RESUMO
Streptococcus (S.) phocae subsp. phocae causes bronchopneumonia and septicemia in a variety of marine mammals. Especially in harbor seals infected with phocine distemper virus it plays an important role as an opportunistic pathogen. This study was initiated by the detection of IgG cleavage products in Western blot analysis after incubation of bacterial supernatant with harbor seal serum. Hence, the objectives of this study were the identification and characterization of a secreted IgG cleaving protease in S. phocae subsp. phocae isolated from marine mammals. To further identify the responsible factor of IgG cleavage a protease inhibitor profile was generated. Inhibition of the IgG cleaving activity by iodoacetamide and Z-LVG-CHN2 indicated that a cysteine protease is involved. Moreover, an anti-IdeS antibody directed against the IgG endopeptidase IdeS of S. pyogenes showed cross reactivity with the putative IgG protease of S. phocae subsp. phocae. The IgG cleaving factor of S. phocae subsp. phocae was identified through an inverse PCR approach and designated IdeP (Immunoglobulin G degrading enzyme of S. phocae subsp. phocae) in analogy to the cysteine protease IdeS. Notably, recombinant (r) IdeP is a host and substrate specific protease as it cleaves IgG from grey and harbor seals but not IgG from harbor porpoises or non-marine mammals. The identification of IdeP represents the first description of a protein in S. phocae subsp. phocae involved in immune evasion. Furthermore, the fact that IdeP cleaves solely IgG of certain marine mammals reflects functional adaption of S. phocae subsp. phocae to grey and harbor seals as its main hosts.
Assuntos
Endopeptidases/metabolismo , Imunoglobulina G/metabolismo , Phoca/microbiologia , Inibidores de Proteases/farmacologia , Infecções Estreptocócicas/veterinária , Streptococcus pyogenes/enzimologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Reações Cruzadas , Endopeptidases/genética , Especificidade de Hospedeiro , Evasão da Resposta Imune , Iodoacetamida/farmacologia , Oligopeptídeos/farmacologia , Proteínas Recombinantes , Análise de Sequência de DNA/veterinária , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/imunologiaRESUMO
Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.
Assuntos
Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Imunoglobulina G/metabolismo , Streptococcus/enzimologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Endopeptidases/classificação , Endopeptidases/genética , Genoma Bacteriano , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina E/metabolismo , Dados de Sequência Molecular , Filogenia , Proteólise , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Streptococcus/genética , Especificidade por Substrato , SuínosRESUMO
Cholera epidemics are caused by Vibrio cholerae serogroups O1 and O139, whereas strains collectively known as non-O1/non-O139 V. cholerae are found in cases of extraintestinal infections and bacteremia. The mechanisms and factors influencing the occurrence of bacteremia and survival of V. cholerae in normal human serum have remained unclear. We found that naturally occurring IgG recognizing V. cholerae outer membrane protein U (OmpU) mediates a serum-killing effect in a complement C1q-dependent manner. Moreover, outer membrane vesicles (OMVs) containing OmpU caused enhanced survival of highly serum-sensitive classical V. cholerae in a dose-dependent manner. OMVs from wild-type and ompU mutant V. cholerae thereby provided a novel means to verify by extracellular transcomplementation the involvement of OmpU. Our data conclusively indicate that loss, or reduced expression, of OmpU imparts resistance to V. cholerae towards serum killing. We propose that the difference in OmpU protein levels is a plausible reason for differences in serum resistance and the ability to cause bacteremia observed among V. cholerae biotypes. Our findings provide a new perspective on how naturally occurring antibodies, perhaps induced by members of the microbiome, may play a role in the recognition of pathogens and the provocation of innate immune defense against bacteremia.
Assuntos
Adesinas Bacterianas/metabolismo , Bacteriemia/imunologia , Cólera/imunologia , Vibrio cholerae/imunologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/sangue , Citotoxicidade Celular Dependente de Anticorpos , Bacteriólise , Clonagem Molecular , Suscetibilidade a Doenças , Engenharia Genética , Humanos , Imunidade Humoral , Imunidade Inata , Imunoglobulina G/sangue , Mutação/genética , Vibrio cholerae/genéticaRESUMO
Streptococcus suisis a major endemic pathogen of pigs causing meningitis, arthritis, and other diseases. ZoonoticS. suisinfections are emerging in humans causing similar pathologies as well as severe conditions such as toxic shock-like syndrome. Recently, we discovered an IdeS family protease ofS. suisthat exclusively cleaves porcine IgM and represents the first virulence factor described, linkingS. suisto pigs as their natural host. Here we report the identification and characterization of a novel, unrelated protease ofS. suisthat exclusively targets porcine IgG. This enzyme, designated IgdE forimmunoglobulinG-degradingenzyme ofS. suis, is a cysteine protease distinct from previous characterized streptococcal immunoglobulin degrading proteases of the IdeS family and mediates efficient cleavage of the hinge region of porcine IgG with a high degree of specificity. The findings that allS. suisstrains investigated possess the IgG proteolytic activity and that piglet serum samples contain specific antibodies against IgdE strongly indicate that the protease is expressedin vivoduring infection and represents a novel and putative important bacterial virulence/colonization determinant, and a thus potential therapeutic target.
Assuntos
Proteínas de Bactérias/metabolismo , Cisteína Proteases/metabolismo , Imunoglobulina G/metabolismo , Infecções Estreptocócicas/veterinária , Streptococcus suis/enzimologia , Suínos/microbiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Domínio Catalítico , Cisteína Proteases/química , Imunoglobulina G/química , Modelos Moleculares , Dados de Sequência Molecular , Proteólise , Infecções Estreptocócicas/metabolismo , Streptococcus suis/metabolismo , Especificidade por Substrato , Suínos/metabolismoRESUMO
The important human gram positive bacterial pathogen Streptococcus pyogenes employs various virulence factors to promote inflammation and to facilitate invasive disease progression. In this study we explored the relation of the secreted streptococcal cysteine proteases IdeS and SpeB, and neutrophil (PMN) proteases. We found that SpeB is resistant to proteolytic attack in an inflammatory environment, emphasizing the importance of SpeB for streptococcal pathogenicity, while PMN enzymes and SpeB itself process the IgG degrading endopeptidase IdeS. Processing occurs as NH2-terminal cleavage of IdeS resulting in reduced immunorecognition of the protease by specific antibodies. While the endopeptidase retains IgG cleaving activity, its ability to suppress the generation of reactive oxygen species is abolished. We suggest that the cleavage of NH2-terminal peptides by SpeB and/or neutrophil proteases is a mechanism evolved to prevent early inactivation of this important streptococcal virulence factor, albeit at the cost of impaired functionality.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Exotoxinas/imunologia , Elastase de Leucócito/imunologia , Infecções Estreptocócicas/imunologia , Tonsilite/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Exotoxinas/genética , Exotoxinas/metabolismo , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunoglobulina G/genética , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Dados de Sequência Molecular , Neutrófilos/enzimologia , Neutrófilos/imunologia , Proteólise , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Infecções Estreptocócicas/enzimologia , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/imunologia , Tonsilite/enzimologia , Tonsilite/genética , Tonsilite/patologiaRESUMO
Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated Ide(Ssuis) . The objective of this study was to characterize the function of Ide(Ssuis) in the host-pathogen interaction. Edman-sequencing revealed that Ide(Ssuis) cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that Ide(Ssuis) is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant Ide(Ssuis) . Furthermore, expression of Ide(Ssuis) reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10Δide(Ssuis). Bactericidal assays confirmed a positive effect of Ide(Ssuis) expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that Ide(Ssuis) is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas do Sistema Complemento/fisiologia , Proteínas Hemolisinas/fisiologia , Imunoglobulina M/fisiologia , Infecções Estreptocócicas/veterinária , Streptococcus suis/fisiologia , Doenças dos Suínos/imunologia , Animais , Cápsulas Bacterianas/fisiologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Suínos , Doenças dos Suínos/microbiologiaRESUMO
Streptococcus suis (S. suis) is a major porcine pathogen causing meningitis, arthritis and several other pathologies. Recently, we identified a highly specific immunoglobulin M degrading enzyme of S. suis, designated IdeSsuis, which is expressed by various serotypes. The objective of this work was to access the immunogenicity and protective efficacy of a recombinant vaccine including IdeSsuis. Vaccination with rIdeSsuis elicited antibodies efficiently neutralizing the IgM protease activity. Importantly, 18 piglets vaccinated with rIdeSsuis alone or in combination with bacterin priming were completely protected against mortality and severe morbidity after S. suis serotype 2 challenge. In contrast, 12 of the 17 piglets either treated with the placebo or primed with the bacterin only, succumbed to S. suis disease. Immunity against IdeSsuis was associated with increased killing of S. suis wt in porcine blood ex vivo leading to a tenfold difference in the bacterial survival factor in blood of placebo-treated and rIdeSsuis-vaccinated piglets. In conclusion, the results of this study indicate that rIdeSsuis is a highly protective antigen in pigs.
Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Peptídeo Hidrolases/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus suis/enzimologia , Streptococcus suis/imunologia , Doenças dos Suínos/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Antígenos de Bactérias/genética , Vacinas Bacterianas/genética , Vacinas Bacterianas/isolamento & purificação , Imunoglobulina M/metabolismo , Peptídeo Hidrolases/genética , Proteólise , Sorogrupo , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/patologia , Infecções Estreptocócicas/prevenção & controle , Streptococcus suis/classificação , Streptococcus suis/genética , Análise de Sobrevida , Sus scrofa , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/patologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
UNLABELLED: The Mac/IdeS protein of group A Streptococcus (GAS) is a secreted cysteine protease with cleavage specificity for IgG and is highly expressed in the GAS serotype M1T1 clone, which is the serotype most frequently isolated from patients with life-threatening invasive infections. While studies of Mac/IdeS with recombinant protein have shown that the protein can potentially prevent opsonophagocytosis of GAS by neutrophils, the role of the protein in immune evasion as physiologically produced by the living organism has not been studied. Here we examined the contribution of Mac/IdeS to invasive GAS disease by generating a mutant lacking Mac/IdeS in the hyperinvasive M1T1 background. While Mac/IdeS was highly expressed and proteolytically active in the hyperinvasive strain, elimination of the bacterial protease did not significantly influence GAS phagocytic uptake, oxidative-burst induction, cathelicidin sensitivity, resistance to neutrophil or macrophage killing, or pathogenicity in pre- or postimmune mouse infectious challenges. We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence. Given the conservation of Mac/IdeS and homologues across GAS strains, it is possible that Mac/IdeS serves another important function in GAS ecology or contributes to virulence in other strain backgrounds. IMPORTANCE: Group A Streptococcus (GAS) causes human infections ranging from strep throat to life-threatening conditions such as flesh-eating disease and toxic shock syndrome. Common disease-associated clones of GAS can cause both mild and severe infections because of a characteristic mutation and subsequent change in the expression of several genes that develops under host immune selection. One of these genes encodes Mac/IdeS, a protease that has been shown to cleave antibodies important to the immune defense system. In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse. These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.
Assuntos
Proteínas de Bactérias/metabolismo , Imunoglobulina G/metabolismo , Fagócitos/microbiologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Deleção de Genes , Humanos , Masculino , Camundongos , Viabilidade Microbiana , Fagocitose , Proteólise , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genética , Virulência , Fatores de Virulência/genéticaRESUMO
Streptococcus pyogenes employs an IgG specific endopeptidase, IdeS, to counteract the effector functions of specific IgG. The physiological significant step in disarming specific IgG is the cleavage of one IgG heavy chain. So far, characterizations of IdeS enzymatic activity have employed techniques that failed to differentiate between the first and the second cleavage step. The present data demonstrate that IdeS is active as a monomer and that IdeS activity follows classical Michaelis-Menten kinetics arguing against the previously proposed formation of a functional IdeS dimer. Our results show that IdeS inactivates IgG 100-fold faster than previously reported.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Proteólise , Cinética , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
The human bacterial pathogen Streptococcus pyogenes has developed a broad variety of virulence mechanisms to evade the actions of the host immune defense. One of the best-characterized factors is the streptococcal cysteine protease SpeB, an important multifunctional protease that contributes to group A streptococcal pathogenesis in vivo. Among many suggested activities, SpeB has been described to degrade various human plasma proteins, including immunoglobulins (Igs). In this study, we show that SpeB has no Ig-cleaving activity under physiological conditions and that only Igs in a reduced state, i.e., semimonomeric molecules, are cleaved and degraded by SpeB. Since reducing conditions outside eukaryotic cells have to be considered nonphysiological and IgG in a reduced state lacks biological effector functions, we conclude that SpeB does not contribute to S. pyogenes virulence through the proteolytic degradation of Igs.
Assuntos
Proteínas de Bactérias/metabolismo , Exotoxinas/metabolismo , Imunoglobulina G/metabolismo , Streptococcus pyogenes/enzimologia , Proteínas de Bactérias/genética , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Células Cultivadas , Exotoxinas/genética , Humanos , Imunoglobulina G/química , Neutrófilos/fisiologia , Plasma , Streptococcus pyogenes/patogenicidade , VirulênciaRESUMO
Streptococcus suis serotype 2 is a highly invasive, extracellular pathogen in pigs with the capacity to cause severe infections in humans. This study was initiated by the finding that IgM degradation products are released after opsonization of S. suis. The objective of this work was to identify the bacterial factor responsible for IgM degradation. The results of this study showed that a member of the IdeS family, designated Ide(Ssuis) (Immunoglobulin M-degrading enzyme of S. suis), is responsible and sufficient for IgM cleavage. Recombinant Ide(Ssuis) was found to degrade only IgM but neither IgG nor IgA. Interestingly, Western blot analysis revealed that Ide(Ssuis) is host specific, as it exclusively cleaves porcine IgM but not IgM from six other species, including a closely related member of the Suidae family. As demonstrated by flow cytometry and immunofluorescence microscopy, Ide(Ssuis) modulates binding of IgM to the bacterial surface. Ide(Ssuis) is the first prokaryotic IgM-specific protease described, indicating that this enzyme is involved in a so-far-unknown mechanism of host-pathogen interaction at an early stage of the host immune response. Furthermore, cleavage of porcine IgM by Ide(Ssuis) is the first identified phenotype reflecting functional adaptation of S. suis to pigs as the main host.
Assuntos
Imunoglobulina M/metabolismo , Peptídeo Hidrolases/metabolismo , Streptococcus suis/enzimologia , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Imunoglobulina A , Imunoglobulina G , Imunoglobulina M/imunologia , Proteínas Opsonizantes , Análise de Sequência de DNA , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Streptococcus suis/imunologia , Suínos/imunologia , Fatores de Virulência/metabolismoRESUMO
SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.
Assuntos
Epiderme/enzimologia , Pé , Regulação da Expressão Gênica , Mãos , Calicreínas/antagonistas & inibidores , Inibidores de Proteases/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Humanos , Calicreínas/química , Calicreínas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Proteases/química , Conformação Proteica , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Inibidores de Serinopeptidase do Tipo Kazal , Especificidade por SubstratoRESUMO
A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes , IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies.
Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Inibidores de Cisteína Proteinase/síntese química , Imunoglobulina G/química , Peptidomiméticos/síntese química , Piperidinas/síntese química , Sequência de Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/química , Inibidores de Cisteína Proteinase/química , Eletroforese em Gel de Poliacrilamida , Ensaios Enzimáticos , Exotoxinas/antagonistas & inibidores , Exotoxinas/química , Dados de Sequência Molecular , Papaína/antagonistas & inibidores , Papaína/química , Peptidomiméticos/química , Piperidinas/química , Estereoisomerismo , Streptococcus pyogenes/enzimologia , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
Survival of the important bacterial pathogen Streptococcus pyogenes relies on its ability to circumvent the antimicrobial actions of innate and specific immune responses and to modulate the inflammatory responses induced during the course of an infection. Inflammatory processes play key roles during streptococcal pathogenesis and streptococcal infections are accompanied by an intense inflammatory state. As an exclusively human pathogen, S. pyogenes has adapted to the various countermeasures employed by its host to fight bacterial infections, in particular to interfere with the effector functions of immunoglobulin G (IgG). For this purpose, S. pyogenes has evolved an IgG-specific endopeptidase, IdeS, which is highly specific for the lower hinge region of IgG. This review summarizes the current knowledge about this intriguing enzyme as well as its role in inflammation and in the attenuation of human immune responses towards streptococcal infection.
Assuntos
Proteínas de Bactérias/imunologia , Inflamação/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Proteínas de Bactérias/metabolismo , Humanos , Imunoglobulina G/metabolismo , Inflamação/enzimologia , Inflamação/microbiologia , Infecções Estreptocócicas/enzimologia , Streptococcus pyogenes/enzimologiaRESUMO
Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG(1) was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG(3) was also cleaved within the heavy chain, but at several sites around the CH2 region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC- and isothermal titration calorimetry-based assays followed by Hill plots, revealed non-Michaelis-Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG(1) cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin-dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance.
Assuntos
Adesinas Bacterianas/farmacologia , Cisteína Endopeptidases/farmacologia , Imunoglobulina G/metabolismo , Periodontite/patologia , Porphyromonas gingivalis/metabolismo , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases Gingipaínas , Interações Hospedeiro-Patógeno , Humanos , Dados de Sequência Molecular , Periodontite/microbiologiaRESUMO
Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR) have been identified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20-30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid). The streptococcal leucine rich (Slr) protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein) and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Colágeno Tipo I/metabolismo , Lipoproteínas/metabolismo , Streptococcus pyogenes/metabolismo , Western Blotting , Parede Celular/metabolismo , Microscopia Eletrônica de Transmissão , Ligação Proteica , Streptococcus pyogenes/ultraestrutura , Ressonância de Plasmônio de SuperfícieRESUMO
Human cystatin C is considered the physiologically most important inhibitor of endogenous papain-like cysteine proteases. We present here an unexpected function of cystatin C. Instead of acting as an inhibitor, cystatin C acts as a facultative, endogenous cofactor for the papain-like IgG-cleaving enzyme IdeS of the human pathogen Streptococcus pyogenes. IdeS activity is not dependent on cystatin C, but is significantly enhanced in the presence of cystatin C. We report a protease inhibitor that accelerates the activity of its putative target protease and a unique example of how a host protease inhibitor is "hijacked" by a bacterial protease to increase its activity. This finding has important implications for the view on protease-inhibitor interactions, and is relevant to consider in the therapeutic use of protease inhibitors.
Assuntos
Proteínas de Bactérias/metabolismo , Cistatinas/metabolismo , Cisteína Endopeptidases/metabolismo , Inibidores de Proteases/metabolismo , Streptococcus/enzimologia , Cistatina C , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Humanos , Hidrólise , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cinética , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
The important human bacterial pathogen Streptococcus pyogenes has evolved a variety of mechanisms to evade the actions of the human immune system. M protein and M-like proteins are major virulence factors that bind with high affinity to the Fc-part of IgG. However, the contribution of non-immune binding of IgG to bacterial virulence is not fully established. Importantly, the capacity of S. pyogenes to bind IgG is limited and due to the presence of large amounts of IgG present in vivo, the majority of IgGFc binding sites at the streptococcal surface are likely to be occupied by non-specific IgG. S. pyogenes also secretes a highly effective IgG-endopeptidase, IdeS that inhibits phagocytic killing by cleavage of specific IgG creating F(ab')2 and 1/2Fc fragments. In the present work, IgG and 1/2Fc binding to the streptococcal surface was studied and correlated to IdeS activity. Binding of IgG to the streptococcal surface is shown to be equilibrium and thus not designed to mediate a lasting protection against specific antibodies. However, non-immune binding of IgG to the bacterial surface is followed by the proteolytic cleavage of the antibody by the IgG-endopeptidase IdeS. IdeS generated 1/2Fc fragments do not compete efficiently with intact IgG in binding to the bacterial surface and rapid dissociation of 1/2Fc allows binding of new IgG. Thus, a correlated binding and proteolytic cleavage of IgG also increases the probability that the bacteria can resist specific IgG, despite the presence of a large excess of non-specific IgG in the circulation. As a consequence of IdeS activity, circulating 1/2Fc fragments are generated. These 1/2Fc fragments were shown to be biological active by acting as priming agents for polymorphonuclear leucocytes, suggesting a new mechanism of immune evasion employed by S. pyogenes.
Assuntos
Proteínas de Bactérias/metabolismo , Fragmentos Fc das Imunoglobulinas/imunologia , Neutrófilos/citologia , Neutrófilos/imunologia , Bacteriólise , Humanos , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/sangue , Viabilidade Microbiana , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pyogenes/imunologiaRESUMO
IdeS, a secreted cysteine protease of the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G (IgG). Two allelic variants of the enzyme have been described, the IgG-specific endopeptidase, IdeS (or Mac-1) and Mac-2, a protein with only weak IgG endopeptidase activity, which has been suggested to interfere with opsonophagocytosis by blocking Fcgamma receptors of phagocytic cells. However, despite the fact that Mac-2 proteins interact with Fcgamma receptors, no inhibition of reactive oxygen species (ROS) production, opsonophagocytosis, or streptococcal killing by Mac-2 has been reported. In the present study, Mac-2 proteins are shown to contain IgG endopeptidase activity indistinguishable from the enzymatic activity exhibited by IdeS/Mac-1 proteins. The earlier reported weak IgG endopeptidase activity appears to be unique to Mac-2 of M28 serotype strains (Mac-2(M28)) and is most likely due to the formation of a disulfide bond between the catalytic site cysteine and a cysteine residue in position 257 of Mac-2(M28). Furthermore, Mac-2 proteins are shown to inhibit ROS production ex vivo, independently of the IgG endopeptidase activity of the proteins. Inhibition of ROS generation per se, however, was not sufficient to mediate streptococcal survival in bactericidal assays. Thus, in contrast to earlier studies, implicating separate functions for IdeS and Mac-2 protein variants, the current study suggests that Mac-2 and IdeS are bifunctional proteins, combining Fcgamma receptor binding and IgG endopeptidase activity. This finding implies a unique role for Mac-2 proteins of the M28 serotype, since this serotype has evolved and retained a Mac-2 protein lacking IgG endopeptidase activity.
Assuntos
Proteínas de Bactérias/metabolismo , Imunoglobulina G/metabolismo , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/fisiologia , Proteínas de Bactérias/genética , Humanos , Viabilidade Microbiana , Mutagênese Sítio-Dirigida , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genéticaRESUMO
IdeS, a recently discovered cysteine proteinase secreted by the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G. The fact that the enzyme targets one of the key molecules of the adapted immune response raised the question of whether an antibody response against IdeS could inhibit, i.e., neutralize, enzyme activity. Paired acute- and convalescent-phase serum samples from patients with pharyngotonsillitis (n = 10), bacteremia (n = 7), and erysipelas (n = 4) were analyzed. Antibodies with the ability to neutralize IdeS enzymatic activity were already found in two-thirds of acute-phase sera. However, patients who seroconverted to IdeS, in particular patients with pharyngotonsillitis and erysipelas, developed specific antibodies during convalescence with an increased capability to efficiently neutralize the enzymatic activity of IdeS. Also, the presence of neutralizing antibodies decreased the ability of IdeS to mediate bacterial survival in human immune blood. In patients with bacteremia, several acute-phase sera contained neutralizing antibodies, but no correlation was found to severity or outcome of invasive infections. Still, 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.