RESUMO
Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18 kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.
Assuntos
Vacina BCG/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vetores Genéticos/genética , Leptospira interrogans/genética , Mycobacterium bovis/genética , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Cricetinae , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/imunologia , Leptospira interrogans/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Mycobacterium bovis/imunologia , Plasmídeos/genética , Plasmídeos/imunologiaRESUMO
Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.
Assuntos
Animais , Cricetinae , Vacina BCG/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vetores Genéticos/genética , Leptospira interrogans/genética , Mycobacterium bovis/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/imunologia , Leptospira interrogans/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Mycobacterium bovis/imunologia , Plasmídeos/genética , Plasmídeos/imunologiaRESUMO
CD4(+) T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an Ag associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell Ag as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4(+) T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate or inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, because recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by alpha-mannosidase, did not activate T cells. This study demonstrates that the universe of bacterial T cell Ags includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.
Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Lipoproteínas/imunologia , Mycobacterium/imunologia , Receptor 2 Toll-Like/imunologia , Antígenos de Bactérias/genética , Carboidratos/química , Carboidratos/genética , Carboidratos/imunologia , Escherichia coli/genética , Escherichia coli/imunologia , Humanos , Lipoproteínas/genética , Ativação Linfocitária/fisiologia , Mycobacterium/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , alfa-Manosidase/químicaRESUMO
An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Lipoproteínas/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Western Blotting , Bovinos , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Interferon gama/sangue , Lipoproteínas/química , Lipoproteínas/isolamento & purificação , Modelos Animais , Dados de Sequência Molecular , Peso Molecular , Mycobacterium avium/genética , Complexo Mycobacterium avium/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium leprae/genética , Mycobacterium scrofulaceum/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Fases de Leitura Aberta , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , OvinosAssuntos
Anticorpos Antifosfolipídeos/imunologia , Anticorpos/imunologia , Lipoproteínas/imunologia , Adulto , Anticorpos Antifosfolipídeos/química , Doenças Autoimunes/imunologia , Feminino , Infecções por HIV/imunologia , Humanos , Hanseníase/imunologia , Masculino , Pessoa de Meia-Idade , Sífilis/imunologiaRESUMO
A novel Mycobacterium leprae lipoprotein LpK (accession no. ML0603) was identified from the genomic database. The 1,116-bp open reading frame encodes a 371-amino-acid precursor protein with an N-terminal signal sequence and a consensus motif for lipid conjugation. Expression of the protein, LpK, in Escherichia coli revealed a 33-kDa protein, and metabolic labeling experiments and globomycin treatment proved that the protein was lipidated. Fractionation of M. leprae demonstrated that this lipoprotein was a membrane protein of M. leprae. The purified lipoprotein was found to induce production of interleukin-12 in human peripheral blood monocytes. The studies imply that M. leprae LpK is involved in protective immunity against leprosy and may be a candidate for vaccine design.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Interleucina-12/biossíntese , Lipoproteínas/imunologia , Proteínas de Membrana/imunologia , Mycobacterium leprae/imunologia , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Escherichia coli , Expressão Gênica , Genes Bacterianos , Humanos , Lipoproteínas/genética , Lipoproteínas/isolamento & purificação , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Monócitos/imunologia , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Análise de Sequência de DNARESUMO
The search for leptospiral antigens (L. interrogans serogroup icterohaemorrhagiae) was carried out in 24 guinea pigs experimentally inoculated with 1 ml of culture containing 10(7)-10(8) leprospires and sequentially sacrificed from the first until the 6th day of infection. Semiquantitative analysis of histopathological variables comprising kidney interstitium, tubules and glomeruli was done in 1 micron sections of tissue embedded in glycolmetacrylate. Leptospiral antigen (LAg) and its glycolipoprotein (GLP) expression were detected through PAP in paraffin embedded tissue. The mild interstitial involvement of the kidney, manifested chiefly by oedema and focal interstitial nephritis seen at the 4th day, progressed to tubular damage at the 6th day, characterized by either swelling or cytoplasmic acidophilia of epithelial cells with loss of cell cohesion and sloughing of cells into the tubular lumina. Brush border alterations and mitochondrial changes were observed. Endothelial cell injury was noted in the interstitial vessels. LAg expression was parallel to the kidney changes: small deposits of elongated forms of LAg were detected at the 4th day either within the vascular lumen or free in the interstitium. A rise in the antigen expression was observed at the 5th day when it was seen either around tubules or in their walls. LAg was detected inside the tubular lumina at the 6th day of infection when granular LAg and GLP were abundant. This sequence reproduces the pathway of leptospires in the kidney and the crescent amounts of antigens detected toward the end of the experiment, with antigen concentration in cases of major tissue damage suggesting a direct action of the microorganisms and/or their products in the pathogesis of the lesions.