RESUMO
The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: Progress and Challenges in the Replacement of HIST' was held on 24 August 2014, in Prague, Czech Republic, as a satellite meeting to the 9th World Congress on Alternatives and Animal Use in the Life Sciences. Participants discussed the progress and challenges associated with the development, validation, and implementation of in vitro assays as replacements for the histamine sensitisation test (HIST) for acellular pertussis vaccines. Discussions focused on the consistency approach, the necessary framework for regulatory acceptance of a harmonised method, and recent international efforts towards the development of in vitro assays to replace the HIST. Workshop participants agreed that acceptable alternatives to the HIST should be based on ADP ribosylation-mediated cell intoxication and therefore that the CHO cell clustering assay, which measures cell intoxication, should be further pursued and developed as a possible replacement for the HIST. Participants also agreed to continue ongoing multinational discussions involving national and international standardisation authorities to reach consensus and to organise collaborative studies in this context for assay characterisation and calibration of reference materials.
Assuntos
Histamina/administração & dosagem , Toxina Pertussis/uso terapêutico , Vacina contra Coqueluche/uso terapêutico , Vacinas Acelulares/uso terapêutico , Coqueluche/prevenção & controle , Animais , Células CHO , Cricetinae , Cricetulus , República Tcheca , Educação/métodos , Educação/tendências , Humanos , Camundongos , Coqueluche/diagnósticoRESUMO
As the Bacillus Calmette-Guérin (BCG) vaccine does not confer long-lasting protection against lung Mycobacterium tuberculosis infection, the development of more efficient vaccines is greatly needed. Here, we used mycobacterial low-molecular weight proteins of the 6-kDa Early Secreted Antigenic Target (ESAT-6) protein family (ESX) antigens for the evaluation of a novel vaccine delivery strategy that enables versatile in vivo targeting of antigens into specialized dendritic cell (DC) subsets. ESX antigens were genetically fused to the tetramerizing core of streptavidin (SA) to form high-affinity complexes with biotin (biot)-conjugated antibodies recognizing DC surface receptors. When directed through the CD11b or CD11c ß2-integrins or diverse C-type lectins, the ESX-SA:biot-antibody complexes were efficiently captured and presented on major histocompatibility complex molecules of DCs to specific T-cell receptors. Robust ESX-specific T-cell responses were induced by immunization with as little as several picomoles of ESX-SA targeted to DC subsets. Moreover, directing of TB10.4-SA to airway CD205(+) cells enabled the induction of mucosal T-cell responses and provided significant protection against virulent M. tuberculosis.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Células Dendríticas/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Apresentação de Antígeno , Antígenos CD/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11c/imunologia , Antígenos CD18/imunologia , Células Cultivadas , Feminino , Humanos , Imunidade Ativa , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Sistema Respiratório/patologia , Linfócitos T/imunologiaRESUMO
Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC(-)) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC(-) constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of γ-IFN-secreting CMV-specific CD8(+) T cells, as well as increase of total IFN-γ and TNF-α production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC(-) constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC(-) constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.
Assuntos
Adenilil Ciclases/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas contra Citomegalovirus/imunologia , Citomegalovirus/imunologia , Epitopos de Linfócito T/imunologia , Fosfoproteínas/imunologia , Proteínas da Matriz Viral/imunologia , Adenilil Ciclases/genética , Sequência de Aminoácidos , Vacinas contra Citomegalovirus/genética , Humanos , Ativação Linfocitária , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologiaRESUMO
Scientific data from family medicine are relevant for the majority of the population. They are therefore essential from an ethical and public health perspective. We need to promote quality research in family medicine despite methodological, financial and logistic barriers. To highlight the strengths and weaknesses of research in family medicine in the French-speaking part of Switzerland we asked practitioners from this region to share their experience, critics and needs in relation to research. This article summarizes their contribution in light of the international literature.
Assuntos
Medicina de Família e Comunidade , Avaliação das Necessidades , Pesquisa , HumanosRESUMO
Family practitioners are well aware of the guidelines for diabetic care yet they often find it difficult to apply them in practice. Experience from the literature as well as our own research provide guidance on ways to address this problem in Primary care: 1) collaboration with a nurse practitioner for the prevention of micro and macro-angiopathic complications, 2) the use of motivational interviewing techniques to motivate patients to lifestyle changes, 3) multidisciplinary collaboration (with specialists, nurses, colleagues, pharmacists, etc) and the support of information technology. Research within the Swiss academic institutes of Primary care should provide further, more concrete, guidance on ways to apply these different options in Switzerland to improve the quality of care for diabetic patients.
Assuntos
Diabetes Mellitus/terapia , Atenção Primária à Saúde , Qualidade da Assistência à Saúde , Benchmarking , Pesquisa Biomédica , Diabetes Mellitus/enfermagem , Angiopatias Diabéticas/prevenção & controle , Humanos , Estilo de Vida , Motivação , Profissionais de Enfermagem , Papel do Profissional de Enfermagem , Estado Pré-Diabético/terapia , Ensaios Clínicos Controlados Aleatórios como Assunto , SuíçaRESUMO
AIM: The aim of this study was to provide a detailed description of the health problems for which primary care services are provided to adolescents in a juvenile detention facility in Europe. METHODS: We reviewed the medical files of all detainees in a juvenile detention centre in Switzerland in 2007. The health problems for which primary care services were provided were coded using the International Classification for Primary Care, version 2. Analysis was descriptive, stratified by gender. RESULTS: A total of 314 adolescents (18% female) aged 11-19 years were included. Most (89%) had a health assessment and 195 (62%) had consultations with a primary care physician; 80% of the latter had a physical health problem, and 60% had a mental health problem. The most commonly managed problems were skin (49.7%), respiratory (23.6%), behavioural (22.6%) and gynaecological problems (females: 23.9%); 13% females (no males) had sexually transmitted infections (STI), and 8.7% were pregnant. Substance abuse was common (tobacco: 64.6%, alcohol: 26.2%, cannabis: 31.3%). CONCLUSION: In addition to health problems known to be more prevalent among young offenders, such as mental health problems and STI, these adolescent detainees required care for a range of common primary care problems. These data should inform the development of comprehensive primary care services in all juvenile detention facilities in Europe.
Assuntos
Serviços de Saúde do Adolescente/estatística & dados numéricos , Nível de Saúde , Atenção Primária à Saúde/estatística & dados numéricos , Prisioneiros , Prisões/organização & administração , Adolescente , Criança , Estudos Transversais , Feminino , Humanos , Delinquência Juvenil , Masculino , Transtornos Mentais/epidemiologia , Gravidez , Prisioneiros/psicologia , Prisioneiros/estatística & dados numéricos , Transtornos Respiratórios/epidemiologia , Infecções Sexualmente Transmissíveis/epidemiologia , Dermatopatias/epidemiologia , Suíça , Adulto JovemRESUMO
To limit drug adverse effects, the use of a limited choice of drugs is desirable. We identified 29 frequent health problems and selected first and second choice medication based on the following criteria: clinical efficacy based on medical evidence or expert consensus, safety profile, and costs. For each substance, adverse effect, contraindication, interaction risk, specific dosing, and safety use during pregnancy and lactation were reviewed. More than seventy substances were identified. This list is available for download at the following address (in French): http://www.hcuge.
Assuntos
Formulários de Hospitais como Assunto , Atenção Primária à Saúde , Humanos , Erros de Medicação/prevenção & controle , SuíçaRESUMO
We report the case of an elderly immunocompetent patient with Aeromonas hydrophila bacteremia without evidence of portal of entry. Despite several risk factors for a poor outcome, such as impaired renal function, two positive blood cultures, and community-acquired infections, the patient survived. Antimicrobial susceptibility was normal. Unknown polycystic liver disease was discovered and misdiagnosed as a hepatic abscess at the time of the bacteremia which was confirmed by repeated CT scans. Because of the absence of other risk factors for Aeromonas bacteremia, hepatic polycystic disease may take part in the onset of Aeromonas sp bacteremia as well as immunosenescence.
Assuntos
Aeromonas/patogenicidade , Bacteriemia/microbiologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/imunologia , Imunocompetência/imunologia , Aeromonas/imunologia , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos/imunologia , Bacteriemia/imunologia , Infecções por Bactérias Gram-Negativas/patologia , Humanos , MasculinoRESUMO
Hypertension is associated with a greater risk of developing a stroke or a coronary heart disease, but remains, for the meanwhile, undertreated in a vast majority of patients. Non pharmacological interventions can reduce blood pressure and should be recommended to every patient suffering from hypertension. Nevertheless, a drug therapy must often be introduced. The threshold at which a treatment should be started depends mostly on the cardiovascular risk of each patient and the benefit of antihypertensive drug therapy depends primarily on the reduction of the blood pressure itself.
Assuntos
Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , HumanosRESUMO
The Neisseria meningitidis FAM20 strain secretes two proteins of unknown function, FrpA and FrpC, which contain typical RTX domains found in cytotoxins from other gram-negative pathogens. To evaluate whether the Frp proteins could be involved in meningococcal virulence, 65 isolates of all serogroups were screened by PCR for the presence of both frp genes. The frpA allele was, however, poorly conserved. A single strain harbored an frpA allele of the previously described size, while large insertions were detected in the frpA loci of 22 isolates (34%), and the 42 remaining isolates (65%) did not contain frpA at all. In contrast, frpC alleles, albeit of variable length, were detected in all invasive and most carrier strains. This suggests that meningococci may produce a family of FrpC proteins of various molecular masses. High levels of both immunoglobulin G (IgG) and IgA class antibodies recognizing recombinant FrpC were indeed detected in convalescent-phase sera of most patients at 2 and 4 to 5 weeks after the first symptoms of meningococcal disease. These results show that FrpC-like proteins are produced and may play a role in invasive meningococcal infections.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Citotoxinas , Proteínas de Membrana , Infecções Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Neisseria meningitidis/metabolismo , Alelos , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Humanos , Proteínas de Ligação ao Ferro , Infecções Meningocócicas/microbiologia , Dados de Sequência Molecular , Proteínas Periplásmicas de Ligação , Polimorfismo Genético/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNARESUMO
CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver its N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells and particularly into professional antigen-presenting cells. We have previously identified within the primary structure of CyaA several permissive sites at which insertion of peptides does not alter the ability of the toxin to enter cells. This property has been exploited to design recombinant CyaA toxoids capable of delivering major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell epitopes into antigen-presenting cells and to induce specific CD8(+) cytotoxic T-lymphocyte (CTL) responses in vivo. Here we have explored the capacity of the CyaA vector carrying several different CD8(+) T-cell epitopes to prime multiple CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from lymphocytic choriomeningitis virus (LCMV), the V3 region of human immunodeficiency virus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hybridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lead to the induction of specific CTL responses for each of the three epitopes, as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying a foreign CD8(+) CTL epitope. These results highlight the potency of the adenylate cyclase vector for induction of protective CTL responses with multiple specificity and/or broad MHC restriction.
Assuntos
Adenilil Ciclases/imunologia , Bordetella pertussis/imunologia , Infecções por Rhabdoviridae/prevenção & controle , Adenilil Ciclases/genética , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Bordetella pertussis/genética , Epitopos/imunologia , Humanos , Imunidade , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Infecções por Rhabdoviridae/virologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Vacinas Virais/farmacologiaRESUMO
The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Assuntos
Toxina Adenilato Ciclase , Adenilil Ciclases/análise , Proteínas de Bactérias/química , Bordetella pertussis/enzimologia , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fatores de Virulência de Bordetella/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Dados de Sequência MolecularRESUMO
The capacity of adenylate cyclase toxin (ACT) to penetrate into target cells depends on post-translational fatty-acylation by the acyltransferase CyaC, which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, the in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic serine 20 and histidine 33 residues ablated acylating activity of CyaC. Conservative replacements of alanine 140 by glycine (A140G) and valine (A140V) residues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi- and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983, or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost exclusively at Lys-983. The respective proportion of toxin molecules acylated at Lys-983 correlated well with the cell-invasive activity of both ACT mixtures, which was about half of that of ACT fully acylated on Lys-983 by intact CyaC. These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary and sufficient.
Assuntos
Aciltransferases/metabolismo , Toxina Adenilato Ciclase , Bordetella pertussis/enzimologia , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Virulência de Bordetella/metabolismo , Fatores de Virulência de Bordetella/toxicidade , Acilação , Aciltransferases/química , Aciltransferases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Eletroforese em Gel Bidimensional , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Hemólise/efeitos dos fármacos , Histidina/genética , Histidina/metabolismo , Lisina/genética , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Ácido Palmítico/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Alinhamento de Sequência , Serina/genética , Serina/metabolismo , Ovinos , Especificidade por Substrato , Fatores de Virulência de Bordetella/química , Fatores de Virulência de Bordetella/genéticaRESUMO
Calreticulin (CRT), a high-affintiy calcium binding protein and chaperone, was recently identified as one of the targets of autoantibodies in coeliac disease. We evaluated the level of IgA and IgG antibodies to CRT in sera from patients with coeliac disease and various autoimmune diseases. The level of antibodies to gliadin (shown previously to cross-react with CTR), isolated enterocytes and tissue transglutaminase were determined for comparison. The mean level of IgA antibodies to CRT was significantly higher (P< 0.001) in sera from coeliac patients with active disease (139.9+/-11.2 AU/+/-SE) than in healthy controls (20.9+/-1.7 AU). In sera of patients with systemic lupus erythematosus (SLE), insulin dependent diabetes mellitus (IDDM), multiple sclerosis (MS) and autoimmune thyroiditis (AT) or inflammatory bowel disease (IBD) the mean level (25.8+/-3.7 to 38.1+/-5.6 AU) did not exceed the cut-off value. A low level of these antibodies, however, was detected in some sera of patients with MS and IBD. The level of IgG anti-CRT antibodies was increased in coeliac patients (mean 125.4+/-8.0 AU, P< 0.001) when compared to that in healthy controls (33.9+/-2.3 AU). The IgG anti-CRT antibodies were also detected in about 30% of SLE patients sera (54.1+/-3.6 AU, P< 0.001), but the mean level reached only half that detected in coeliac patients.
Assuntos
Autoanticorpos/sangue , Doenças Autoimunes/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Doença Celíaca/imunologia , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Ribonucleoproteínas/imunologia , Adolescente , Adulto , Idoso , Western Blotting , Calreticulina , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Humanos , Pessoa de Meia-IdadeRESUMO
The adenylate cyclase (CyaA) of Bordetella pertussis delivers the N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells, in particular, professional antigen-presenting cells. This allows the delivery of CD8(+) T-cell epitopes to the major histocompatibility complex class I presentation pathway. We have previously shown that immunization of mice with CyaA carrying a single CD8(+) T-cell epitope leads to antiviral protection as well as to protective and therapeutic antitumor immunity associated with the induction of specific cytotoxic T-lymphocyte (CTL) responses. Here, we evaluated the capacity of CyaA carrying one to four copies of the CD8(+) CD4(+) T-cell epitope from the nucleoprotein of the lymphocytic choriomeningitis virus to induce T-cell responses. Both CTL and Th1-like specific responses were detected in mice immunized with recombinant CyaA with or without adjuvant. Although the insertion of the larger peptides resulted in partial loss of the invasive capacity of recombinant CyaA, insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser degree, CTL responses. These results underscore the potency of CyaA for vaccine design with a new impact on diseases in which the Th1 response has been described to have a beneficial effect.
Assuntos
Adenilil Ciclases/imunologia , Antígenos Virais/genética , Proteínas de Bactérias/imunologia , Bordetella pertussis/enzimologia , Bordetella pertussis/imunologia , Precursores de Proteínas/imunologia , Células Th1/imunologia , Toxina Adenilato Ciclase , Adenilil Ciclases/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Bordetella pertussis/genética , Epitopos/genética , Feminino , Interferon gama/biossíntese , Ativação Linfocitária , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Precursores de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8(+) T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8(+) T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8(+) T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.
Assuntos
Adenilil Ciclases/imunologia , Adenilil Ciclases/metabolismo , Apresentação de Antígeno , Bordetella pertussis/enzimologia , Bordetella pertussis/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/administração & dosagem , Antígenos de Histocompatibilidade Classe I/metabolismo , Toxina Adenilato Ciclase , Adenilil Ciclases/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Bordetella pertussis/genética , Primers do DNA/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Proteínas Hemolisinas/metabolismo , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/imunologia , Fatores de Virulência de Bordetella/metabolismoRESUMO
The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in target cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.
Assuntos
Toxina Adenilato Ciclase , Ácido Glutâmico/metabolismo , Canais Iônicos/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Cátions , Membrana Celular/metabolismo , Primers do DNA , Ácido Glutâmico/genética , Hemólise/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Ovinos , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Bordetella pertussis adenylate cyclase toxin (ACT) is one of the few known protein toxins penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This capacity of ACT was recently exploited for in vivo delivery of single viral CD8(+) T-epitopes into MHC class I-presenting cells and induction of protective antiviral cytotoxic T-cell (CTL) responses. Here, we have explored the potential of the cell-invasive adenylate cyclase domain of the toxin to deliver larger antigens by evaluating the epitope-specific CTL responses induced by constructs bearing one to four copies of the CD8(+) T-epitope from the nucleoprotein of the lymphocytic choriomeningitis virus. The increase in the number of copies of the epitope was accompanied by a moderate decrease of the specific cell invasiveness of the ACT protein and did not lead to further enhancement of the level of induced epitope-specific CTL cells in mice, as compared to ACT with a single copy of the epitope. These results demonstrate the capacity of ACT to deliver larger heterologous antigens comprising several epitopes for antigenic presentation in vivo.
Assuntos
Toxina Adenilato Ciclase , Bordetella pertussis/química , Antígenos CD8/genética , Linfócitos T/imunologia , Fatores de Virulência de Bordetella/imunologia , Animais , Citotoxicidade Imunológica , Epitopos , Escherichia coli/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinação , Fatores de Virulência de Bordetella/biossíntese , Fatores de Virulência de Bordetella/genéticaRESUMO
Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.
Assuntos
Adenilil Ciclases/imunologia , Anticorpos Antibacterianos/genética , Anticorpos Monoclonais/genética , Proteínas de Bactérias/imunologia , Bordetella pertussis/enzimologia , Bordetella pertussis/imunologia , Proteínas de Escherichia coli , Precursores de Proteínas/imunologia , Fatores de Virulência de Bordetella/imunologia , Toxina Adenilato Ciclase , Adenilil Ciclases/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Bordetella pertussis/genética , Reações Cruzadas , Primers do DNA/genética , Mapeamento de Epitopos , Proteínas Hemolisinas/imunologia , Camundongos , Mutação , Precursores de Proteínas/genética , Fatores de Virulência de Bordetella/genéticaRESUMO
The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the epsilon-amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta9 C16:1) fatty-acyl groups. Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin. These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status.