RESUMO
Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.
Assuntos
Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Chaperonina 60/administração & dosagem , Chaperonina 60/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Lactococcus lactis/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Mycobacterium leprae/enzimologia , Administração Oral , Animais , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Feminino , Inflamação/tratamento farmacológico , Inflamação/imunologia , Lactococcus lactis/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Background: Leprosy is a complex infectious and neurological disease caused by Mycobacterium leprae. Nerve damage is related to immunological hypersensitivity responses known as leprosy reactions (LRs). Diagnostic tools to predict LRs are not available. We hypothesized that natural octyl disaccharide-leprosy IDRI diagnostic (NDO-LID) would be helpful as an indicator of LRs and neuritis. Methods: To assess the utility of NDO-LID in indicating reactions, ELISA were used to detect specific antibodies in serum samples from 80 Colombian leprosy patients (40 with and 40 without history of LRs). Responses were detected using a range of detection reagents detecting IgG, IgM or both isotypes. Results: Patients with a history of LRs had an increased seropositivity rate for anti-NDO-LID antibodies compared to patients without (anti-NDO-LID protein A [p=0.02], IgG anti-NDO-LID [p=0.01] and IgM anti-NDO-LID [p=0.01]). Further analyses of patients with a history of LRs indicated that both seropositivity rate and magnitude of responses were elevated among patients with neuritis versus those without neuritis (anti-NDO-LID protein A [p=0.03], IgG anti-NDO-LID [p=0.001] and IgM anti-NDO-LID [p=0.06]). Conclusions: Our data indicate that testing for serum anti-NDO-LID antibodies can be a useful screen to identify patients at risk of developing LRs and neuritis.
Assuntos
Anticorpos Antibacterianos/sangue , Hanseníase/sangue , Mycobacterium leprae/enzimologia , Neurite (Inflamação)/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Colômbia , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G , Imunoglobulina M , Hanseníase/imunologia , Hanseníase/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/imunologia , Neurite (Inflamação)/imunologia , Neurite (Inflamação)/fisiopatologia , Valor Preditivo dos Testes , Testes Sorológicos , Adulto JovemRESUMO
Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.
Assuntos
Humanos , Mycobacterium leprae/enzimologia , Serina Endopeptidases/biossíntese , Sequência de Bases , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Mycobacterium leprae/genética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40 degrees C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.
Assuntos
Mycobacterium leprae/enzimologia , Serina Endopeptidases/biossíntese , Sequência de Bases , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , Mycobacterium leprae/genética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Proteases are commonly involved in bacterial pathogenesis and their inhibition has represented a successful therapeutic approach to treat infectious diseases. However, there is little information on the role of proteases in the pathogenesis of Mycobacteria. Five of these genes, three coding for putative secreted proteases, were selected in the present study to investigate their expression in Mycobacterium leprae isolated from skin biopsies of multibacillary leprosy patients. Via nested-PCR, it was demonstrated that mycP1 or ML0041, htrA2 or ML0176, htrA4 or ML2659, gcp or ML0379 and clpC or ML0235 are transcribed in vivo during the course of human infection. Moreover, the expression of Gcp in leprosy lesions was further confirmed by immunohistochemistry using a specific hyperimmune serum. This observation reinforces the potential role of mycobacterial proteases in the context of leprosy pathogenesis.
Assuntos
Hanseníase/enzimologia , Mycobacterium leprae/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Anticorpos Antibacterianos , Antígenos de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Hanseníase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium leprae/genética , Mycobacterium leprae/patogenicidade , Pele/microbiologiaRESUMO
The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Chaperoninas/metabolismo , Endopeptidases/metabolismo , Escherichia coli/enzimologia , Proteínas de Choque Térmico , Mutagênese Sítio-Dirigida , Mycobacterium leprae/enzimologia , Serina Endopeptidases , Proteases Dependentes de ATP , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Aminoácidos/análise , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Caseínas/metabolismo , Catálise , Chaperonina 60 , Chaperoninas/genética , Chaperoninas/isolamento & purificação , Endopeptidases/química , Hidrólise , Dados de Sequência Molecular , Mycobacterium leprae/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoAssuntos
Dados de Sequência Molecular , Dapsona/farmacologia , Di-Hidropteroato Sintase/genética , Genes Bacterianos , Hansenostáticos/farmacologia , Hanseníase/microbiologia , Mutação , Mycobacterium leprae , Mycobacterium leprae/enzimologia , Mycobacterium leprae/genética , Resistência Microbiana a Medicamentos/genéticaAssuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fito-Hemaglutininas/farmacologia , Hanseníase Tuberculoide/imunologia , Linfócitos T/imunologia , Mycobacterium leprae/enzimologia , Peso Molecular , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Sequência de Aminoácidos , Superóxido Dismutase/imunologia , Superóxido Dismutase/isolamento & purificação , Vacinas BacterianasRESUMO
El ensayo del ATP ha sido utilizado para evaluar la viabilidad de M. leprae obtenidos de pacientes lepromatosos en tratamiento con DDS. Los resultados obtenidos inmediatamente fueron subsecuentemente confirmados (8-11 meses más tarde) usando la técnica de la almohadilla plantar del ratón. Aquella misma técnica puede ser usada para identificar casos resistentes a los 3-4 meses de iniciar el tratamiento antileproso. La rapidez técnica y el bajo costo del método del ATP son comparados con aquellos del método de la almohadilla plantar del ratón
Assuntos
Humanos , Adenosina Trifosfatases/metabolismo , Dapsona/uso terapêutico , Hanseníase/tratamento farmacológico , Mycobacterium leprae/enzimologia , Dapsona/farmacologia , Mycobacterium leprae/efeitos dos fármacos , Resistência Microbiana a MedicamentosRESUMO
El ensayo del ATP ha sido utilizado para evaluar la viabilidad de M. leprae obtenidos de pacientes lepromatosos en tratamiento con DDS. Los resultados obtenidos inmediatamente fueron subsecuentemente confirmados (8-11 meses más tarde) usando la técnica de la almohadilla plantar del ratón. Aquella misma técnica puede ser usada para identificar casos resistentes a los 3-4 meses de iniciar el tratamiento antileproso. La rapidez técnica y el bajo costo del método del ATP son comparados con aquellos del método de la almohadilla plantar del ratón (AU)