RESUMO
Mycobacterium leprae, a major human pathogen, grows poorly at 37 degrees C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacterial sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium leprae/fisiologia , Pseudogenes , Fator sigma/genética , Proteínas de Bactérias/biossíntese , Chaperonina 60/biossíntese , Deleção de Genes , Teste de Complementação Genética , Proteínas de Choque Térmico HSP70/biossíntese , Transtornos de Estresse por Calor , Temperatura Alta , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Fator sigma/biossíntese , alfa-Cristalinas/genética , alfa-Cristalinas/fisiologiaRESUMO
Gene expression analysis in Mycobacterium leprae, an obligate intracellular pathogen and the etiologic agent of leprosy, has been hampered by the lack of an efficient method to purify RNA from leprosy lesions. Therefore to date, transcripts for only a few genes have been identified. We report the use of a single-tube homogenization/RNA extraction method that produces enough RNA to study the expression of 30 genes from a single skin biopsy specimen of a multibacillary leprosy patient and demonstrate that RNA can be purified after fixation of biopsies in 70% ethanol for up to a year. This represents a major advancement in the ability to study M. leprae gene expression directly from biopsy material and should help to define genes that are associated with intracellular survival of this human pathogen.
Assuntos
Biópsia/métodos , Perfilação da Expressão Gênica/métodos , Hanseníase/microbiologia , Hanseníase/patologia , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Testes Genéticos/métodos , Humanos , Mycobacterium leprae/genética , Análise de Sequência de RNA/métodos , Pele/microbiologia , Pele/patologiaRESUMO
Gene expression analysis in Mycobacterium leprae, an obligate intracellular pathogen and the etiologic agent of leprosy, has been hampered by the lack of an efficient method to purify RNA from leprosy lesions. Therefore to date, transcripts for only a few genes have been identified. We report the use of a single-tube homogenization/RNA extraction method that produces enough RNA to study the expression of 30 genes from a single skin biopsy specimen of a multibacillary leprosy patient and demonstrate that RNA can be purified after fixation of biopsies in 70% ethanol for up to a year. This represents a major advancement in the ability to study M. leprae gene expression directly from biopsy material and should help to define genes that are associated with intracellular survival of this human pathogen.
Assuntos
Humanos , Análise de Sequência de RNA , Biópsia , Hanseníase , Mycobacterium leprae , Pele , Perfilação da Expressão Gênica , RNA Bacteriano , Testes GenéticosRESUMO
Millions of patients with leprosy suffer from nerve damage resulting in disabilities as a consequence of Mycobacterium leprae infection. However, mechanisms of nerve damage have not been elucidated because of the lack of a model that maintains M. leprae viability and mimics disease conditions. A model was developed using viable M. leprae, rat Schwann cells, and Schwann cell-neuron cocultures incubated at 33 degrees C. M. leprae retained 56% viability in Schwann cells for 3 weeks after infection at 33 degrees C, compared with 3.6% viability at 37 degrees C. Infected Schwann cells had altered morphology and expression of genes encoding cellular adhesion molecules at 33 degrees C but were capable of interacting with and myelinating neurons. Cocultures, infected after myelination occurred, showed no morphological changes in myelin architecture after 1 month of incubation at 33 degrees C, and M. leprae retained 53% viability. This article describes a new model for studying the effects of M. leprae on Schwann cells.
