Asunto(s)
Terapia Genética/métodos , Leishmaniasis/terapia , Proteínas de la Membrana/antagonistas & inhibidores , Interferencia de ARN/fisiología , ARN Interferente Pequeño/antagonistas & inhibidores , Regulación hacia Abajo/genética , Humanos , Leishmaniasis/genética , Leishmaniasis/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Peroxinas , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genéticaRESUMEN
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.
Asunto(s)
Lepra/metabolismo , Mycobacterium leprae , Osteoblastos/enzimología , Células de Schwann/enzimología , Regulación hacia Abajo/genética , Citometría de Flujo , Regulación de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Lepra/genética , Lepra/patología , Endopeptidasa Neutra Reguladora de Fosfato PHEX/genética , Endopeptidasa Neutra Reguladora de Fosfato PHEX/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/genéticaRESUMEN
BACKGROUND: Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG. RESULTS: M. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1-4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA. CONCLUSION: This study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.
Asunto(s)
Apoptosis/fisiología , Mycobacterium leprae/fisiología , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Letal Asociada a bcl/genética , Apoptosis/genética , Línea Celular , Supervivencia Celular/genética , Regulación hacia Abajo/genética , Electroforesis en Gel de Agar/métodos , Regulación de la Expresión Génica/genética , Humanos , Microscopía Fluorescente/métodos , Mycobacterium bovis/patogenicidad , Mycobacterium bovis/fisiología , Mycobacterium leprae/patogenicidad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/genética , VirulenciaRESUMEN
In order to understand the role of the upstream region of the Mycobacterium leprae 18-kDa gene on the gene regulation, the region was divided into two at the -50 position from the first start codon of the gene and their effect on transcription was examined by using a LacZ transcriptional reporter gene assay. The presence of each of these two regions conferred transcription repression not only on its cognate M. lepraerae 18-kDa gene promoter, but also on a heterologous promoter such as the Mycobacterium bovis BCG hsp65 gene promoter. Moreover, it was found that these regions could confer transcription repression activity in both cases in an orientation-independent manner. Thus, these results indicate that the upstream region of the M. leprae 18-kDa gene harbors transcription repression responsive element(s) acting as an operator and can be further divided into two separately functional regions, suggesting a bipartite structure of the element(s). The identification of transcription repression activity of the upstream region in the M. leprae 18-kDa gene will contribute greatly for the understanding of the 18-kDa gene regulation mechanism, and provide also useful information for the manipulation of mycobacterium gene expression.
Asunto(s)
Proteínas Bacterianas/genética , Regulación hacia Abajo/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium leprae/genética , Elementos de Respuesta/genética , Transcripción GenéticaRESUMEN
PURPOSE OF REVIEW: Exposure to certain environmental microorganisms can promote the induction of T regulatory cells via the innate immune system. This review explores the possibility that reduced exposure to such organisms is leading to increased immunoregulatory disorders in a subset of individuals in whom this regulatory T-cell-inducing pathway is less efficient. We concentrate on mycobacteria and on asthma, because these are well documented. RECENT FINDINGS: The blood cells of the children of farmers, who are partly protected from allergies, express increased levels of messenger RNA encoding CD14 and TLR2, and polymorphisms of CD14 are linked to allergic manifestations in some studies. Polymorphisms of TLR2 (which recognizes mycobacterial components in concert with CD14) are involved in the pattern of response to mycobacteria, and in the type of leprosy that develops. Similarly, polymorphisms of Nramp1, which affect the response to mycobacteria, are linked with the diseases of immunodysregulation that are increasing in parallel with allergic disorders. Moreover, congenic mice bearing different variants of Nramp1 differ in their allergic responses. These parallels are suggestive, in view of the observation that a saprophytic environmental mycobacterium is a potent inducer of regulatory T cells, and has shown significant effects in several phase I/II studies in man. SUMMARY: The components of the innate immune system that are involved in responses to mycobacteria overlap with those implicated in allergic disorders. Polymorphisms might define the subset of individuals who develop immunoregulatory disorders. Understanding the role of the innate immune system will facilitate the design of clinical trials using microbial products.