RESUMO
Bordetella pertussis is the etiologic agent of whooping cough, an illness whose incidence has been increasing over the last decades. Pertussis reemergence despite high vaccination coverage, together with the recent isolation of circulating strains deficient in some of the vaccine antigens, highlight the need for new vaccines. Proteins induced under physiological conditions, such as those required for nutrient acquisition during infection, might represent good targets for better preventive strategies. By mean of serological proteome analysis we identified two novel antigens of B. pertussis potentially involved in iron acquisition during host colonization. We had previously demonstrated that one of them, designated IRP1-3, is protective against pertussis infection in mice. In the present study, we show that the other antigen, named AfuA (BP1605), is a highly antigenic protein, exposed on the bacterial surface, conserved among clinical isolates and expressed during infection. Immunization of mice with the recombinant AfuA induced opsonophagocytic antibodies which could explain the protection against B. pertussis infection conferred by mice immunization with rAfuA. Importantly, we found that the addition of rAfuA and rIRP1-3 proteins to the commercial three pertussis components acellular vaccine significantly increased its protective activity. Taken together, our results point at these two antigens as potential components of a new generation of acellular vaccines.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Bordetella pertussis/imunologia , Proteína 1 Reguladora do Ferro/imunologia , Vacina contra Coqueluche/imunologia , Coqueluche/imunologia , Animais , Anticorpos Antibacterianos/sangue , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos , Proteínas Opsonizantes/imunologia , Vacina contra Coqueluche/química , Vacinação , Coqueluche/microbiologia , Coqueluche/prevenção & controleRESUMO
Antigenic proteins whose expression is induced under iron starvation, an environmental condition that bacterial pathogens have to face during colonization, might be potential candidates for improved vaccine. By mean of immune proteomics we identified novel antigens of Bordetella pertussis maximally expressed under iron limitation. Among them, Bp1152 (named as IRP1-3) showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals. Computer analysis showed IRP1-3 as a dimeric membrane protein potentially involved in iron uptake. Experimental data revealed the surface-exposure of this protein and showed its increase under iron starvation to be independent of bacterial virulence phase. Immunization of mice with the recombinant IRP1-3 resulted in a strong antibody response. These antibodies not only recognized the native protein on bacterial surface but also promote effective bacterial phagocytosis by human PMN, a key protecting activity against this pathogen. Accordingly, IRP1-3 proved protective against B. pertussis infection in mouse model. Expression of IRP1-3 was found conserved among clinical isolates of B. pertussis and positively regulated by iron starvation in these strains. Taken together these results suggest that this protein might be an interesting novel vaccine candidate.
Assuntos
Antígenos de Bactérias/imunologia , Bordetella pertussis/imunologia , Proteínas de Membrana/imunologia , Vacina contra Coqueluche/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/administração & dosagem , Feminino , Proteínas de Membrana/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Vacina contra Coqueluche/administração & dosagem , Fagocitose , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.
Assuntos
Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/metabolismo , Trypanosoma/enzimologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Chlorocebus aethiops , Clonagem Molecular , Desintoxicação Metabólica Fase I/genética , Redes e Vias Metabólicas/genética , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo/genética , Homologia de Sequência , Trypanosoma/genética , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Células VeroRESUMO
Parvulins are a conserved group of peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze the cis/trans isomerization of proline-preceding peptide bonds. Parvulin-class PPIases are structurally unrelated to cyclophilins and FK506-binding proteins that are defined as receptors for immunosuppressive drugs. In Trypanosoma cruzi we identified parvulin TcPIN1 as a homolog of the human hPin1 PPIase. The 117 amino acids of the TcPIN1 display 40% identity with the catalytic core of hPin1 and exhibit prolyl cis/trans isomerase activity. TcPIN1 lacks the WW domain at the N-terminus, and is able to rescue the temperature-sensitive phenotype on a mutation in the Saccharomyces cerevisiae hPin1 homolog, ESS1/PTF1. Western blot analysis revealed that the enzyme was present both in dividing and non-dividing forms of T. cruzi. In epimastigote cells neither cell growth kinetics nor cell morphology was affected by the overexpression of the small parvulin TcPIN1. These results suggest the occurrence of a supplementary conserved level of post-translational control in trypanosomatids.