Extracellular Products of Streptococcus pyogenes and Their Involvement in Pathogenesis Debbie Soefie Retnoningrum School of Pharmacy, Institut Teknologi Bandung, Bandung e-mail: [email protected] Received 24 March 2008, accepted for publication 14 May 2009 Abstract Streptococcus pyogenes or group A streptococcus (GAS) is an exclusive human pathogen. To be a successful pathogen, this pathogen is equipped with various surface-exposed and secreted virulence factors. The functions of secreted virulence factors are particularly important since they interact with host components to establish infections and cause diseases in human. They include a number of proteases, DNase, superantigens, and plasminogen activator. How these secreted factors interact with host protein(s) define this pathogen’s ability to bring out various diseases. Several proteases act independently to target immunoglobulin molecules in order to evade host defense system and modulate host proteins to induce host-mediated damages. Besides producing proteases, many pathogenic strains of GAS also produce DNase; however, its involvement in host pathogenesis remains elusive. Superantigen, another secreted protein is responsive for serious host-destruction by bypassing antigen presentation to induce massive production of cytokines. GAS also secretes a plaminogen activator, streptokinase that is crucial for invasiveness. All together, secreted products of this pathogen work in concert to pinpoint different targets in order to destroy and or to disable human defense system and cause host damages. Keywords: Streptococcus pyogenes, Secreted virulence factors, Bacterial colonization, Host damage Abstrak Streptococcus pyogenes atau streptokokus kelompok A (group A streptococcus, GAS) adalah pathogen yang hanya menginfeksi manusia. Untuk menjadi pathogen yang berhasil, GAS dilengkapi dengan faktor virulensi, baik berupa molekul permukaan maupun yang disekresikan. Faktor virulensi yang disekresikan mempunyai fungsi penting karena berinteraksi dengan komponen inang untuk menyebabkan infeksi dan penyakit pada manusia. Faktor virulensi tersebut mencakup sejumlah protease, DNase, superantigen dan aktivator plasminogen. Bagaimana faktor virulensi tersebut berinteraksi dengan protein inang menentukan kemampuan pathogen untuk menyebabkan berbagai penyakit. Beberapa protease bekerja secara independen untuk mentarget molekul imunoglobulin dengan tujuan menghindari sistem pertahanan inang dan mengubah protein inang, sehingga menginduksi kerusakan yang diperantarai inang. Selain menghasilkan protease, banyak galur GAS pathogenik juga menghasilkan DNase; tetapi, keterlibatannya dalam pathogenesis inang masih belum dimengerti. Superantigen, protein lain yang disekresi, bertanggung jawab untuk menimbulkan kerusakan inang yang parah dengan memotong jalur presentasi antigen sehingga menimbulkan produksi sitokin dalam jumlah besar. GAS juga mensekresi aktivator plasminogen, streptokinase yang penting untuk invasi. Secara bersama-sama, produk yang disekresi oleh pathogen ini bekerja bersama-sama untuk menyerang target berbeda dengan tujuan menghancurkan dan atau melumpuhkan sistem pertahanan manusia dan menyebabkan kerusakan inang. Kata kunci: Streptococcus pyogenes, disekresi faktor virulensi, bakteri penjajahan, Host kerusakan mediated damages that are responsible for disease manifestation. GAS is equipped by various surface displayed and secreted virulence factors. Both virulence factors collaborate to ensure pathogen establishment in our body. Surface-associated virulence factors have been discussed previously (Retnoningrum, 2006). Culture supernatants of the GAS contain at least 16 proteins with identifiable export sequence (Lyon and Caparon, 2004). Mostly, their genes have been cloned and their gene products were studied at protein level for their in vitro and or in vivo activities including their proven or putative role(s) in disease pathogenesis using animal models or patients suffering from GAS infections or sequelae. The secreted

1. Introduction Streptococcus pyogenes or group A streptococcus (GAS) is a Gram positive bacterium and is the causative agent of numerous suppurative infections, strep throat, impetigo (skin infection) and soft tissues (impetigo, cellulitis, and necrotizing fasciitis), as well as several systemic diseases that can result toxigenic processes (scarlet fever and toxic shock syndrome) and sequelae (rheumatic fever and glomerulonephritis). To be a successful pathogen, GAS should overcome or modify our host defense mechanisms in order to colonize our body. After colonization, this bacterium causes bacterial or host33

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virulence factors play a vital role in streptococcal colonization, invasion, dissemination and host damage. This mini review focuses on several secreted virulence factors of GAS and covers proteases, DNases, plasminogen activators, and superantigens and will discuss how these secreted products interact with human components and their involvement in streptococcal pathogenesis. Virulence genes in genomes of GAS To date, 13 genomes of GAS strain have been completely sequenced, two of M1 serotypes (SF370 and MGAS5005), M2 (MGAS10270), two of M3 (MGAS315 and SSI-1), M4 (MGAS10750), M5 (Malfredo), M6 (MGAS10394), two of M12 serotypes (MGAS2096 and MGAS9429), M18 (MGAS8232), M28 (MGAS6180) and M49 (NZ131). The genome size is varied in the range of 1,815,783 base pairs (bp) for M49 and 1,937,111 bp for M4 (McShan et al., 2008). By comparing genomes of four strains (MGAS315, SF370, SSI-1 and MGAS8232), the number of putative and predicted virulence genes are between 42 and 43. They are classified as proteinases, adhesins, hemolisins, enzymes involved capsule synthesis and degradation, DNases, superantigens, immunoreactive antigens, regulators, bacteriocins, NADase, hyaluronidase, streptolysin S and secreted inhibitor of complement (sic). Most virulence genes are chromosomally encoded; however some of them are located in phage sequences, several to mention are some hyaluronidase genes, mitogenic factor (MF)-like gene, and a number of superantigen genes encoding for streptococcal pyrogenic exotoxin A (SpeA), SpeC, SpeH, SpeI, SpeK and streptococcal superantigen (SSA). The presence of virulence genes are vary between serotypes, for instance, gene encoding for DNases such as MF-like gene are absent in M1 (SF370) and M5 (MGAS8232), MF2 gene is not found in M3 (SSI-1 and MGAS315), MF3 gene is not present in M3 and M5 and streptodornase (sda) gene is missing in M1. With regards to superantigen genes, so far ten different superantigens are discovered in GAS and their presence is also varied between serotypes. For instance, M1 serotype is lacking speA gene, M3 does not posses speC gene, M3 and M5 is missing speH gene but speG gene is present in M1, M3 and M5 serotypes (Nakagawa et al., 2003; Ferreti et al., 2001). Taken together this information, the genome of GAS exhibits some plasticity especially in terms of their virulence genes and this explains the diversity of diseases they cause in human. The existence of gene encoding for secreted virulence factors is also diverse between strains and this explains unique strategy that GAS strains to deal with our defense mechanism and to cause host damage.

Evasion of host defense mechanisms Some GAS strains, mostly M1 and M3 are reported to be associated with systemic diseases. Their survival and multiplication in human requires clever strategy to combat innate and adaptive immunity. GAS avoids innate immunity for example by inhibiting leukocyte recruitment and complement activation as well as killing immune cells. Moreover, this pathogen develops mechanisms to cleave immunoglobulins (Igs) to circumvent adaptive immunity. Here, a number of strategies of GAS to avoid human defense mechanisms by secreted virulence factors will be addressed. From their genome information, it was revealed that GAS evolves a number of secreted proteinases with C3b and Ig degrading ability, and some of them belong to serine or cysteine proteinase. GAS must evade complement system which acts as protective shield at early phase of infection. C3b is a component produced upon complement activation and acts as an opsonin that ultimately involved in C3bdriven phagocytosis which results in bacterial elimination. GAS secretes virulence factors that interact with these molecules. SpeB, a cysteine protease, is a virulence factor that affects many proteins of immune system. One recent investigation strongly indicated that wild type GAS, but not GAS lacking SpeB production had ability for C3b cleavage (Terao et al., 2008). By cleaving C3b, GAS avoids C3b-mediated phagocytosis and hence survives in human blood. The gene encoding for SpeB is highly conserved and is present in all GAS isolates (Yu and Ferretti, 1991). Our results showed that at the amino acid level, SpeB in various strains exhibits 97 –– 98% identity to SpeB from M49 strain NZ131 indicating that its amino acid conservation is required for its functions. Using motif searching tools (NCBI, www.ncbi.nlm.nih.gov), SpeB contains a putative conserved domain showing that it belongs to peptidase C10 family. This protease is secreted as a 371 amino acids (40 kDa) zymogen, an inactive precursor at early stationary phase and processed by autolytic cleavage to remove its first 118 amino acid into a 28 kDa mature protease that is accumulated at late stationary phase. The precursor form of SpeB has been crystallized and its three dimensional structure was determined at high resolution by x-ray crystallography. This study revealed that the folding of SpeB is similar to that of papain which is also a cysteine protease (Kagawa et al., 2000). Of the streptococcal secreted proteases, SpeB is the most studied; therefore, this mini review discusses SpeB more extensive than other proteases. SpeB is also shown to have a broad human Igdegrading activity for all types of Ig. This protein is a specific IgG-protease and partially or totally degrades them. It is interesting to point out that SpeB cleaves IgG in a similar manner to IgA protease produced by

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Neisseria gonorhheae where IgG is cleaved at a defined site in the hinge region into stable monomeric Fab fragments and one Fc fragment. Although the degradation pattern resembles that of papain, the cleavage site is different. SpeB cleaves between two glycines located between residues 236 and 237, whereas papain cuts between residues histidines and threonine at positions 224 –– 225 (Collins and Olsen, 2003). SpeB also cleaves the C-terminal part of the heavy chain of IgA, IgM, and IgD into low-molecular weight fragments, whereas the heavy chains of IgE are completely degraded (Collin and Olsen, 2001). Nevertheless, the role of Ig-degrading activity in pathogenesis and disease manifestation in GAS awaits further investigation. SpeB is not the only enzyme that acts on human Ig. Another enzyme also degrades IgG and is designated as IgG-degrading enzyme of S. pyogenes or IdeS (Åkesson et al., 2006). Like SpeB, IdeS is a secreted cysteine protease that cleaves human IgG in the hinge region at the same position as SpeB does. Both cystein proteases cleave human IgG between two glycine residues at positions 236 and 237 (Collin and Olsen, 2003). This cleavage inhibits antibody-mediated phagocytosis; therefore by expressing IdeS, GAS is able to evade phagocytosis even in the presence of Ig. Unlike SpeB that degrades all types of Ig, IdeS shows high degree of specificity since it only cleaves IgG. The IdeS genes are present in many isolates and the protein is secreted by several serotypes. Patients infected with GAS produce antibody against IdeS indicating that IdeS is expressed in vivo during infection. Our in silico study indicates that amino acid sequences of IdeS of M49 (strain NZ131) has 99% identity to those of MGAS315 and SSI-1, while only shows 84 –– 84% identity to those of MGA92429, 10270, 6180 and 10394 (M6 serotype). The SpeB of NZ131 shows 63% identity to IdeZ, an IgG-degrading protease of Streptococcus equi subsp. zooepidemicus. One recent research raised the question whether specific IgG are able to neutralize IdeS and the presence of neutralizing antibodies correlates with disease manifestations. The investigation showed that neutralizing antibodies against IdeS enzymatic activity were detected in two-thirds of acute-phase sera. The presence of neutralizing antibodies reduced IdeS ability to mediate bacterial survival in immune human blood. In patients with bacteremia, it was found that there was no correlation between the presence of neutralizing antibodies to severity or outcome of invasive infections. The fact that the human immune response targets the enzymatic activity of IdeS supports the view that the enzyme plays an important role during streptococcal infection (Åkesson et al., 2006). Another protease that takes part in immune modulation is EndoS. It is a big protein of various sizes

and from published sequences; their size is in the range of 834 –– 953 amino acids. Due to its size variations, the identity is rather low (41%) between different strains. It has an endo-beta-N-acetylglucosaminidase activity and specifically degrades native but not denatured IgG by hydrolyzing the glycan moeity leaving an Nacetylglucosamine with a core fucose. It was reported that EndoS does not remove any major glycans from IgA and IgM. EndoS recognizes two identical N-linked glycans attached to asparagine 297 in the heavy chain of IgG and hydrolyzes the chitibiose core of the glycan between 4 GlcNAc ȕ1 and 4 GlcNac, leaving the intermost GlcNAc with a core fucose (Collins and Olsen, 2001). By performing motif searching, it is revealed that EndoS belongs to G18-chitinase-like superfamily and the active site is located at the amino terminus half of the protein. The significance of EndoS size variation is currently unknown. This evidence pointed out that GAS has evolved three different proteases i.e SpeB, IdeS and EndoS that targets Ig molecules (Figure 1). Both SpeB and IdeS cleaves Ig at hinge region and SpeB cleaves all five Igs, while IdeS specializes on IgG. On the other hand, EndoS acts on the carbohydrate group of native IgG. Carbohydrate moeity of Fc portion of Ig has been accounted for complement activation, binding to Fc receptor on macrophages and inducing antibodydependent cellular cytotoxicity. In addition, antibodycomplexes produced from carbohydrate-lacking Ig failed to be rapidly cleared from the circulation system (Nose and Wigzell, 1983). It is therefore removal of carbohydrate moeity of Ig molecules has a profound impact on their biological activity. IgG especially IgG1 and IgG3 has strong moderate ability for opsonization and complement activation, consequently by removing their carbohydrate group, EndoS secreted by GAS will inactivate the function of IgG. They collaborate to attack by different mechanisms and or speficity to disable one of our humoral immunity. These findings contribute to the understanding of the role of secreted enzymes in the pathogenesis of GAS at molecular level. The role of human proteins on SpeB production Human serum albumin (HSA) is the most abundant protein in the blood; therefore we expected that HSA would affect the expression of virulence gene(s). The effect of human serum and HSA to the expression of speB gene was investigated using a GAS CS101, an M49 strain carrying a luciferase (luc) reporter gene located downstream of speB promoter.

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it seems that HSA signals streptococcal cells to produce more SpeB. Modulation of host proteins by SpeB

Figure1. Interaction of several important secreted products of GAS with human components and their consequences in streptococcal pathogenesis. Using this system, we were able to investigate the level of speB gene transcription by measuring luciferase (Luc) activity. Our result demonstrated that in the presence of individual or pooled human plasma from three different people in the growth medium, the Luc activity produced by CS101 was significantly higher than in its absence. The maximal Luc activity was reached at 5 hours growth in Todd Hewith Broth Yeast Extract (THY) and O.D.600 nm ~ 1.2 (transition from exponential to stationary phase). This result was in agreement with previous work showing that SpeB precursor was produced at early stationary phase (Kagawa et al., 2000). To determine the human plasma component(s) responsible for speB upregulation, fractionated human plasma (fractions < 3 kDa, 3 –– 10 kDa, and > 10 kDa) was used in the experiment. Only fraction > 10 kDa, but not other fractions increased the Luc activity and the activity in the presence of > 10 kDa fraction was considerably higher than that in the presence of pooled human plasma. The Luc activity in the presence of fraction < 3 kDa and 3 –– 10 kDa was similar as that in THY and THY - NaCl solution as negative controls. It is obvious that HSA with molecular weight of 65 kDa was present in fraction > 10 kDa; however to ensure that HSA is certainly responsible for speB upregulation, the same experiment was performed in the presence of HSA. The result showed that the addition of HSA in growth medium, the Luc activity increased significantly compared to the addition of NaCl solution as negative control (Retnoningrum, 1999 unpublished data). This evidence supports the notion that SpeB production is regulated by human serum component, HSA in particular. As previously discussed, SpeB has a number of functions while GAS is present in human blood and

Our body consists of many different proteins with unique functions. SpeB was demonstrated to affect human proteins by activating or inactivating them. SpeB activates a 66-kDa host matrix metalloproteinase and this is proposed to contribute to the extensive soft tissue destruction observed in many patients bearing streptococcal infection. SpeB was first found to have fibrinolytic activity in 1940 and found to cause myocardial necrosis in rabbits and trigger apoptosis (Elliot, 1945; Kagawa et al., 2000). How relevant this finding with damage in human is uncertain since GAS exclusively causes diseases in human. Most strains of GAS associated with severe invasive disease such as streptococcal toxic shock syndrome (STTS) and necrotizing faciitis express a variant of SpeB with integrin-binding activity, which may be linked to their pathogenesis (Kagawa et al., 2000). Integrin-binding activity has been shown to be essential in internalization of host cells by GAS and this process might be crucial in establishment of invasive diseases. SpeB also has another immunomodulating activity on several host molecules or cells. It was shown to act on H-kininogen results in bradykinin release and bradykinin causes vasodilatation that will lower the blood pressure (Heward et al., 1996). SpeB also activates a cytokine, IL-1ȕ which is an important mediator of inflammatory response (Kapur et al., 1993). Moreover, it causes histamine release from mast cells and histamine is a protein that involves in many allergic reactions (Watanabe et al., 2002). It is obvious that by interacting with host proteins or cells, SpeB causes host-mediated damages. Antibacterial peptide is a component of innate immunity and it works mostly by inserting bacterial membrane leading to cell leakage. GAS defeats this human defense by degrading an antibacterial peptide LL-37 and affecting decorin that releases dermatan sulfate which is an inhibitor for antibacterial peptides (Schmidtchen et al., 2002). SpeB as a virulence factor: how good is the evidence? It is undoubltful that SpeB functions as an important virulence factor and its production should contribute to GAS pathogenesis. SpeB is found predominantly in total secreted proteins (Kagawa et al., 2000). The role of SpeB in virulence of GAS was proven in a mouse model using a pair of isogenic strains with speB gene inactivated by genetic approach. Intraperitoneal challenge of isogenic speB mutant strains was significantly less lethal to mice (Lukomski et al., 1997) and subcutaneous infection caused less mortality and tissue damage (Kuo et al., 1998;

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Lukomski et al., 1997). Another study showed that GAS mutants lacking SpeB were less resistant to phagocytosis possibly due to SpeB-mediated cleavage of M protein and did not spread into internal organs as the wild-type bacteria did (Lukomski et al., 1998). Animal studies have suggested that SpeB plays an important role in the balance of the host-pathogen interaction. SpeB also plays a role in host tissue tropism, since SpeB increased the bacterial reproduction in mouse impetigo model an acts synergistically with cell wall antigens and streptolysin O (SLO) to induce injury in rats (Shanley et al., 1996). Relevance of animal model is questionable, since GAS only infects human. However, there is ample evidence supporting the role of SpeB in pathogenesis in human. Patients with invasive disease caused by different serotypes of GAS seroconverted to SpeB indicating that SpeB is produced in vivo during infections (Terao et al., 2008). Patients suffering from severe invasive disease have low antibody titer against SpeB, suggesting that these patients failed to produce Spe-specific antibody which is supposed to neutralize SpeB and this contributes to the development of serious condition. Highly invasive M1 strains from STSS patients are associated with the production of SpeB. Another study showed that there is an inverse relationship between SpeB production and disease severity, possibly due to a sparing of the M protein on the surface. SpeB has been suggested to play a role in the development of Acute Poststreptococcal Glomerulonephritis and these patients have elevated antibody levels against SpeB, and SpeB can be detected in glomerulonephritis biopsies (Kuo et al., 2004). These data undoubtedly demonstrated that SpeB involves in streptococcal pathogenesis. How does serine protease contribute to other virulence factors? GAS produces another type of protease, a serine protease. In Gram negative bacteria, serine proteases have been demonstrated to involve in the degradation of misfolded or aggregated exported proteins. However, numerous investigations have provided significant finding about the importance of HtrA, a serine protease in bacterial pathogenesis. GAS serine protease is a 407 amino acid polypeptide, and is high conserved with identity of 99% at their amino acid level to serine protease of M49 NZ131. It has been reported that GAS lacking HtrA reduced its virulence in a mouse model of systemic infection. This strongly indicates a possible role for HtrA in the biogenesis of secreted virulence factors. Two important secreted virulence factors, SpeB and the hemolysin streptolysin S (SLS) need extensive processing for the generation of their active forms. One recent study showed that mutation in gene encoding for HtrA affects the

production of at least two secreted virulence factors, SpeB and SLS. Mutant lacking HtrA is unable to secrete active form of SpeB. On the other hand, the mutant increases the hemolytic activity caused by SLS. Interestingly, mutation in gene encoding for HtrA did not result in attenuation of GAS in a murine model of subcutaneous infection (Lyon and Caparon, 2004). This finding pointed out that HtrA is vital for systemic, but not for subcutaneous infections in animal model. The relevance of this finding to human is unclear. What is the role of DNase in GAS pathogenesis? Besides secreting enzymes that attack protein, GAS also produces DNase. Many pathogenic bacteria produce extracellular DNase, but the role of this enzyme in the pathogenesis is unknown. GAS has been reported to produce four types of DNase: DNase A, B, C and D. Of the four DNases, DNaseB is the dominant DNase produced in many strains (Matsumoko et al., 2005). All GAS genomes sequenced contain several genes encoding putative or proven secreted DNases. For example, an M1 strain, MGAS5005 has three genes that encode for putative secreted DNases. Two of these genes (spd3 and sdaD2) are encoded by prophages and one is chromosomally encoded (spd). Three indirect lines of evidence indicate the role of DNase in GAS pathogenesis. First, all strains of the GAS produce at least one secreted DNase, and most strains make several different enzymes. Second, DNase is produced more when GAS interacts with human epithelial cells, polymorphonuclear leukocytes, and oropharyngeal cells. Third, patients with GAS infections generate antibody againts DNases, indicating that these enzymes are immunogenic and produced in vivo during infections. To provide direct evidence on the role of DNase in GAS pathogenesis, a DNase-lacking mutant strain was generated using genetic approach and this mutant showed significantly less virulent in mouse models. Compared to the wild-type strain, the mutant strain was eliminated from the skin injection site considerably faster. The mutant strain has significantly less ability to cause experimental pharyngeal disease in cynomolgus macaques. Another result strongly suggested that the prophage-encoded SdaD2 enzyme is the major DNase that contributes to virulence. Extracellular DNases produced by GAS play role in disease progression, although the association between their ability to hydrolyze DNA and GAS pathogenesis is unclear (Sumby et al., 2005). Whether activity to hydrolyze DNA or other unrelated activity of DNAse indeed contributes to pathogenesis is presently unknown.

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How do streptococcal superantigens damage our body? Certain strains of GAS produce a number of secreted virulence factors that enable them to cause host damage (Fig. 1). Among these are exotoxins designated collectively as superantigens (SSA). These molecules belong to a protein family that bypasses normal route of T cell activation. These molecules bind to major histocompatibility complex class II molecules and outside part of T-cell receptor. This nonspecific binding results in the activation of a large proportion of antigen-presenting cells and T cells, with subsequent release of high level of cytokines in the blood that is responsible for undesirable effects in human and can lead to death due to multiorgan failure (Nooh et al., 2006). A number of SSA such as SpeA, SpeC, SpeH, and SpeG are produced by GAS. These SSA have been associated in STSS and the involvement of multiorgan failure in this syndrome suggests that toxins might be involved in pathogenesis. However, other virulence factors such as streptolysin O and various cell wall antigens can also cause toxic shock. Some reports indicated that SpeB also functions as a SSA and this activity is independent of its proteolytic activity (Erikson and Norgren, 1999; Leonard et al., 1991). Other reports on the contrary found that SpeB does not have any superantigenic property and the observed activity originates from contaminating SSA such as SpeA, SpeC or other unknown mitogens (Gerlach et al., 1994). A number novel SSAs have been identified, including streptococcal mitogenic exotoxin Z (SmeZ, SmeZ-2), SpeG, SpeH, SpeJ, and Spe-I. They share typical SSA features and are highly mitogenic on T cells. These findings strongly indicate that, in addition to SpeA and SpeC, one or more of these novel SSAs might be involved in STSS. Recently, SpeB was reported to completely degrade SmeZ. SpeG was more resistant to SpeB cleavage, while SpeA was not affected by SpeB (Nooh et al., 2006). This evidence supports previous finding that SpeB is not an SSA, but it affects the degradation of several SAgs. Plasminogen activator is an important virulence factor for streptococcal invasiveness GAS has been long known to activate plasminogen (Pg) and this ability has been suggested as a critical component in the establishment of invasive streptococcal infections. A virulence factor that activates Pg is streptokinase (SK). SK is a secreted protein produced by many GAS strains. It facilitates lyses of blood clots by activating Pg to plasmin, the fibrinolytic enzyme.

SK is a 414 amino acids long polypeptide with three domains, Į domain ranging from amino acids 1 –– 150, ȕ domain from 151 –– 287 and Ȗ domain 288 –– 414. The Į and Ȗ domains show the highest conservation among SK with • 85% sequence identity and these two domains provide most of the contact sites with the plasmin and exhibit a synergistic effect on Pg activation. The ȕ domain provides no direct contact sites with the plasmin active-site, although it is required to place Pg in a region located from amino acids 251 and 262. A region between amino acids 144 and 218 which spans the second structural loop of the ȕ domain is the most diverse sequence, and contains a region of sequence diversity associated with infection and disease in GAS. One study reported that mutagenesis of this polymorphic region does not alter Pg activation, which suggests an alternative function for this molecular motif in streptococcal disease (Lizano and Johnston, 2005). GAS shows preference for human Pg. Previous studies have shown that streptococcal isolates that can activate mouse Pg are more invasive when injected into the skin. SK and Pg are suggested to play a key role in invasion process. 2. Conclusions GAS has evolved several strategies to overcome our human defense and cause host damages by expressing a number of secreted virulence factors. Three secreted proteases i.e. SpeB, IdeS and EndoS by using different mechanisms target immune proteins, Ig molecules to enhance streptococal blood survival. SpeB modulates several non-immune host proteins and all together this interaction results in host-mediated damages. SSA on the other hand specializes on T cell and activates large population of them in order to cause severe host damage that may lead to mortality. SK helps streptococcal dissemination by increasing invasion. By putting the information together, secreted products involve in bacterial colonization and host damage. References Åkesson, P. L. Moritz, M. Truedsson, B. Christensson, and U. P. -Rammingen, 2006, IdeS, a Highly Specific Immunoglobulin G (IgG)-Cleaving Enzyme from Streptococcus pyogenes, Is Inhibited by Specific IgG Antibodies Generated during Infection, Infect. Immun., 74:1, 497-503. Collin, M., and A. Olsen, 2001, Effect of SpeB and EndoS from Streptococcus pyogenes on Human Immunoglobulins, Infect. Immun. 69:11, 7187-7189. Collin, M,. and A. Olsen, 2003, Extracellular Enzymes with Immunomodulating Activities: Variations

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