function of the protein. Similarly, little is known about any role of this form of the protein in pathogenesis, although NADase has been associated with cytotoxic activity (Michos, et al., 2006); no association is seen between NADase subtype (active or inactive) and particular disease category or invasiveness (Riddle, Bessen, & Caparon, 2010). Additionally, no association was found between the NADase-inactive nga allele and virulence in a mouse infection model (Tatsuno, Isaka, & Hasegawa, 2013). Notably, there does appear to be an association between tissue tropism and NADase subtype where emm-types that act as generalists (those that infect both throat and skin) tend to be NADase-active, while the specialists (those that infect either throat or skin) are associated with production of NADase-inactive forms (Riddle, Bessen, & Caparon, 2010).
Delivery of NAD-glycohydrolase to the cytoplasm of human cells results in major changes in host cell biology that enhance streptococcal pathogenicity and intracellular survival (Bricker, Cywes, Ashbaugh, & Wessels, 2002; O'Seaghdha & Wessels, 2013). Mutants deficient in SLO and/or NADase also showed impaired survival in macrophages, with both proteins being necessary for resistance to macrophage-mediated killing; survival was mediated by preventing the acidification of the phagolysosome (Bastiat-Sempe, Love, Lomayesva, & Wessels, 2014). When taken up by a macrophage, the phagosome that contains bacteria fuses with a cellular lysosome, creating a phagolysosome, which normally results in destruction of an invading bacterium. With S. pyogenes, SLO and NADase are secreted into the phagolysosome; SLO prevents acidification, while the NADase is translocated into the macrophage cytosol, via a specific translocation mechanism that involves the N-terminal domain of the protein, where it hydrolyzes NAD and thereby interferes with the cell’s ability to repair damage to the phagolysosome membrane (Bastiat-Sempe, Love, Lomayesva, & Wessels, 2014).
In addition to acting as a means of introducing NADase into host cells, other roles for SLO in S. pyogenes pathogenesis have been reported. SLO mutants appear attenuated for virulence (Walker, et al., 2014), with mutants varying in their ability to have an effect or cause disease, with higher expression levels being seen in invasive isolates compared to non-invasive isolates (Ato, Ikebe, Kawabata, Takemori, & Watanabe, 2008). SLO activity enhances mucosal inflammation through tissue destruction (Brosnahan & Schlievert, 2011; Reglinski & Sriskandan, 2014); it also induces co-aggregation of platelets and neutrophils, which may be important in tissue viability in severe infections that involve tissue destruction (Bryant, et al., 2005). Expression of SLO is also required for induction of caspase-1-dependent release of IL-1β, which is important in the development of an inflammatory and immune response (Harder, et al., 2009). Additionally, SLO activates human polymorphonuclear neutrophils that can result in an exaggerated host response (Nilsson, et al., 2006), as well as modulates cytokine synthesis in human peripheral blood mononuclear cells (Stevens & Bryant, 1997). In addition, unlike the other major streptococcal hemolysin (SLS), SLO induces an immune response during infection, and antistreptolysin O antibodies are still used to confirm streptococcal infections (Sheeler, Houston, Radke, Dale, & Adamson, 2002). SLO is extremely toxic, but its immunogenicity could make it useful in vaccine development. Mutated SLO that lacks hemolytic activity has been shown to reduce virulence and has a decreased capacity to destroy immune cells in mouse models of infection. When the mutated version of the toxin was used to immunize mice, the toxoid provided protection against the wild-type strain by antibody-mediated neutralization (Chiarot, et al., 2013).
S. pyogenes can be internalized by epithelial cells as part of the host defense; SLO has been shown to interfere with this internalization process through membrane perturbation and disruption of the clathrin-dependent uptake pathway (Logsdon, Håkansson, Cortés, & Wessels, 2011). Mutation of single amino acids within the structural domains of SLO can affect activity; a double mutant was found to have no toxicity with an impaired ability to bind to eurkayotic cells and was unable to form the required oligomeric structures in the membrane (Chiarot, et al., 2013).
Full expression of SLO and the NADase in non-immune human blood requires the regulator CodY, with decreased transcript levels of each protein being seen in a CodY mutant in an incubation-time–dependent manner (Malke & Ferretti, 2007).
Streptolysin S, SLS, is the second type of hemolysin produced by S. pyogenes, and was originally extracted from streptococcal cells grown in the presence of serum, hence its name. It is a member of a family of proteins known as the thiazole/oxazole-modified microcins, which are produced by a number of pathogens that show hemolytic exotoxin activity (Molloy, Cotter, Hill, Mitchell, & Ross, 2011). SLS is an oxygen stable cytotoxin that forms hydrophilic pores in a variety of cell types from both the innate and adaptive immune systems, including erythrocytes, leukocytes, and platelets. In addition, pores are formed in sub-cellular organelles (Ginsburg, 1972; Miyoshi-Akiyama, et al., 2005; Molloy, Cotter, Hill, Mitchell, & Ross, 2011; Ofek, Bergner-Rabinowitz, & Ginsburg, 1970). However, while protoplasts and spheroplasts are lysed by SLS, bacteria with intact cell walls are not (Bernheimer, 1966). Although its toxicity is not fully understood, it is believed that SLS acts through the accumulation of proteins in the membrane, which leads to transmembrane pore formation that results in osmotic lysis, in a mechanism similar to that mediated by complement (Carr, Sledjeski, Podbielski, Boyle, & Kreikemeyer, 2001).
SLS is a 2.7 kDa ribosomally synthesized, post-translationally, and extensively modified (prior to export) peptide (Molloy, Cotter, Hill, Mitchell, & Ross, 2011) encoded by a nine-gene operon locus (sagA to sagI) (Nizet, et al., 2000). The modifications result in the formation of a peptide with an unusual structure (a heterocyclic compound) that is only cytolytic when associated with a cell surface or in the presence of “carrier” molecules (Molloy, Cotter, Hill, Mitchell, & Ross, 2011). It has recently been shown that some HIV protease inhibitors are able to stop SLS production by blocking a proteolytic cleavage, by SagE, in SLS production (Maxson, et al., 2015). Agents such as these can provide reversible control of production, which may allow for investigation of the role of SLS in virulence without the need for genetic manipulation of the bacteria. The peptide, either because of its small size or modified structure, is not immunogenic when produced during an infection (Dale, Chiang, Hasty, & Courtney, 2002), however, antibodies raised to a synthetic peptide were able to neutralize the hemolytic activity of SLS (Dale, Chiang, Hasty, & Courtney, 2002).
Streptolysin S contributes to the pathogenesis of S. pyogenes in a variety of ways, including cytotoxicity, activation inflammatory response, and inhibition phagocytosis (Ginsburg, 1999). SLS-deficient mutants generated from clinical isolates by transposon mutagenesis showed reduced virulence, as compared to the parental strains (Betschel, Borgia, Barg, Low, & De Azavedo, 1998). Both of the streptolysins (SLO and SLS) have been reported to enhance virulence, with SLS enhancing the virulence of acapsular S. pyogenes (Sierig, Cywes, Wessels, & Ashbaugh, 2003). Although SLS mutants show reduced virulence in lab situations (tissue culture or animal models), non-hemolytic mutants that result from naturally occurring mutations or deletions in the SLS biosynthetic operon are still able to cause severe soft tissue infections (Jantsch, et al., 2013), pharyngeal infections, and otitis media (Yoshino, et al., 2010).
SLS is considered to be one of the two major secreted virulence factors of S. pyogenes, along with the cysteine protease, SpeB. The role of each of these in virulence is still somewhat unclear, but recent results have indicated that they act synergistically in mouse models; SLS is important to mouse mortality, while SpeB is more important to local tissue damage (Hung, et al., 2012). One possible function for SLS is facilitation of invasion of the pathogen through degradation of epithelial intercellular junctions (Sumitomo, et al., 2011); this degradation would occur with the assistance of streptococcal proteinases.
The CAMP factor of S. pyogenes, encoded by the cfa gene, was first identified during the sequencing of the genome of an M1 strain (Ferretti, et al., 2001), and was confirmed to be an active CAMP factor (Gase, Ferretti, Primeaux, & McShan, 1999). The CAMP reaction is a synergistic process that involves a sphingomyelinase from Staphylococcus aureus and a cohemolysin (CAMP factor) from various streptococci; this reaction was first reported to occur with S. agalactiae (Podbielski, Blankenstein, & Lütticken, 1994). The CAMP factor appears to be widespread among the streptococci, as it is present in groups A, B, C, G, M, P, R, and U (Gase, Ferretti, Primeaux, & McShan, 1999). The group A streptococcal CAMP factor is widespread among isolates; 82% of strains tested produced an active CAMP factor with 99% of the strains that contained the gene (Gase, Ferretti, Primeaux, & McShan, 1999). The CAMP factor is at least partially controlled by CodY, with its expression being decreased in a CodY mutant during growth in blood (Malke & Ferretti, 2007). The carbon catabolite repressor protein, CcpA, also has a regulatory effect on cfa regulation, as it is growth-phase regulated with increased expression in the stationary phase (Kietzman & Caparon, 2010). Another nutritional related regulatory protein, CvfA, can also affect cfa expression (Kang, Caparon, & Cho, 2010).
S. pyogenes is known to produce up to four DNases as extracellular products. A potent DNase produced by the M1T1 globally disseminated clone (Nasser, et al., 2014; Walker, et al., 2014) is the bacteriophage-encoded SdaD2, and the major DNase that contributes to virulence (Sumby, et al., 2005). This enzyme protects S. pyogenes from neutrophil-mediated killing by degrading DNA-based neutrophil extracellular traps (Buchanan, et al., 2006; Walker, et al., 2007). These traps are an innate response that binds bacteria to prevent them from spreading, and in doing so, ensures the presence of antimicrobial agents to kill or degrade the bacteria and their virulence factors (Brinkmann, et al., 2004). The SdaI DNase may play a role in S. pyogenes hyper-invasiveness in some genetic backgrounds, but not all, which indicates that severe diseases can result from the production of a balance of different virulence factors (Cole, Barnett, Nizet, & Walker, 2011; Venturini, et al., 2013). SdaI also shows a novel innate immune system evasion mechanism, where the enzyme destroys CpG-rich DNA and suppresses the TLR-9 mediated response (Uchiyama, Andreoni, Schuepbach, Nizet, & Zinkernagel, 2012).
The expression of sda1 is negatively regulated by CovRS (Walker, et al., 2014); it is upregulated in a covRS mutant (Sumby, Whitney, Graviss, Deleo, & Musser, 2006). However, under oxidative stress, sda1 expression is PerR-dependent (Wang, et al., 2013); PerR, the peroxide regulator, is a transcription factor involved in metal homeostasis and oxidative stress in S. pyogenes (Brenot, Weston, & Caparon, 2007; Wang, et al., 2013).
Another bacteriophage-encoded DNase is SpdI1, a type 1 extracellular, non-specific metal-dependent nuclease expressed during prophage induction (Korczynska, Turkenburg, & Taylor, 2012). This DNase is able to degrade both single- and double-stranded DNA, as well as RNA (Korczynska, Turkenburg, & Taylor, 2012). It is also co-expressed with the phage-encoded streptococcal pyrogenic exotoxin C (SpeC) (Broudy, Pancholi, & Fischetti, 2002). The prophage-encoded Spd1 was found in emm12 strains isolated from an ongoing scarlet fever outbreak in Hong Kong (Tse, et al., 2012; Walker, et al., 2014).
Other nucleases include streptodornase B or mitogenic factor 1 (SdaB or Mf-1) and the bacteriophage encoded Spd-3 found in the chromosome of the M49 strain NZ131 (McShan, et al., 2008). Note that with these genes that if the global regulator CodY is deleted, SdaB is more abundant, but Spd-3 is less abundant (McDowell, Callegari, Malke, & Chaussee, 2012). Another transcriptional regulator, Rgg (also known as RopB) represses transcription of streptodornase B and spd-3 by directly controlling the expression of these genes (Anbalagan & Chaussee, 2013; Anbalagan, McShan, Dunman, & Chaussee, 2011). The role of DNases in S. pyogenes infections remains to be fully investigated.
SpnA (Streptococcus pyogenes nuclease A) is a cell-bound nuclease (Hasegawa, et al., 2010) reported to be involved in the escape of the bacteria from neutrophil extracellular traps (Chang, Khemlani, Kang, & Proft, 2011). This nuclease, like the others, plays a role in streptococcal survival and appears to be expressed during infection, since antibodies to the protein are detected in convalescent serum (Chang, Khemlani, Kang, & Proft, 2011).
After years of declining streptococcal infections, a resurgence of severe infections occurred in the late 1980s and early 1990s. This resurgence was the result of a global dissemination of an M1T1 clone, which accounted for a significant portion of isolates from developed countries (Walker, et al., 2014). This invasive clone was derived from a progenitor strain by acquisition of bacteriophages containing an extracellular DNase, which were then acquired in a stepwise manner by the superantigen A1 variant of the streptococcal pyrogenic exotoxin (SpeA); this was followed by evolution to the A2 variant of the toxin, prior to acquisition of a 36 kb region encoding genes for SLO and NAD-glycohydrolase (Cole, Barnett, Nizet, & Walker, 2011; Maamary, et al., 2012; Nasser, et al., 2014; Venturini, et al., 2013).
S. pyogenes encodes proteins that are classified as hyaluronidases. These include the chromosomally-encoded hyaluronate lyase, HylA, and the bacteriophage-encoded hyaluronidases, HylP (Hynes, 2004); these phage-encoded enzymes are also hyaluronate lyases (El-Safory, Lee, & Lee, 2011). The hyaluronidase-type enzymes have been classified as virulence factors, based on their ability to aid in the spread of the organism or its proteins and toxins. Another protein that was originally classified as a hyaluronidase, Spy1600, in the genome of M1 strain SF370 (Ferretti, et al., 2001) and other sequenced strains, is a β-N-acetylglucosaminidase and not a hyaluronidase (Sheldon, et al., 2006).
Certain strains of S. pyogenes express and secrete an active hyaluronate lyase (Hynes & Walton, 2000). Production of active hyaluronate lyase was originally reported to be associated with certain serotypes, particularly M-types 4 and 22 (Crowley, 1944). Subsequent work indicated that the production was strain-associated, rather than being related to serotype (Benchetrit, Avelino, & de Oliveira, 1984). Genomic studies showed that the hylA gene is present in all S. pyogenes strains sequenced; however, not all of these produce an enzymatically active product (Hynes, Dixon, Walton, & Arigides, 2000; Hynes, Hancock, & Ferretti, 1995; Starr & Engleberg, 2006). There are differences in the hylA genes, with three gene structures having been reported: full length, truncated, and those that contain a deletion (Hynes, Johnson, & Stokes, 2009); this is different from the enzymatically active N-terminal degradation products seen with S. agalactiae (Gase, Ozegowski, & Malke, 1998). An additional difference reported in the group A streptococcal hyaluronate lyases is a point mutation that changes amino acid 199 of HylA from an aspartic acid residue to a valine residue; this change results in a loss of enzymatic activity (Hynes, Johnson, & Stokes, 2009). The aspartic acid residue is present in emm4 and emm22 HylA protein; namely, those serotypes that produce active enzymes. Of interest is that both these serotypes lack the ability to produce a hyaluronic acid capsule, due to the loss of the hasABC genes required for capsule production (Flores, Jewell, Fittipaldi, Beres, & Musser, 2012; Henningham, et al., 2014). This could partially address the often-asked question as to why an organism would produce both a protective shield (hyaluronic acid capsule) and an enzyme (hyaluronate lyase) capable of destroying that protection. It has been suggested that hyaluronate lyase may be an anti-virulence factor for those organisms involved in severe invasive diseases by making it more susceptible to phagocytosis (Hynes, Johnson, & Stokes, 2009; Starr & Engleberg, 2006). Despite the loss of the capsule-producing ability by serotypes 4 and 22, they are still pathogenic and are able to proliferate in blood (Flores, Jewell, Fittipaldi, Beres, & Musser, 2012; Henningham, et al., 2014). It appears that these serotypes have developed alternative methods of virulence and that a hyaluronic acid capsule is not needed to colonize the upper respiratory tract, to cause mucosal infections, or even to cause invasive infections (Flores, Jewell, Fittipaldi, Beres, & Musser, 2012). In these cases, the hyaluronate lyase activity may be more important for the diffusion of bacterial toxins and other proteins, rather than for the overall spread of the organism (Starr & Engleberg, 2006). Recent evidence suggests that hyaluronate lyase may also limit infections because of increased uptake of bacterial cells by macrophages; an effect not only of the bacterial capsule, but also controlled by host hyaluronan (Schommer, Muto, Nizet, & Gallo, 2014). Additionally, Henningham et al. found a mutual exclusivity of capsule and hyaluronate lyase activity (Henningham, et al., 2014). If an active hylA gene was introduced into an M1 strain, capsule expression was abolished (Henningham, et al., 2014); if capsule genes were added to an M4 isolate, the amount of capsule was significantly less in the presence of an active hylA gene. Currently, there is no evidence of a direct relationship between hyaluronate lyase activity and disease.
In addition to being a possible virulence factor, hyaluronate lyase may be important for supplying nutritional needs to a producing cell, at least in those strains that are capable of producing active enzyme (Starr & Engleberg, 2006). Regulation of genes in S. pyogenes in response to nutritional stress is in part controlled by CodY and this gene, at least in part, regulates hylA expression; levels of HylA being decreased in a CodY mutant (McDowell, Callegari, Malke, & Chaussee, 2012). In Streptococcus pneumoniae, hyaluronidase production is regulated by the global LacI/GalR family regulator, RegR (Chapuy-Regaud, et al., 2003). A similar RegR gene appears to control the expression of hylA in S. pyogenes (Sloan & Hynes, 2015).
Many (if not all) strains of S. pyogenes carry one or more prophage genomes that encode a number of potential virulence factors (Suvorov, Polyakova, McShan, & Ferretti, 2009). The prophage genomes encode a hyaluronate lyase, which, when produced, is associated with the phage tail fibers. The role of the phage hyaluronate lyases may be to allow the phage to penetrate the hyaluronic acid capsule and allow for its attachment to appropriate receptors (Baker, Dong, & Pritchard, 2002; Hynes & Ferretti, 1989; Hynes, Hancock, & Ferretti, 1995; Niemann, Birch-Andersen, Kjems, Mansa, & Stirm, 1976). The fact that temperate phages are able to establish infections in encapsulated strains may have played an intriguing role in the development of streptococcal virulence. These phages would be capable of infecting, lysogenizing, and laterally transferring virulence factors into encapsulated strains, resulting in increased virulence as proposed for the evolutionary events that led to the development of the epidemic M1 clone (Nasser, et al., 2014). In contrast to their lysogenic cousins, virulent phages are unable to infect encapsulated strains of group A streptococci unless the capsule is removed (McClean, 1941). This leaves the virulent phages capable of infecting, and killing, unencapsulated and less virulent isolates.
The phage-encoded hyaluronate lyase enzymes have no similarity to the chromosomally encoded lyases. Bacteriophage hyaluronate lyases from S. pyogenes show polymorphisms resulting at least in part from intragenic recombinational events (Marciel, Kapur, & Musser, 1997). One major area of variation between the phage lyases is the presence or absence of the Gly-X-Y motif (Hynes & Ferretti, 1989; Hynes, Hancock, & Ferretti, 1995). This repeating motif has been suggested to play a role in stabilizing the enzyme structure (Stern & Stern, 1992), while structures of the lyases with and without the repeat motif have recently been resolved (El-Safory, Lee, & Lee, 2011; Singh, Malhotra, & Akhtar, 2014). Whether or not the phage-encoded hyaluronate lyases play a direct role in pathogenesis is unclear. Antibodies to the lyases have been detected following infection (Halperin, Ferrieri, Gray, Kaplan, & Wannamaker, 1987), which indicates that these lyases are produced. The phage enzyme may add to the overall virulence of the bacteria through functioning as an additional spreading factor (Hynes, 2004).
Streptococcal inhibitor of complement (SIC) is a 31 kDa protein found in M1 strains of S. pyogenes (Akesson, Sjöholm, & Björck, 1996; Frick, Akesson, Rasmussen, Schmidtchen, & Björck, 2003). The sic gene and resulting protein are highly polymorphic within the M1 strains examined (Hoe, et al., 2001; Stockbauer, et al., 1998). The protein inhibits complement-mediated lysis by inhibiting the formation and function of the membrane attack complex (Akesson, Sjöholm, & Björck, 1996; Fernie-King, Seilly, Davies, & Lachmann, 2002; Fernie-King, et al., 2001), as well as the activity of other immune response antibacterial proteins involved in bacterial clearance, including lysozyme, LL-37, defensins (Fernie-King, Seilly, Binks, Sriprakash, & Lachmann, 2007; Frick, Akesson, Rasmussen, Schmidtchen, & Björck, 2003), the chemokine MIG/CXCL9 (Egesten, et al., 2007), and some bacterial antimicrobial products (Minami, et al., 2009). Recently, SIC was shown to interfere with the activation of the contact system of the innate immune system (Frick, et al., 2011); when activated, this system generates antimicrobial peptides (Frick, et al., 2006).
The ability to interfere with the complement and contact systems of the host indicate that in those strains that produce SIC, this protein will enhance both virulence and dissemination of the bacteria (Frick, et al., 2011). In addition, SIC contributes to adherence, colonization, and bacterial survival by altering cellular processes that are critical to efficient streptococcal contact, internalization, and killing (Hoe, et al., 2002; Lukomski, et al., 2000). The variation seen in the SIC gene during an epidemic indicates a rapid selection (Hoe, et al., 1999), and suggests that exposure to the immune response during infection results in changes that will enhance bacterial survival.
In addition to SIC, some closely related variants have been reported; CRS (closely related to SIC) variants have been reported in M57 isolates (Binks, McMillan, & Sriprakash, 2003), and distantly related to SIC (DRS) variants in the emm12 and emm55 (Binks & Sriprakash, 2004). DRS has also been reported to have similar functions in S. dysgalactiae subsp. equisimilis (Smyth, et al., 2014).
Secretion of extracellular products by S. pyogenes, and particularly toxins, plays a major role in pathogenesis. In many cases, the secretion of these toxins requires factors in addition to the Sec translocation pathway; however, SIC may not require additional factors (Vega & Caparon, 2012). Cationic antimicrobial peptides are able to inhibit the secretion of SpeB and SLO through the ExPortal, while the secretion of SIC, which protects the streptococci from such peptides, was unaffected.
S. pyogenes produces a metalloprotein, superoxide dismutase (SodA), the function of which is to convert superoxide anions to oxygen and hydrogen peroxide; the hydrogen peroxide can then be detoxified by peroxidases (Grifantini, Toukoki, Colaprico, & Gryllos, 2011). SodA has been found on both streptococcal cell surfaces and in culture supernatants (McMillan, Davies, Good, & Sriprakash, 2004). As S. pyogenes does not produce a catalase, SodA plays a vital role in detoxifying the oxidative burst produced by host white cells in response to the detection of these pathogenic bacteria. It has recently been shown that these bacteria also produce a glutathione peroxidase (GpoA), which enhances the organism’s ability to survive oxidative stress (Kwinn & Nizet, 2007) during phagocytosis.
S. pyogenes encodes a number of surface-associated, immunoglobulin-binding proteins, including M proteins, M-related proteins, and M-like proteins. These proteins bind the Fc portion of the immunoglobulin molecules: M and M-like proteins bind Fc region IgA, while the M-related proteins bind the Fc portion of IgG (Carlsson, Berggård, Stålhammar-Carlemalm, & Lindahl, 2003; Stenberg, O'Toole, & Lindahl, 1992; Walker, et al., 2014). The role of these surface-bound molecules is unclear, but they most likely play a role in immune system evasion.
A highly conserved secreted immunoglobulin-binding protein, SibA, has been reported to be present in most strains of group A streptococci from a variety of different serotypes (Fagan, Reinscheid, Gottschalk, & Chhatwal, 2001). The SibA product is a 45 kDa protein that binds the Fc and Fab region of IgA, IgG, and IgM (Fagan, Reinscheid, Gottschalk, & Chhatwal, 2001; Walker, et al., 2014). Sequence-wise, it lacks homology to M protein, although structurally it has the N-terminal alpha-helical secondary structure that is implicated in Ig binding by M protein (Fagan, Reinscheid, Gottschalk, & Chhatwal, 2001).
The immunoglobulin-degrading enzyme of S. pyogenes, IdeS, is a secreted cysteine proteinase that specifically cleaves the hinge region of IgG (Akesson, Moritz, Truedsson, Christensson, & von Pawel-Rammingen, 2006; von Pawel-Rammingen, Johansson, & Björck, 2002); this proteinase is also known as Mac-1, Sib35, and MspA (Walker, et al., 2014). The proteinase is not essential for phagocyte resistance or virulence in mice (Okumura, et al., 2013). The proteinase preferentially cleaves Fab-bound IgG while allowing non-specifically bound IgG to remain attached to M protein. In doing so, this may aid the streptococcus in resisting phagocytosis and cytotoxicity through antibody-mediated processes (Su, et al., 2011). Mac-2 is a related IgG endopeptidase that prevents the recognition of IgG bound to S. pyogenes by competitively blocking IgG from recognition by Fc receptors on host cells (Agniswamy, Lei, Musser, & Sun, 2004). Given that IdeS/Mac have homologs across group A streptococcal strains, it is possible that these proteinases play some other role in streptococcal survival, and under different conditions or in other strains, the proteinases do contribute to virulence (Okumura, et al., 2013). Like a number of the other virulence factors, IdeS is regulated by CodY; when grown in the presence of blood, its expression is reduced in CodY mutants (Malke & Ferretti, 2007).
Another extracellular anchorless immunoglobulin protein, Sib35, has been reported to be present in all strains of S. pyogenes examined, but not in other streptococci (Kawabata, et al., 2002). This protein was 35 kDa in size and different from the extracellular SibA (Kawabata, et al., 2002); Sib35 was found extracellularly, as well as on the surface of the cell. The protein was shown to bind IgG, IgA, and IgM, and had similarity to IdeS, the IgG-degrading enzyme (Okamoto, Tamura, Terao, Hamada, & Kawabata, 2005). In addition to the binding of immunoglobulins, this protein was shown to induce B-cell proliferation, as well as differentiation into immunoglobulin-producing plasma cells (Okamoto, Terao, Tamura, Hamada, & Kawabata, 2008). Patients with streptococcal infections had a higher antibody titer to Sib35 than healthy volunteers, which indicates that the protein is produced during an infection. When used to vaccinate mice, those that received Sib35 were found to have enhanced survival rates, as compared to controls, when challenged with group A streptococci (Okamoto, Tamura, Terao, Hamada, & Kawabata, 2005).
EndoS is a large (108 kDa) endoglycosidase Ig-degrading enzyme that removes carbohydrates from immunoglobulin G in a highly specific manner (Trastoy, et al., 2014; Walker, et al., 2014). The activity of the enzyme is such that it only hydrolyzes the glycan moiety on native, but not denatured, IgG (Collin & Olsén, 2001a; Collin & Olsén, 2001b). This enzyme enhances survival by reducing binding of IgG to the Fc receptors and impairing complement activation (Collin, et al., 2002). The crystal structure of EndoS has recently been determined (Trastoy, et al., 2014), which will allow for its investigation as a potential therapeutic agent. EndoS aids S. pyogenes in evading the host response; understanding the structure of the protein aids in determining potential inhibitors of the activity that can be used to inhibit activity, and therefore potentially improve clinical outcomes. One possible novel use of EndoS is as a treatment in the reduction of the proinflammatory properties of immune complexes in systemic lupus erythematosus patients (Lood, et al., 2012).
A variant of EndoS has been reported to be found exclusively in serotype M49 strains; this is referred to as EndoS2 (Sjögren, et al., 2013). The ndoS gene from M1 and ndoS2 gene from M49 are 53% identical, and the proteins are 37% identical (Sjögren, et al., 2013). EndoS2 activity differs from EndoS activity by hydrolysis of N-linked glycans on native IgG heavy chains and α1-acid glycoproteins (Sjögren, et al., 2013); EndoS is specific for the native form of IgG (Collin & Olsén, 2001a). The hydrolysis of two immune system components by EndoS2 suggests a possible role in host immunomodulation and pathogenesis during M49 serotype infections.
In mutagenesis studies in which the EndoS gene was knocked out in an M1T1 isolate, no difference in bacterial survival was seen with immune cell-killing assays or in a systemic mouse model of infection (Sjögren, Okumura, Collin, Nizet, & Hollands, 2011). However, an increased resistance to killing by neutrophils and monocytes in vitro was observed. If EndoS was introduced and expressed in an M49 isolate of group A streptococci, there was an observed increase in virulence in a mouse infection model (Sjögren, Okumura, Collin, Nizet, & Hollands, 2011). These results suggest that in the virulent M1T1 serotype, EndoS has minimal impact on pathogenicity; however, in certain strains, high levels of expression or local accumulation may contribute to virulence.
M protein is a surface protein of group A streptococci, although it can be released through the action of bacterial- or host-derived proteases (Oehmcke, Shannon, Mörgelin, & Herwald, 2010; Shannon, et al., 2007), and can influence streptococcal virulence in that extracellular state. The M protein released from the bacteria into circulation can contribute to the systemic activation of the coagulation cascade during the infectious process (Oehmcke, Shannon, Mörgelin, & Herwald, 2010; Shannon, et al., 2007). Platelets can be activated by soluble M protein to form complexes with neutrophils and monocytes, which results in the activation of these cells, and can evoke additional inflammatory responses in doing so (Oehmcke, Shannon, Mörgelin, & Herwald, 2010; Shannon, et al., 2007). Binding fibrinogen to soluble M1 results in aggregates that are capable of activating neutrophil β2 integrins (Macheboeuf, et al., 2011). This triggers a release of heparin-binding protein, an inflammatory mediator that induces vascular leakage (Reglinski & Sriskandan, 2014). Soluble M protein has been shown to be an inducer of neutrophils, monocytes, and a T-cell activator (Herwald, et al., 2004; Påhlman, et al., 2006; Påhlman, et al., 2008) being as potent as other streptococcal superantigens, which suggests that soluble M1 protein is a novel superantigen (Påhlman, et al., 2008).
The coagulation pathway can be initiated by both intrinsic and extrinsic pathways, with activation of the intrinsic pathway occurring on the cell surface and the extrinsic pathway being activated through the interaction with soluble M protein (Oehmcke, et al., 2012; Walker, et al., 2014). With the coagulation system and early immune response to a streptococcal infection being tightly linked, any dysregulation will contribute to the invasive pathogenesis of the organism (Walker, et al., 2014).
Group A streptococci are successful pathogens capable of causing significant morbidity, as well as mortality. The diverse arsenal of virulence factors they produce to establish colonization and overcome host defense mechanisms are an important contribution to the disease process. Many of these virulence factors are extracellular and cell bound, as well as those discussed here that are released into the external environment. In addition to their role in virulence, many of the extracellular products produced by group A streptococci, such as proteinases, hyaluronate lyases, DNases, NADase, esterases, and others, as well as extracellular enzymes that are not considered virulence factors (such as amylase) can also function as digestive enzymes that produce nutrients from the host tissue for assimilation by the infecting bacteria. Other extracellular proteins, in particular those that function as virulence factors (streptolysins and pyrogenic exotoxins, among others) act as agents of host tissue damage and provide the opportunity for the digestive enzymes mentioned to obtain essential growth nutrients for the infecting bacteria.
Information on the physical properties of the virulence factors, the regulation, and the role of the released extracellular products is part of the equation that will lead to an understanding of the relationship that exists between the bacteria and its host. With the advent of modern molecular technologies, vast quantities of data and information have been generated, but there is also a need to interpret the meaning of this data. Recent research has moved beyond looking at a single virulence factor in isolation; now it will be necessary to take a broader approach, as these virulence factors do not work in isolation and are not independently regulated. In many cases, regulatory factors control expression of more than one virulence factor; sometimes in the same manner, sometimes inversely, with one factor being up-regulated while another is down regulated. What this says about virulence factors and their role in the disease process still needs to be determined. There is also a need to examine the role of multiple virulence factors in vivo; what happens in culture media may not be indicative of what actually occurs during an infection.
Even though S. pyogenes has been studied for over 130 years, there are still many unanswered questions related to pathogenicity and virulence. Infections caused by S. pyogenes have widespread effects, both personally to the affected and economically to their communities. The overall control and prevention of streptococcal infections needs to be an area of emphasis for future research in order to reduce morbidity and mortality. Vaccines would be one answer, and understanding the role of the extracellular products in the disease process may provide new ways for looking at the development of such vaccines.
Except where otherwise noted, this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC-BY-NC-ND 4.0). To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/
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