ABSTRACT
Mycobacterium leprae, the causative agent of leprosy, is uncultivable in defined media. Development of new diagnostic tools which do not depend on growth of bacteria is needed for the early detection of M. leprae and for monitoring the effectiveness of chemotherapy. We used a real-time PCR-based assay to quantify the copy number of bacterial DNA and hsp18 mRNA from 47 leprosy patients using paraffin-embedded biopsy samples. The assay used was specific, sensitive and reproducible. The applicability of this approach in monitoring the chemotherapy of leprosy was examined. A reduction in DNA and mRNA during chemotherapy was observed and hsp18 mRNA could not be detected in patients who underwent 2 years of multidrug therapy (MDT). However, a considerable amount of M. leprae DNA could be detected even after 2 years of MDT. A significant amount of hsp18 mRNA was found in reactional cases as well. This raises important questions regarding the role of bacterial antigens in leprosy reactions and the rationale of omitting antibiotics in the treatment of reactional cases. Results in this study show that real-time PCR could be a better tool for the careful monitoring of bacillary DNA and mRNA in lesions, which will help to improve diagnosis, disease progression and the treatment regimen.
Subject(s)
Biopsy , DNA, Bacterial/analysis , Leprosy , Mycobacterium leprae/isolation & purification , Polymerase Chain Reaction/methods , RNA, Messenger/analysis , Skin/microbiology , Bacterial Proteins/genetics , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Heat-Shock Proteins/genetics , Humans , Leprostatic Agents/therapeutic use , Leprosy/diagnosis , Leprosy/drug therapy , Leprosy/microbiology , Leprosy/physiopathology , Mycobacterium leprae/classification , Mycobacterium leprae/drug effects , Mycobacterium leprae/genetics , Paraffin , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Reproducibility of Results , Sensitivity and Specificity , Tissue Embedding/methods , Treatment OutcomeABSTRACT
BACKGROUND: Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty. RESULTS: The gene encoding Mycobacterium leprae small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in E. coli. The localization and in vitro characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of E. coli. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes SmaI and NdeI. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under in vitro conditions as is demonstrated for several small heat shock proteins. CONCLUSION: The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.
Subject(s)
Bacterial Proteins/metabolism , Heat-Shock Proteins, Small/metabolism , Leprosy/microbiology , Mycobacterium leprae/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Heat-Shock Proteins, Small/chemistry , Heat-Shock Proteins, Small/genetics , Humans , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mycobacterium leprae/chemistry , Mycobacterium leprae/genetics , Protein Binding , Protein TransportABSTRACT
Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of slashed circleC31. Transformation of this plasmid into Mycobacterium smegmatis mc2 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5'TTG disrupting the gatA gene (Glu-tRNA(Gln) amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient's biopsy showed that mce locus is transcribed as an operon in the pathogen also.
