RESUMO
BACKGROUND: The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials of this genome should increase our understanding of their impact on M. leprae physiology. RESULTS: Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Transcribed pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational "silencing" of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno (SD) sequences. Transcribed pseudogenes also contained multiple "in-frame" stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. CONCLUSION: Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational "silencing" mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts.
Assuntos
Perfilação da Expressão Gênica , Genoma Bacteriano , Mycobacterium leprae/genética , Pseudogenes , Sequência de Bases , Códon de Iniciação , Códon de Terminação , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Genes Bacterianos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Transcrição GênicaRESUMO
Mycobacterium leprae, the etiological agent of leprosy, is noncultivable on axenic media. Therefore, the viability of M. leprae for clinical or experimental applications is often unknown. To provide new tools for M. leprae viability determination, two quantitative reverse transcriptase PCR (RT-PCR) assays were developed and characterized. M. leprae sodA mRNA and 16S rRNA were used as RNA targets, and M. leprae repetitive element (RLEP) DNA was used to determine relative bacterial numbers in the same purified bacterial preparations or from crude biological specimens. Results demonstrated that both assays were good predictors of M. leprae viability during short-term experiments (48 h) involving rifampin (rifampicin) treatment in axenic medium, within rifampin-treated murine macrophages (MPhi), or within immune-activated MPhi. Moreover, these results strongly correlated those of other M. leprae viability assays, including radiorespirometry-based and Live/Dead BacLight viability assays. The 16S rRNA/RLEP assay consistently identified the presence of M. leprae in eight multibacillary leprosy patient biopsy specimens prior to multidrug therapy (MDT) and demonstrated a decline in viability during the course of MDT. In contrast, the sodA/RLEP assay was able to detect the presence of M. leprae in only 25% of pretreatment biopsy specimens. In conclusion, new tools for M. leprae viability determination were developed. The 16S rRNA/RLEP RT-PCR M. leprae viability assay should be useful both for short-term experimental purposes and for predicting M. leprae viability in biopsy specimens to monitor treatment efficacy, whereas the sodA/RLEP RT-PCR M. leprae viability assay should be limited to short-term experimental research purposes.
Assuntos
DNA Bacteriano/genética , Hanseníase/microbiologia , Viabilidade Microbiana , Mycobacterium leprae/fisiologia , Reação em Cadeia da Polimerase/métodos , Animais , Proteínas de Bactérias/genética , Primers do DNA/genética , Humanos , Hanseníase/tratamento farmacológico , Macrófagos/microbiologia , Camundongos , Mycobacterium leprae/genética , RNA Ribossômico 16S/genética , Superóxido Dismutase/genéticaRESUMO
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/fisiologiaAssuntos
Proteínas de Bactérias/genética , Técnicas Bacteriológicas , Técnicas Biossensoriais , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/métodos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , RNA Polimerases Dirigidas por DNA , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
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.