Assuntos
Proteínas de Bactérias/imunologia , Deleção de Genes , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Pulmão/microbiologia , Camundongos Endogâmicos C57BL , Mycobacterium bovis/genética , Análise de Sobrevida , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
In this study, a Mycobacterium bovis knockout strain in phoP-phoR and mce2 operons was tested as an antituberculosis experimental vaccine in animal models. The double mutant strain was significantly more attenuated than the wild type strain in inmunocompetent and inmunodeficient mice. Vaccination with the double mutant protected mice against challenge with a virulent M. bovis strain.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Mycobacterium bovis/genética , Vacinas contra a Tuberculose/genética , Tuberculose Bovina/prevenção & controle , Animais , Bovinos , Contagem de Colônia Microbiana , Citocinas/biossíntese , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Camundongos Endogâmicos BALB C , Camundongos Nus , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/patogenicidade , Células Th1/imunologia , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/toxicidade , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/toxicidade , Virulência/genéticaRESUMO
A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.
Assuntos
Proteínas de Bactérias/genética , Lipoproteínas/genética , Proteínas de Membrana Transportadoras/genética , Mycobacterium bovis/genética , Tuberculose/imunologia , Animais , Vacina BCG/uso terapêutico , Bovinos , Cobaias , Humanos , Interleucina-2/imunologia , Interleucina-2/metabolismo , Camundongos , Modelos Animais , Mycobacterium bovis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Óperon/genética , Óperon/imunologia , Tuberculose/genética , Tuberculose/prevenção & controleRESUMO
BACKGROUND: In many regions of the world, wild mammals act as reservoir of Mycobacterium bovis, a situation that prevents the eradication of bovine tuberculosis. In order to observe whether a strain isolated from a wild boar, previously tested as highly virulent in a mice model, is also virulent in cattle, we performed cattle experimental inoculation with this strain RESULTS: Groups of Friesian calves were either infected with the wild boar strain M. bovis 04-303 or with the bovine strain NCTC10772 as a control. We found that antigen-specific IFN-γ release in whole blood samples occurred earlier in animals infected with M. bovis 04-303. Both M. bovis strains resulted in a positive skin test, with animals infected with the wild boar isolate showing a stronger response. These results and the presence of more severe organ lesions, with granuloma and pneumonic areas in cattle demonstrate that the wild boar isolate is more virulent than the NCTC10772 strain. Additionally, we tested the infectivity of the M. bovis strains in guinea pigs and found that M. bovis 04-303 had the highest pathogenicity. CONCLUSIONS: M. bovis strains isolated from wild boars may be pathogenic for cattle, producing TB lesions.
Assuntos
Reservatórios de Doenças/veterinária , Mycobacterium bovis/imunologia , Sus scrofa/microbiologia , Tuberculose Bovina/microbiologia , Animais , Argentina/epidemiologia , Bioensaio/veterinária , Bovinos , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Reservatórios de Doenças/microbiologia , Feminino , Cobaias , Histocitoquímica/veterinária , Interferon gama/sangue , Fígado/microbiologia , Pulmão/microbiologia , Linfonodos/microbiologia , Masculino , Mycobacterium bovis/genética , Mycobacterium bovis/patogenicidade , Reação em Cadeia da Polimerase/veterinária , Tuberculose Bovina/epidemiologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/transmissão , VirulênciaRESUMO
BACKGROUND: The P27-P55 (lprG-Rv1410c) operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis. METHOD: In order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions. RESULTS: The mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant. CONCLUSIONS: The results clearly indicate that P27 and P55 are functionally connected in processes that involve the preservation of the cell wall and the transport of toxic compounds away from the cells.
Assuntos
Proteínas de Bactérias/genética , Lipoproteínas/genética , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/genética , Óperon , Anti-Infecciosos/farmacologia , Western Blotting , Membrana Celular/química , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/química , Resistência Microbiana a Medicamentos , Eletroforese em Gel de Poliacrilamida , Etambutol/farmacologia , Etídio/farmacologia , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Corantes de Rosanilina/farmacologia , Dodecilsulfato de SódioRESUMO
BACKGROUND: The exported repetitive protein (erp) gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS) repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice. RESULTS: In order to elucidate the function of Erp we searched for Erp-binding proteins from M. tuberculosis by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several Actinomycetales genera, orthologues of Rv2617c are exclusively present in members of the M. tuberculosis complex (MTC). The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from Mycobacterium smegmatis and Mycobacterium leprae were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism. CONCLUSION: We identified interactive partners of Erp, an M. tuberculosis protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Escherichia coli/genética , Perfilação da Expressão Gênica , Mycobacterium leprae/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: mce3 is one of the four virulence-related mce operons of Mycobacterium tuberculosis. In a previous work we showed that the overexpression of Mce3R in Mycobacterium smegmatis and M. tuberculosis abolishes the expression of lacZ fused to the mce3 promoter, indicating that Mce3R represses mce3 transcription. RESULTS: We obtained a knockout mutant strain of M. tuberculosis H37Rv by inserting a hygromycin cassette into the mce3R gene. The mutation results in a significant increase in the expression of mce3 genes either in vitro or in a murine cell macrophages line as it was determined using promoter-lacZ fusions in M. tuberculosis. The abundance of mce1, mce2 and mce4 mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR. The mce3R promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the in vitro culture of M. tuberculosis. CONCLUSION: Mce3R repress the transcription of mce3 operon and self regulates its own expression but does not affect the transcription of mce1, mce2 and mce4 operons of M. tuberculosis.