RESUMO
Pulmonary alveolar proteinosis (PAP) is a group of rare diseases with disturbed homeostasis of alveolar surfactant. While 90% of the primary adult forms are caused by granulocyte-macrophage colony-stimulating factor autoantibodies, the underlying cause of the juvenile form remains unknown. In order to distinguish primary from secondary effects in the pathogenesis of these two forms, the present authors studied the surfactant protein processing proteases napsin A and cathepsin H. In total, 16 controls, 20 patients with juvenile PAP and 13 adults with idiopathic PAP were enrolled. Amounts and activities of the proteases in the bronchoalveolar lavage fluid (BALF) were determined by immunoblotting and specific substrate cleavage. Both proteases were present and active in BALF from controls and increased in juvenile and adult PAP patients. The amount of active cathepsin H in relation to total cathepsin H was increased in PAP patients compared with controls. Cystatin C, the physiological inhibitor of cathepsin H in the alveolar space, was not increased to the same degree as cathepsin H, resulting in an imbalance of inhibitor to protease in the alveolar space. A general defect in napsin A or cathepsin H expression or activity was not the specific cause for abnormal surfactant accumulation in juvenile pulmonary alveolar proteinosis.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Proteinose Alveolar Pulmonar/enzimologia , Adulto , Líquido da Lavagem Broncoalveolar/química , Estudos de Casos e Controles , Catepsina H , Pré-Escolar , Cistatina C , Cistatinas/metabolismo , Humanos , LactenteRESUMO
Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions.
Assuntos
Cisteína Endopeptidases/metabolismo , Infecções Estreptocócicas/enzimologia , Streptococcus pyogenes/enzimologia , Animais , Cisteína Endopeptidases/genética , Humanos , Masculino , Camundongos , Mutagênese , Mutação , Plasmídeos , Infecções Estreptocócicas/mortalidade , Streptococcus pyogenes/patogenicidade , Virulência/genéticaRESUMO
Ten novel streptococcal shuttle vectors for genomic integration and allelic replacements have been constructed based on plasmid pSF152. These vectors can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The basic vector pFW5 (2.8 kb, aad9 spectinomycin-resistance marker) carries two multiple cloning sites MCS-I and MCS-II (10 and 15 restrictions sites, respectively) to either side of the aad9 resistance gene. Each MCS is flanked by transcription termination sites for stabilization of recombinant plasmids. In vector pFW6 the transcription terminator between aad9 and MCS-II was deleted. Plasmids pFW7 through pFW10 carry resistance genes for kanamycin, chloramphenicol, erythromycin, and tetracyclin instead of aad9. Vectors pFW11 and pFW12 are pFW5/6 derivatives harboring an improved synthetic aad9 promoter. In pFW-phoA and pFW-gfp, promoterless alkaline phosphatase and green fluorescent protein boxes were integrated into MCS-I. If streptococcal DNA fragments are cloned into MCS-I and MSC-II, these vectors can be used for specific allelic replacements in streptococci via double-crossover recombinations. Depending on the vector used, this event will not lead to polar effects, facilitating mutagenesis within operons. The vectors containing reporter boxes allow in vivo studies of gene expression and promoter activity in pathogenic streptococci and potentially, also in other Gram-positive bacteria.
Assuntos
Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Streptococcus pyogenes/genética , Fosfatase Alcalina/genética , Alelos , Sequência de Bases , Clonagem Molecular/métodos , DNA Bacteriano , Escherichia coli/genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Dados de Sequência MolecularRESUMO
Previous studies of recent clinical isolates of serotype M1 group A streptococci indicated that they display two patterns of non-immune human IgG subclass binding reactivity associated with their M1 protein. One group reacted with all four IgG subclasses (type IIo), while the second group expressed an M1 protein reacting preferentially with human IgG3 (type IIb). In this study, we have demonstrated that a cysteine protease, SpeB, present in culture supernatants of M1 serotype group A streptococcal isolates expressing type IIb IgG binding protein, can convert a recombinant Emm1 protein from a type IIo functional profile to a type IIb profile by removal of 24 amino acids from the N-terminus of the mature M1 protein. Furthermore, SpeB can convert bacteria expressing IgG binding proteins of the type IIo phenotype into those expressing type IIb proteins. The role of the cysteine protease as the central bacterial enzyme in this posttranslational modification event was confirmed by generation of an isogenic SpeB-negative mutant.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Cisteína Endopeptidases/metabolismo , Imunoglobulina G/metabolismo , Streptococcus pyogenes/enzimologia , Sequência de Aminoácidos , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Humanos , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/imunologiaRESUMO
The vir regulon of group A streptococci (GAS) organizes the expression of several bacterial virulence factors under the control of the Mga regulator. Previously, the genes encoding the Mga regulator (mga), M and M-related proteins (emm, mrp, enn) and C5a peptidase (scpA) were reported to be clustered on the streptococcal genome in a core vir regulon. In the present study, the genomic regions of a serotype M49 strain upstream of mga and downstream of scpA were sequenced to assess the boundaries of the vir regulon. In the upstream region, an operon was identified that may be potentially involved in substrate transport and is independent from Mga regulation. In the downstream region, another Mga-controlled, scpA-cotranscribed gene was detected. This gene termed orfX encoded a 385-amino acid (aa) potential surface protein of unknown function. No binding of serum proteins to a recombinant ORFX was detectable and phagocytosis resistance of an orfX mutant remained unchanged. Downstream of orfX, another Mga-independent gene determined the 3' end of the core vir regulon. Utilizing the M49 wild type, a mga- mutant and comparative Northern blot hybridization, genes encoding the capsule synthesis machinery, streptokinase and streptolysin O, as well as erythrogenic toxin A and DNase C were found to be Mga independent. In contrast, expression of the genes encoding the cysteine protease SpeB, streptococcin A and the oligopeptide permease was reduced in the mga- mutant. This indicated that in addition to the core vir regulon, Mga directly or indirectly controls a number of genes dispersed throughout the GAS genome.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Regulon , Streptococcus pyogenes/genética , Fatores de Virulência , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/química , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/análise , Streptococcus pyogenes/patogenicidade , VirulênciaRESUMO
The human pathogen Streptococcus pyogenes expresses several virulence factors that are required for the pathogens survival within the host and the concomitant development of disease. To examine the influence of one virulence factor, the extracellular cysteine protease SpeB, on the expression of other virulence factors, the speB structural gene of a serotype M3 and M49 strain was inactivated. Morphologic examination, quantification of extracellular hyaluronic acid capsule, and Northern blot analysis of the isogenic speB -mutants revealed a strain-dependent decrease of hyaluronic acid capsule production and an increase in superoxide dismutase transcription. The transcription of streptolysin O (slo), di- and oligo-peptide permease (dpp, opp), hyaluronidase (hyl), streptokinase (ska) and streptococcal pyrogenic exotoxin A (speA) was unaffected.
Assuntos
Cápsulas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Ácido Hialurônico/metabolismo , Streptococcus pyogenes/metabolismo , Proteínas de Bactérias , Cisteína Endopeptidases/genética , Humanos , Mutação , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , VirulênciaRESUMO
Many of the virulence factors associated with fulminant group A streptococci (GAS) infection are expressed under in vitro exponential growth conditions. However, the survival of GAS in tissue and intracellularly, as well as colonization of asymptomatic carriers, has been reported for GAS. The bacteria associated with these niches may encounter high-density, low-nutrient-flowthrough conditions that may more closely mimic in vitro stationary-phase conditions than exponential growth. Therefore, the behavior of GAS in stationary-phase culture was examined. We observed that after 24 h in stationary phase, GAS serotypes M49 and M2 developed a unstable colony dimorphism of typical large and atypical small colonies. Between days 4 and 5, we isolated stabilized atypical small colonies which remained stable for up to nine passages (approximately 200 generations) on fresh medium before fully reverting to the large-colony phenotype. Upon analysis, the small colonies showed no difference in cell number per colony, growth rate, survival in prolonged stationary-phase culture, or antibiotic sensitivity. However, the small colonies showed decreased transcription of hyaluronic acid capsule, the global positive virulence factor regulator gene mga, the mga-regulated emm mRNA (M-protein structural gene), and speB (cysteine protease). Accordingly, the small colonies were completely sensitive in a traditional phagocytosis assay. The production of virulence factors and phagocytosis resistance of the small-colony isolates was recovered when, after several passages on fresh medium, the colony morphology began to revert.
Assuntos
Antígenos de Bactérias , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/biossíntese , Proteínas de Transporte , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Cisteína Endopeptidases/biossíntese , Variação Genética , Fagocitose , Recombinases Rec A/biossíntese , Streptococcus pyogenes/crescimento & desenvolvimento , Fatores de Tempo , VirulênciaRESUMO
Staphylococcal scalded skin syndrome (SSSS) is an exfoliative dermatitis that results from infection with exfoliative toxin-producing Staphylococcus aureus. SSSS is seen primarily in infants and children. Here we ask if there is a specific maturation process that protects healthy adults from this syndrome. For these studies, an active recombinant exfoliative toxin A (rETA) was used in a neonatal mouse model. A time course generated on the susceptibility to the toxin as a function of mouse age indicated that BALB/c mice developed the characteristic symptoms of SSSS until day 7 of life. Between day 7 and day 8 of life there was a dramatic decrease in susceptibility, such that mice at day 9 of life were resistant to the effects of the toxin. This time course corresponds approximately to the time needed for maturation of the adaptive immune response, and SSSS in adults is often identified with immunocompromised states. Therefore, mice deficient in this response were examined. Adult mice thymectomized at birth and adult SCID mice did not develop the symptoms of SSSS after injection with the toxin, indicating that the adaptive immune response is not responsible for the lack of susceptibility observed in the older mice. SSSS in adults is also associated with renal disorders, suggesting that levels of toxin in serum are important in the development of the disease. rETA was not cleared as efficiently from the serum of 1-day-old mice compared to clearance from 10-day-old mice. Ten-day-old mice were given repeated injections of toxin so that the maximal level of toxin was maintained for a sustained period of time, and exfoliation occurred in these mice. Thus, whereas the adaptive immune response is not needed for protection of adult mice from SSSS, efficient clearance of the toxin from the bloodstream is a critical factor.
Assuntos
Exfoliatinas/sangue , Síndrome da Pele Escaldada Estafilocócica/imunologia , Staphylococcus aureus/imunologia , Envelhecimento/imunologia , Animais , Modelos Animais de Doenças , Exfoliatinas/administração & dosagem , Exfoliatinas/imunologia , Imunidade Ativa , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/imunologia , Síndrome da Pele Escaldada Estafilocócica/fisiopatologia , SíndromeRESUMO
Staphylococcal scalded-skin syndrome is an exfoliative dermatitis characterized by the separation of the epidermis at the stratum granulosum. This disruption is mediated by one of two Staphylococcus aureus exotoxins, exfoliative toxins A and B (ETA and ETB). Both ETA and ETB have been reported to be bacterial superantigens. A controversy exists, however, as other data indicate that these exotoxins are not superantigens. Here we demonstrate that recombinant exfoliative toxins produced in Escherichia coli do not act as T-cell mitogens and thus are not bacterial superantigens. These data fit the clinical profile of the disease, which is not associated with the classic symptoms of a superantigen-mediated syndrome.
Assuntos
Exfoliatinas , Síndrome da Pele Escaldada Estafilocócica/patologia , Staphylococcus aureus , Superantígenos , Animais , Exfoliatinas/genética , Exfoliatinas/isolamento & purificação , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Síndrome da Pele Escaldada Estafilocócica/fisiopatologiaRESUMO
During sequencing of an 11.5 kb genomic region of a serotype M49 group A streptococcal (GAS) strain, a series of genes were identified including nra(negative regulator of GAS). Transcriptional analysis of the region revealed that nra was primarily monocistronically transcribed. Polycistronic expression was found for the three open reading frames (ORFs) downstream and for the four ORFs upstream of nra. The deduced Nra protein sequence exhibited 62% homology to the GAS RofA positive regulator. In contrast to RofA, Nra was found to be a negative regulator of its own expression and that of the two adjacent operons by analysis of insertional inactivation mutants. By polymerase chain reaction and hybridization assays of 10 different GAS serotypes, the genomic presence of nra, rofA or both was demonstrated. Nra-regulated genes include the fibronectin-binding protein F2 gene (prtF2) and a novel collagen-binding protein (cpa). The Cpa polypeptide was purified as a recombinant maltose-binding protein fusion and shown to bind type I collagen but not fibronectin. In accordance with nra acting as a negative regulator of prtF2 and cpa, levels of attachment of the nra mutant strain to immobilized collagen and fibronectin was increased above wild-type levels. In addition, nra was also found to regulate negatively (four- to 16-fold) the global positive regulator gene, mga. Using a strain carrying a chromosomally integrated duplication of the nra 3' end and an nra-luciferase reporter gene transcriptional fusion, nra expression was observed to reach its maximum during late logarithmic growth phase, while no significant influence of atmospheric conditions could be distinguished clearly.
Assuntos
Adesinas Bacterianas , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores/genética , Streptococcus pyogenes/genética , Fatores de Transcrição , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Colágeno/metabolismo , Regulação para Baixo , Fibronectinas/metabolismo , Genes Bacterianos , Integrinas/genética , Dados de Sequência Molecular , Receptores de Colágeno , Streptococcus pyogenes/metabolismo , Transativadores/genéticaRESUMO
Group A streptococcal (GAS) cysteine protease is a major virulence factor involved in the pathogenesis of purulent and invasive infections. The secreted enzyme cleaves a number of different bacterial and host proteins which could contribute to different stages of the infective processes. It has been proposed that, among these functions, SpeB plays a role in obtaining nutrients during late growth phases. In the present study, speB mutants of various GAS serotypes were found to exhibit unaltered growth characteristics in several complex and chemically defined media (CDM). When amino acid-depleted CDM was prepared, neither SpeB activity on whole proteins added to the medium during incubation nor the addition of SpeB-digested proteins was able to support bacterial growth. SpeB also was unable to liberate iron from iron-containing protein sources added to iron-deficient CDM. However, SpeB levels in culture supernatants changed in response to the protein and glucose content of the media. Using a speB promoter-luciferase reporter, speB expression levels were found to correspond to peptide concentrations in the culture media. The effect appeared to be specific for peptides since addition of peptides derived from various proteins had an affect on expression, while addition of the whole proteins had no effect. Addition of glucose to CDM had no effect on speB expression, while glucose addition to complex medium decreased speB expression. Overall, SpeB did not appear to be directly involved in providing the bacteria with nutritional factors but expression of the speB gene responded to ratios of peptides and carbohydrates in the culture medium.
Assuntos
Proteínas de Bactérias/biossíntese , Meios de Cultura/farmacologia , Cisteína Endopeptidases/biossíntese , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Streptococcus pyogenes/enzimologia , Aminoácidos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/fisiologia , Indução Enzimática , Precursores Enzimáticos/metabolismo , Genes Reporter , Glucose/farmacologia , Ferro/metabolismo , Ferro/farmacologia , Peptídeos/farmacologia , Proteínas/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Sorotipagem , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , VirulênciaRESUMO
Bacterial oligopeptide permeases are membrane-associated complexes of five proteins belonging to the ABC-transporter family, which have been found to be involved in obtaining nutrients, cell-wall metabolism, competence, and adherence to host cells. A lambda library of the strain CS101 group A streptococcal (GAS) genome was used to sequence 10,192 bp containing the five genes oppA to oppF of the GAS opp operon. The deduced amino acid sequences exhibited 50-84% homology to pneumococcal AmiA to AmiF sequences. The operon organization of the five genes was confirmed by transcriptional analysis and an additional shorter oppA transcript was detected. Insertional inactivation was used to create serotype M49 strains which did not express either the oppA gene or the ATPase genes, oppD and oppF. The mutation in oppA confirmed that the additional shorter oppA transcript originated from the opp operon and was probably due to an intra-operon transcription terminator site located downstream of oppA. While growth kinetics, binding of serum proteins, and attachment to eukaryotic cells were unaffected, the oppD/F mutants showed reduced production of the cysteine protease, SpeB, and a change in the pattern of secreted proteins. Thus, the GAS opp operon appears to contribute to both protease production and export/processing of secreted proteins.