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• W2022233387 abstract "Group A Streptococcus (GAS) is an important human pathogen that possesses an ability to translocate across the epithelial barrier. In this study, culture supernatants of tested GAS strains showed proteolytic activity against human occludin and E-cadherin. Utilizing various types of protease inhibitors and amino acid sequence analysis, we identified SpeB (streptococcal pyrogenic exotoxin B) as the proteolytic factor that cleaves E-cadherin in the region neighboring the calcium-binding sites within the extracellular domain. The cleaving activities of culture supernatants from several GAS isolates were correlated with the amount of active SpeB, whereas culture supernatants from an speB mutant showed no such activities. Of note, the wild type strain efficiently translocated across the epithelial monolayer along with cleavage of occludin and E-cadherin, whereas deletion of the speB gene compromised those activities. Moreover, destabilization of the junctional proteins was apparently relieved in cells infected with the speB mutant, as compared with those infected with the wild type. Taken together, our findings indicate that the proteolytic efficacy of SpeB in junctional degradation allows GAS to invade deeper into tissues. Group A Streptococcus (GAS) is an important human pathogen that possesses an ability to translocate across the epithelial barrier. In this study, culture supernatants of tested GAS strains showed proteolytic activity against human occludin and E-cadherin. Utilizing various types of protease inhibitors and amino acid sequence analysis, we identified SpeB (streptococcal pyrogenic exotoxin B) as the proteolytic factor that cleaves E-cadherin in the region neighboring the calcium-binding sites within the extracellular domain. The cleaving activities of culture supernatants from several GAS isolates were correlated with the amount of active SpeB, whereas culture supernatants from an speB mutant showed no such activities. Of note, the wild type strain efficiently translocated across the epithelial monolayer along with cleavage of occludin and E-cadherin, whereas deletion of the speB gene compromised those activities. Moreover, destabilization of the junctional proteins was apparently relieved in cells infected with the speB mutant, as compared with those infected with the wild type. Taken together, our findings indicate that the proteolytic efficacy of SpeB in junctional degradation allows GAS to invade deeper into tissues. Streptococcus pyogenes (group A Streptococcus; GAS) 3The abbreviations used are: GASgroup A StreptococcusTJtight junctionAJadherence junctionTERtransepithelial electrical resistanceMOImultiplicity of infectionSLSstreptolysin S. is well known as a human-specific pathogen responsible for numerous diseases, ranging from pharyngitis and impetigo to life-threatening invasive diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome (1Carapetis J.R. Steer A.C. Mulholland E.K. Weber M. The global burden of group A streptococcal diseases.Lancet Infect. Dis. 2005; 5: 685-694Abstract Full Text Full Text PDF PubMed Scopus (1946) Google Scholar, 2Tart A.H. Walker M.J. Musser J.M. New understanding of the group A Streptococcus pathogenesis cycle.Trends Microbiol. 2007; 15: 318-325Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Serious postinfectious immune sequelae such as rheumatic fever and acute glomerulonephritis occasionally develop following repeated GAS exposure. Despite the availability of sequence information for several GAS genomes and detailed characterization of their virulence factors, a safe and effective commercial GAS vaccine has yet to be developed (3Cole J.N. Henningham A. Gillen C.M. Ramachandran V. Walker M.J. Human pathogenic streptococcal proteomics and vaccine development.Proteomics Clin. Appl. 2008; 2: 387-410Crossref PubMed Scopus (37) Google Scholar). group A Streptococcus tight junction adherence junction transepithelial electrical resistance multiplicity of infection streptolysin S. The pharynx and skin are thought to be the primary sites of GAS infection. Pharyngeal epithelia and keratinocytes are highly specialized physical barriers apposed tightly by tight junctions (TJs) and adherence junctions (AJs), which protect underlying sterile tissues from the external environment. Loss of cell-cell adhesion caused by bacteria has been reported to be associated with several clinical manifestations. Desmoglein 1, which plays a key role in maintaining the structure and barrier function of the epidermis, is targeted by exfoliative toxins released by Staphylococcus aureus in infectious skin diseases such as bullous impetigo and staphylococcal scalded skin syndrome (4Amagai M. Desmoglein as a target in autoimmunity and infection.J. Am. Acad. Dermatol. 2003; 48: 244-252Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Development of GAS skin infections with bullous lesions also seems to be related to loss of cell-cell adhesion and inoculation of GAS into intradermal space (5Ferrieri P. Dajani A.S. Wannamaker L.W. Chapman S.S. Natural history of impetigo. I. Site sequence of acquisition and familial patterns of spread of cutaneous streptococci.J. Clin. Invest. 1972; 51: 2851-2862Crossref PubMed Scopus (101) Google Scholar). Invasive GAS disease requires successful colonization in the pharynx or skin, followed by overcoming the host epithelial barrier together with evasion of host defense mechanisms. Multiple in vitro studies have demonstrated that GAS isolates associated with invasive diseases efficiently invade epithelial cells (6Terao Y. Kawabata S. Kunitomo E. Murakami J. Nakagawa I. Hamada S. Fba, a novel fibronectin-binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator.Mol. Microbiol. 2001; 42: 75-86Crossref PubMed Scopus (147) Google Scholar, 7Bisno A.L. Brito M.O. Collins C.M. Molecular basis of group A streptococcal virulence.Lancet Infect. Dis. 2003; 3: 191-200Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Although programmed cell death is an essential part of host defense against pathogens, it is considered that internalized GAS exploits this process to access the underlying sterile tissues (8Nakagawa I. Nakata M. Kawabata S. Hamada S. Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells.Cell. Microbiol. 2001; 3: 395-405Crossref PubMed Scopus (70) Google Scholar, 9Cywes Bentley C. Hakansson A. Christianson J. Wessels M.R. Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signaling.Cell. Microbiol. 2005; 7: 945-955Crossref PubMed Scopus (54) Google Scholar). Meanwhile, some studies that investigated the direct interactions of bacteria with epithelial junctions also elucidated the underlying mechanisms of GAS pathogenesis, with interaction of the hyaluronic acid capsule with CD44 implicated in this process (10Cywes C. Wessels M.R. Group A Streptococcus tissue invasion by CD44-mediated cell signaling.Nature. 2001; 414: 648-652Crossref PubMed Scopus (168) Google Scholar). Furthermore, our recent study identified streptolysin S (SLS) as a novel factor that facilitates GAS translocation via degradation of intercellular junctions in concert with the host cysteine protease calpain (11Sumitomo T. Nakata M. Higashino M. Jin Y. Terao Y. Fujinaga Y. Kawabata S. Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier.J. Biol. Chem. 2011; 286: 2750-2761Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, the precise mechanism by which GAS disrupts the epithelial barrier has yet to be completely elucidated. During infection, GAS produces numerous secreted and cell-associated proteins, including toxins, superantigens, and proteases (12Cunningham M.W. Pathogenesis of group A streptococcal infections.Clin. Microbiol. Rev. 2000; 13: 470-511Crossref PubMed Scopus (1754) Google Scholar, 13Olsen R.J. Shelburne S.A. Musser J.M. Molecular mechanisms underlying group A streptococcal pathogenesis.Cell. Microbiol. 2009; 11: 1-12Crossref PubMed Scopus (87) Google Scholar). Although extracellular proteins from GAS have been extensively investigated and shown to be important for pathogenesis, its participation in epithelial barrier dysfunction is as yet unproven. Herein, we provide the first direct evidence that SpeB (streptococcal pyrogenic exotoxin B), a broad spectrum secreted cysteine protease, effectively cleaves transmembrane proteins associated with the epithelial barrier to permit bacterial penetration. Our results reveal a new mechanism to explain how GAS directly disrupts the epithelial barrier. Invasive GAS clinical isolates, strains NIH35 (serotype M28), SSI-1 (serotype 3), SSI-9 (serotype M1), and #30 (serotype M12), were isolated from patients with streptococcal toxic shock syndrome. Other GAS clinical isolates, strains SF370 (serotype M1), TW3358 (serotype M3), TW3337 (serotype M12), TW3339 (serotype M28), NZ131 (serotype M49), and 591 (serotype M49), were used as noninvasive GAS strains. Escherichia coli XL10-Gold (Stratagene) served as a host for plasmids pAT18 and pSET4s (14Trieu-Cuot P. Carlier C. Poyart-Salmeron C. Courvalin P. Shuttle vectors containing a multiple cloning site and a lacZ α gene for conjugal transfer of DNA from Escherichia coli to Gram-positive bacteria.Gene. 1991; 102: 99-104Crossref PubMed Scopus (171) Google Scholar, 15Takamatsu D. Osaki M. Sekizaki T. Thermosensitive suicide vectors for gene replacement in Streptococcus suis.Plasmid. 2001; 46: 140-148Crossref PubMed Scopus (237) Google Scholar). GAS strains and E. coli strains were cultured at 37 °C in Todd-Hewitt broth (Becton, Dickinson and Company) supplemented with 0.2% yeast extract (Becton Dickinson) (THY medium). E. coli strains were cultured in LB medium (Sigma-Aldrich) at 37 °C with agitation. For selection and maintenance of the mutants, antibiotics were added to the media at the following concentrations: ampicillin, 100 μg/ml for E. coli; erythromycin, 150 μg/ml for E. coli and 1 μg/ml for GAS; and spectinomycin, 100 μg/ml for E. coli and GAS. Preparation of recombinant SpeB has been previously described (16Terao Y. Mori Y. Yamaguchi M. Shimizu Y. Ooe K. Hamada S. Kawabata S. Group A streptococcal cysteine protease degrades C3 (C3b) and contributes to evasion of innate immunity.J. Biol. Chem. 2008; 283: 6253-6260Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). An in-frame speB deletion mutant, its complemented strain, and sagA-speB double mutant were constructed using pSET4s, as previously reported (11Sumitomo T. Nakata M. Higashino M. Jin Y. Terao Y. Fujinaga Y. Kawabata S. Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier.J. Biol. Chem. 2011; 286: 2750-2761Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 17Nakata M. Kimura K.R. Sumitomo T. Wada S. Sugauchi A. Oiki E. Higashino M. Kreikemeyer B. Podbielski A. Okahashi N. Hamada S. Isoda R. Terao Y. Kawabata S. Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes.J. Biol. Chem. 2011; 286: 37566-37577Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Primers speBkoF1 (5′-GCGGATCCTGTTTAATCGAAATGTTTTTTGAATGC-3′), speBkoR1 (5′-ACTTTGGTAACCGTTGAAGCCCATTTTTTTTATACCTCTTTC-3′), speBkoF2 (5′-GAAAGAGGTATAAAAAAAATGGGCTTCAACGGTTACCAAAGT-3′), and speBkoR2 (5′-AACTGCAGGTCTTAAAGGATGTACCGTATTGG-3′) were used for deletion of speB gene. For construction of EGFP-expressing GAS strains, a pAT18-EGFP vector was transformed into the GAS strains by electroporation (8Nakagawa I. Nakata M. Kawabata S. Hamada S. Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells.Cell. Microbiol. 2001; 3: 395-405Crossref PubMed Scopus (70) Google Scholar). Caco-2 cells (Riken Cell Bank) were maintained in minimum essential medium (Invitrogen) supplemented with 20% fetal bovine serum (SAFC Biosciences) and 20 μg/ml gentamicin, 17.75 mm NaHCO3 (Wako), and 15 mm HEPES (Dojindo) at pH 7.4. HaCaT cells were cultured in Dulbecco's modified Eagle's medium (Wako) supplemented with 10% fetal bovine serum (SAFC Biosciences), 20 μg/ml gentamicin. Detroit 562 cells (ATCC CCL-138; American Type Culture Collection) were maintained in minimum essential medium-α (Wako) supplemented with 10% fetal bovine serum (SAFC Biosciences) and 20 μg/ml gentamicin. For translocation assays, Caco-2 cells were seeded at 2 × 105 cells/well onto polycarbonate Millicell culture plate inserts (12-mm diameter, 3-μm pore size; Millipore) and cultured for 5 days at 37 °C under a 5% CO2 atmosphere, as described previously (11Sumitomo T. Nakata M. Higashino M. Jin Y. Terao Y. Fujinaga Y. Kawabata S. Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier.J. Biol. Chem. 2011; 286: 2750-2761Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Transepithelial electrical resistance (TER) of the filter-grown monolayers was measured using a Millicell-ERS device (Millipore), and monolayers exhibiting TER values of 450–500 Ω·cm2 were used in the experiments. GAS strains were grown to the exponential phase (A600 = 0.4) and centrifuged at 7000 × g for 5 min. Pelleted cells were washed with PBS and then resuspended in cell growth medium. Polarized monolayers were infected with GAS at an multiplicity of infection (MOI) of 10. The ability of GAS strains to translocate into monolayers was assessed by quantitative cultures of media obtained from the lower chambers at various times after infection as described previously (11Sumitomo T. Nakata M. Higashino M. Jin Y. Terao Y. Fujinaga Y. Kawabata S. Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier.J. Biol. Chem. 2011; 286: 2750-2761Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Caco-2 cells were grown on Millicell filters and infected with GAS at an MOI of 10 for 2 h. To remove nonadherent bacteria, the medium in the upper chamber was replaced with fresh medium at 2 h after infection. After removing nonadherent bacteria, FITC-dextran with a molecular mass of 4, 10, or 70 kDa (Sigma) was added to the apical surface of the cell monolayers. At 8 h after infection, the amount of FITC-dextran in the basolateral medium was measured using a Wallac 1420 ARVOsx fluorometer (excitation, 485 nm; emission, 535 nm; PerkinElmer Life Sciences). Overnight cultures of GAS clinical isolates were centrifuged at 7000 × g for 5 min, and then the supernatants were incubated with 0.5 μg of occludin (Abnova) or E-cadherin (R&D Systems or Advanced BioMatrix) for 6 h at 37 °C. To search for a bacterial protease that cleaves E-cadherin, GAS supernatants were individually pretreated for 30 min at room temperature with the following protease inhibitors; N-ethylmaleimide (1 mm), E-64 (10 μm), chymostatin (330 μm), leupeptin (100 μm), AEBSF (1 mm), aprotinin (800 nm), benzamidine HCl (1 mm), trypsin inhibitor (100 μm), 6-aminohexanoic acid (38 mm), pepstatin (1.5 μm), phosphoramidon (10 μm), bestatin (1 mm), or EDTA (1 mm). All protease inhibitors were purchased from Sigma-Aldrich. For infection assays, epithelial cells were seeded at 2 × 105 cells/well (35-mm diameter; Corning), cultured for 3 days, and then infected with GAS strains at an MOI of 10. At the end of the infection period, the infected cells were lysed with Laemmli gel loading buffer containing 6% 2-mercaptoethanol. Cleavage of intercellular junctions was detected by Western blot analysis (11Sumitomo T. Nakata M. Higashino M. Jin Y. Terao Y. Fujinaga Y. Kawabata S. Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier.J. Biol. Chem. 2011; 286: 2750-2761Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Western blot signals were quantified using Scion Image 4.0.3.2 software (Scion). For identification of the cleavage site in E-cadherin, E-cadherin was incubated with DTT-activated recombinant SpeB (200 nm) for 6 h at 37 °C. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. N-terminal amino acid sequencing was performed using the Edman degradation method with an ABI protein sequencer model 491HT (Applied Biosystems). For quantitative assays of SpeB activity, casein hydrolysis was analyzed using FITC-labeled casein (18Hauser A.R. Schlievert P.M. Nucleotide sequence of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor.J. Bacteriol. 1990; 172: 4536-4542Crossref PubMed Scopus (116) Google Scholar). Filter-sterilized supernatants from overnight GAS cultures or recombinant SpeB were activated in assay buffer (0.1 m phosphate buffer, pH 7.6, 0.01 mm EDTA) supplemented with 10 mm DTT and incubated for 30 min at 37 °C. The activated supernatants were added to an equal volume of FITC-labeled casein (Sigma-Aldrich) prepared with or without 10 μm E-64. At 6 h after incubation, the reactions were stopped by adding 5% trichloroacetic acid, and then the mixtures were stored overnight at 4 °C. Following centrifugation (15,000 × g for 5 min), the resultant supernatants were diluted with 0.5 m Tris buffer (pH 8.5), and fluorescence intensity was measured using a Wallac 1420 ARVOsx fluorometer (excitation wavelength, 485 nm; emission wavelength, 535 nm; PerkinElmer Life Sciences). Statistical analysis was performed using a Mann-Whitney U test. A confidence interval with a p value of < 0.05 was considered to be significant. Culture supernatants from several serotypes of GAS strains recovered from invasive (strains SSI-9, SSI-1, #30, and NIH35) and noninvasive (strains SF370, TW3358, TW3337, TW3339, NZ131, and 591) diseases were incubated with the extracellular domain of human E-cadherin fused to the Fc region of human IgG1, after which cleavage was analyzed using Western blot analysis (Fig. 1). Utilizing an antibody against the extracellular domain of E-cadherin, the intact protein was detected as an ∼120-kDa band under a reducing condition. Loss of the 120-kDa band and appearance of cleavage products with apparent molecular masses of 60–100 kDa were detected in culture supernatant samples obtained from SF370, SSI-1, TW3358, #30, TW3337, NIH35, TW3339, and 591. Interestingly, cleavage was completely abrogated by pretreatment with a protease inhibitor mixture (data not shown). On the other hand, neither the culture supernatant from strain SSI-9 nor that from strain NZ131 was capable of cleaving the E-cadherin fragment. These results indicate that the culture supernatants from strains SF370, SSI-1, TW3358, #30, TW3337, NIH35, TW3339 and 591 include proteases that cleave the extracellular domain of E-cadherin. We also noted that the ability of GAS culture supernatants to cleave E-cadherin was not related to disease severity, i.e., strains from invasive as compared with those from noninvasive diseases. To search for bacterial proteases that contribute to the cleavage of E-cadherin, the culture supernatant of strain NIH35, a prominent degrader, was pretreated with several types of protease inhibitors before co-incubation with recombinant E-cadherin protein (Fig. 2). The cysteine protease inhibitors N-ethylmaleimide, E-64, and chymostatin completely inhibited cleavage of E-cadherin, whereas protease activity partially remained in the leupeptin-treated GAS supernatant. In contrast, protease inhibitors that target serine protease, aspartate protease, and metalloprotease had no detectable effect on E-cadherin cleavage, whereas EDTA did have an acceleratory effect. This result may reflect a chelating effect of EDTA on stabilization of the E-cadherin structure, because E-cadherin forms a homodimer in the extracellular domain in a Ca2+-dependent manner. In experiments with strain SSI-1, even though the degradation pattern of E-cadherin was slightly different from that observed with other strains (Fig. 1), the appearance of cleavage products was completely inhibited by cysteine protease inhibitors (data not shown). It has been reported that GAS secretes two major cysteine proteases, SpeB and immunoglobulin G-degrading enzyme of S. pyogenes (IdeS or Mac-1). E-64, a cysteine protease, specifically inhibits the proteolytic activity of SpeB, but not that of IdeS (19von Pawel-Rammingen U. Johansson B.P. Björck L. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.EMBO J. 2002; 21: 1607-1615Crossref PubMed Scopus (328) Google Scholar). In the present study, supernatant-induced cleavage of E-cadherin was completely inhibited by E-64 (Fig. 2). These results suggest that SpeB is a proteolytic factor for cleavage of E-cadherin. Moreover, restoration of the full-length band was observed in a leupeptin concentration-dependent manner, whereas the cleavage product did not disappear even in the presence of leupeptin at 500 μm (supplemental Fig. S1). Leupeptin is an arginine-dependent protease inhibitor. Because Cys at position 192, His at position 340, and Trp at position 357 play critical roles in the enzymatic activity of SpeB (20Carroll R.K. Musser J.M. From transcription to activation. How group A streptococcus, the flesh-eating pathogen, regulates SpeB cysteine protease production.Mol. Microbiol. 2011; 81: 588-601Crossref PubMed Scopus (60) Google Scholar), leupeptin might be less effective for inhibition of SpeB activity as compared with those other cysteine proteases. Consequently, we investigated whether SpeB is involved in cleavage of E-cadherin. To examine whether SpeB is responsible for GAS supernatant-induced cleavage of intercellular junctions, an in-frame speB deletion mutant and its complemented strain were constructed. Equivalent growth rates of wild type and mutant strains were observed in conventional liquid medium (data not shown). GAS culture supernatant-induced cleavage of E-cadherin was nearly abolished by mutagenesis of the speB gene, whereas it was completely restored by the complementation (Fig. 3A). A similar phenomenon was observed with strains #30, TW3337, and TW3339 (supplemental Fig. S2), indicating that SpeB-dependent protease activity toward E-cadherin is conserved among clinical isolates with no relation to disease severity. These observations prompted us to compare the SpeB-specific protease activities in the culture supernatants of the previously examined GAS strains (Fig. 3B). High caseinolytic activities were detected in culture supernatants from strains #30, TW3337, NIH35, TW3339, and 591, which are findings consistent with its ability to cleave recombinant E-cadherin (Fig. 1). In contrast, lower levels of SpeB activity were observed in supernatants from strains SSI-9, SF370, SSI-1, TW3358, and NZ131. Together, these results indicate that SpeB-associated cysteine protease activity is correlated with the cleavage of intercellular junctions. SpeB is secreted as a 42-kDa zymogen and autocatalyzed into an active 28-kDa cysteine protease (18Hauser A.R. Schlievert P.M. Nucleotide sequence of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor.J. Bacteriol. 1990; 172: 4536-4542Crossref PubMed Scopus (116) Google Scholar, 20Carroll R.K. Musser J.M. From transcription to activation. How group A streptococcus, the flesh-eating pathogen, regulates SpeB cysteine protease production.Mol. Microbiol. 2011; 81: 588-601Crossref PubMed Scopus (60) Google Scholar), and its substrate specificity is similar to that of the papain family of proteases (21Nomizu M. Pietrzynski G. Kato T. Lachance P. Menard R. Ziomek E. Substrate specificity of the streptococcal cysteine protease.J. Biol. Chem. 2001; 276: 44551-44556Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Active SpeB cleaves the host extracellular matrix (22Kapur V. Topouzis S. Majesky M.W. Li L.L. Hamrick M.R. Hamill R.J. Patti J.M. Musser J.M. A conserved Streptococcus pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin.Microb. Pathog. 1993; 15: 327-346Crossref PubMed Scopus (186) Google Scholar), immunoglobulins (23Collin M. Olsén A. Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins.Infect. Immun. 2001; 69: 7187-7189Crossref PubMed Scopus (153) Google Scholar, 24Eriksson A. Norgren M. Cleavage of antigen-bound immunoglobulin G by SpeB contributes to streptococcal persistence in opsonizing blood.Infect. Immun. 2003; 71: 211-217Crossref PubMed Scopus (48) Google Scholar), and complement components (16Terao Y. Mori Y. Yamaguchi M. Shimizu Y. Ooe K. Hamada S. Kawabata S. Group A streptococcal cysteine protease degrades C3 (C3b) and contributes to evasion of innate immunity.J. Biol. Chem. 2008; 283: 6253-6260Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In addition to its ability to cleave host proteins, SpeB exerts proteolytic activity toward streptococcal surface proteins (25Berge A. Björck L. Streptococcal cysteine proteinase releases biologically active fragments of streptococcal surface proteins.J. Biol. Chem. 1995; 270: 9862-9867Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 26Chaussee M.S. Cole R.L. van Putten J.P. Streptococcal erythrogenic toxin B abrogates fibronectin-dependent internalization of Streptococcus pyogenes by cultured mammalian cells.Infect. Immun. 2000; 68: 3226-3232Crossref PubMed Scopus (32) Google Scholar, 27Walker M.J. Hollands A. Sanderson-Smith M.L. Cole J.N. Kirk J.K. Henningham A. McArthur J.D. Dinkla K. Aziz R.K. Kansal R.G. Simpson A.J. Buchanan J.T. Chhatwal G.S. Kotb M. Nizet V. DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection.Nat. Med. 2007; 13: 981-985Crossref PubMed Scopus (319) Google Scholar). Numerous investigations have reported that this proteolytic activity contributes to bacterial evasion from the host defense system and systemic dissemination (reviewed in Refs. 28Chiang-Ni C. Wu J.J. Effects of streptococcal pyrogenic exotoxin B on pathogenesis of Streptococcus pyogenes.J. Formos. Med. Assoc. 2008; 107: 677-685Crossref PubMed Scopus (41) Google Scholar and 29Nelson D.C. Garbe J. Collin M. Cysteine proteinase SpeB from Streptococcus pyogenes. A potent modifier of immunologically important host and bacterial proteins.Biol. Chem. 2011; 392: 1077-1088Crossref PubMed Scopus (108) Google Scholar). Therefore, we postulated that SpeB directly cleaves intercellular junctional proteins, and GAS translocates through the opening of paracellular junctions. To examine whether SpeB directly cleaves transmembrane junctions, recombinant E-cadherin was treated with various concentrations of recombinant SpeB (Fig. 3C). An SpeB-dependent loss of the full-length band and appearance of several cleaved products, which reacted with an antibody against the extracellular domain of E-cadherin, were detected. Thus, five N-terminal residues in the cleavage products of E-cadherin in the presence of 200 nm SpeB were determined by peptide sequence analysis, which identified the N-terminal sequence of fragments with molecular masses of ∼80 and 65 kDa as DWVIP, which is identical to the N terminus of the EC1 domain, i.e., the N terminus of the recombinant protein. SpeB has been reported to cleave IgG in the hinge region (23Collin M. Olsén A. Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins.Infect. Immun. 2001; 69: 7187-7189Crossref PubMed Scopus (153) Google Scholar). Because recombinant E-cadherin is the extracellular domain fused to the IgG Fc region at the C terminus, it is likely that an 80-kDa band represents a fragment with intact extracellular E-cadherin and a truncated Fc fragment. Deduced from this size, the 65-kDa band represents a fragment cleaved within the EC domains, possibly between EC4 and EC5. Meanwhile, the sequence of cleavage fragments with molecular masses of ∼55 and 35 kDa revealed VTDTN, which corresponds to the neighboring sequence of the calcium-binding site between the EC2 and EC3 domains. Speculating from these sizes, the C terminus of the 55- and 35-kDa fragments would be within the Fc region and within the region between EC4 and EC5, respectively. Because each EC domain of E-cadherin contains DXD or DXNDN motifs responsible for mediating calcium-dependent adhesion, SpeB may cleave the regions neighboring these motifs between each EC domain. Furthermore, SpeB-mediated cleavage of occludin, the most prominent member of TJ, was examined using an antibody against the C terminus of occludin (Fig. 3D). Cleavage of the full-length form of occludin, which was recognized as an 85-kDa band, and distinct cleavage products were detected in an SpeB concentration-dependent manner. These findings suggest that SpeB directly cleaves occludin at several sites. To investigate destabilization of the intercellular junctions in GAS-infected epithelial Caco-2 cells, a typical epithelial in vitro model, cells were infected with GAS strains, and the cleavage of junctional proteins was detected using Western blot analysis (Fig. 4). Marked cleavage of E-cadherin and occludin was detected in Caco-2 cells infected with the wild type strain, whereas that was significantly repressed by mutagenesis of the speB gene and restored by complementation. Similar results were observed in keratinocyte HaCaT and pharyngeal epithelial Detroit 562 cells, which are human cell lines derived from primary anatomical sites of GAS infection. On the other hand, the cleavage of JAM-1, a member of the group of TJ proteins, was not detected in cells infected with any of the strains. These findings indicate that SpeB" @default.
• W2022233387 created "2016-06-24" @default.
• W2022233387 creator A5019924283 @default.
• W2022233387 creator A5046070504 @default.
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• W2022233387 date "2013-05-01" @default.
• W2022233387 modified "2023-10-18" @default.
• W2022233387 title "Group A Streptococcal Cysteine Protease Cleaves Epithelial Junctions and Contributes to Bacterial Translocation" @default.
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