RESUMO
The C-type lectin DC-SIGN is involved in early interactions between human innate immune cells and a variety of pathogens. Here we sought to evaluate whether DC-SIGN interacts with the leprosy bacillus, Mycobacterium leprae, and whether DC-SIGN genetic variation influences the susceptibility and/or pathogenesis of the disease. A case-control study conducted in a cohort of 272 individuals revealed no association between DC-SIGN variation and leprosy. However, our results clearly show that DC-SIGN recognizes M. leprae, indicating that mycobacteria recognition by this lectin is not as narrowly restricted to the Mycobacterium tuberculosis complex as previously thought. Altogether, our results provide further elucidation of M. leprae interactions with the host innate immune cells and emphasize the importance of DC-SIGN in the early interactions between the human host and the infectious agents.
Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Hanseníase/epidemiologia , Hanseníase/genética , Mycobacterium leprae/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Adulto , Feminino , Frequência do Gene , Humanos , Lectinas/genética , Lectinas/metabolismo , Masculino , Epidemiologia Molecular , Paquistão/epidemiologia , População/genéticaRESUMO
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 , OvinosRESUMO
Erp (exported repetitive protein), also known as P36, Pirg and Rv3810, is a member of a mycobacteria-specific family of extracellular proteins. These proteins consist of three domains, the N- and C-terminal domains are similar in all mycobacterial species, however, the central domain contains a repeated PGLTS module and differs considerably between species. The erp knockout mutant of Mycobacterium tuberculosis displays very low levels of multiplication both in macrophage cell lines and in vivo in a mouse model of infection. The high interspecies variability of the central repeated region of the Erp protein led us to investigate whether these orthologous proteins were functionally equivalent in a mouse model of tuberculosis. We expressed a gene fusion with the erp gene of Mycobacterium smegmatis, Mycobacterium leprae or M. tuberculosis in trans in an erp-M. tuberculosis mutant and found that these three alleles restored multiplication to similar levels in the spleen of infected mice. However, these alleles gave different levels of colonization in the lung, for the early time-points. Quantitative histological analyses of the lungs of infected animals showed that the nature of the erp allele strongly affected the number and the size of lung lesions, demonstrating the importance of surface determinants for virulence and tissue damage.
Assuntos
Alelos , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Mycobacterium tuberculosis/patogenicidade , Tuberculose Pulmonar/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Células Cultivadas , Feminino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose Pulmonar/patologiaRESUMO
Erp (exported repeated protein) was originally characterized as a virulence factor in Mycobacterium tuberculosis and was thought to be present only in Mycobacterium leprae and members of the TB complex. Here it is shown that Erp is a ubiquitous extracellular protein found in all of the mycobacterial species tested. Erp proteins have a modular organization and contain three domains: a highly conserved amino-terminal domain which includes a signal sequence, a central variable region containing repeats based on the motif PGLTS, and a conserved carboxy-terminal domain rich in proline and alanine. The number and fidelity of PGLTS repeats of the central region differ considerably between mycobacterial species. This region is, however, identical in all of the clinical M. tuberculosis strains tested. In addition, it is shown here that a Mycobacterium smegmatis erp::aph mutant displays altered colony morphology which is complemented by all the Erp orthologues tested. The genome sequence flanking the erp gene includes cell-wall-related ORFs and displays extensive conservation between saprophytic and pathogenic mycobacteria.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mycobacterium/classificação , Mycobacterium/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada , Genoma Bacteriano , Humanos , Dados de Sequência Molecular , Mutação , Mycobacterium/genética , Fenótipo , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
In order to determine whether a human homolog (NRAMP1) to a murine candidate gene for resistance to mycobacteria influences susceptibility to human disease, we analyzed data from seven multicase leprosy families (84 individuals) from French Polynesia for linkage markers within the NRAMP1 gene and leprosy per se. Individual family members were typed at nine polymorphic loci within NRAMP1. In addition, three physically linked, polymorphic microsatellite markers-D2S104, D2S173 and D2S1471-were also typed. Linkage analyses were done using affected sibpair and LOD score methods employing different modes of inheritance with full and reduced penetrance. The results of this study strongly suggest that NRAMP1 is not linked to leprosy susceptibility in the French Polynesian families tested.