RESUMO
Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.
Assuntos
Humanos , Hanseníase , Mycobacterium leprae , Trifosfato de Adenosina , Colesterol , LipídeosRESUMO
Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX) is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14) and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.
Assuntos
Humanos , Hanseníase , Mycobacterium leprae , Osteoblastos/enzimologia , Células de Schwann/enzimologia , Regulação para Baixo , Citometria de Fluxo , Regulação da Expressão Gênica , Imuno-Histoquímica , Hanseníase , Hanseníase/patologia , Endopeptidase Neutra Reguladora de Fosfato PHEX , Endopeptidase Neutra Reguladora de Fosfato PHEX , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
In this study, we compared the level of TNF-alpha secretion induced in monocytic THP-1 cells after phagocytosis of Mycobacterium leprae, the causative agent of leprosy, and M. bovis BCG, an attenuated strain used as a vaccine against leprosy and tuberculosis. The presence of M. leprae and BCG was observed in more than 80 percent of the cells after 24 h of exposure. However, BCG but not M. leprae was able to induce TNF-alpha secretion in these cells. Moreover, THP-1 cells treated simultaneously with BCG and M. leprae secreted lower levels of TNF-alpha compared to cells incubated with BCG alone. M. leprae was able, however, to induce TNF-alpha secretion both in blood-derived monocytes as well as in THP-1 cells pretreated with phorbol myristate acetate. The inclusion of streptomycin in our cultures, together with the fact that the use of both gamma-irradiated M. leprae and heat-killed BCG gave similar results, indicate that the differences observed were not due to differences in viability but in intrinsic properties between M. leprae and BCG. These data suggest that the capacity of M. leprae to induce TNF-alpha is dependent on the stage of cell maturation and emphasize the potential of this model to explore differences in the effects triggered by vaccine strain versus pathogenic species of mycobacteria on the host cell physiology and metabolism
Assuntos
Humanos , Animais , Bovinos , Vacinas Bacterianas/imunologia , Vacina BCG/imunologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Monócitos/microbiologia , Mycobacterium bovis/imunologia , Fator de Necrose Tumoral alfa/biossínteseAssuntos
Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Ativação Linfocitária , Dados de Sequência Molecular , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/química , Especificidade da Espécie , Genoma Bacteriano , Hanseníase Tuberculoide/diagnóstico , Hanseníase Tuberculoide/imunologia , Hanseníase Tuberculoide/microbiologia , Linfócitos T/imunologia , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Mycobacterium leprae/química , Peptídeos/imunologia , Peptídeos/síntese química , Sequência de AminoácidosAssuntos
Anticorpos Monoclonais , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Biblioteca Gênica , Cosmídeos , Dados de Sequência Molecular , Genes Bacterianos , Genes Dominantes , Hanseníase/imunologia , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Mapeamento por Restrição , Mycobacterium leprae/genética , Mycobacterium/genética , Mycobacterium/metabolismo , Peso Molecular , Primers do DNA , Proteínas Recombinantes/biossíntese , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Sequência de BasesRESUMO
The study of the relationship between Mycobacterium leprae and its human host, by investigating the bacterial components and the host immune response, will certainly provide the basis for a better treatment and control of leprosy. In addition, leprosy represents an attractive model from where important aspects regarding pathogen survival strategies, and the basic mechanisms involved in the regulation of the immune system can be learned. This review describes the definition of a major protein of the leprosy bacillus with a potential role in its virulence. The involvement of key cytokines and leukocyte subsets in protection and pathophysiology of this disease is also presented.