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Journal of Bacteriology Nov 1999The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA...
The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosis dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of Escherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. leprae dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both M. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.
Topics: Cloning, Molecular; Dihydropteroate Synthase; Escherichia coli; Genes, Bacterial; Molecular Sequence Data; Mycobacterium leprae; Mycobacterium tuberculosis; Phylogeny; Recombinant Proteins
PubMed: 10542185
DOI: 10.1128/JB.181.21.6814-6821.1999 -
Clinical Microbiology and Infection :... Oct 2013Leprosy continues to be a significant health problem in certain pockets in developing countries. Better understanding of the transmission and source of the infection...
Leprosy continues to be a significant health problem in certain pockets in developing countries. Better understanding of the transmission and source of the infection would help to decipher the transmission link, leading to control of the spread of the disease. The nose is considered to be a portal of entry, suggesting an aerial route for transmission through droplet infection. The evidence suggests that many individuals from endemic countries carry Mycobacterium leprae in their nasal cavities without having obvious symptoms of leprosy. The objective of the present study was to assess the presence of M. leprae on the nasal mucosa in the general population from a leprosy-endemic pocket. M. leprae detection was carried out using PCR targeting RLEP. Four hundred subjects from an area highly endemic for leprosy were included in the study and followed up during three different seasons--winter, summer, and monsoon--for evidence of nasal exposure to M. leprae. PCR positivity for M. leprae was observed in 29%, 21% and 31% of the samples collected in winter, summer and the monsoon season, respectively. Twenty-six individuals from the cohort showed amplification for M. leprae for all seasons. Our results are consistent with reports in the literature showing widespread exposure to M. leprae in the endemic community. The results also suggest possible association of the environmental conditions (climate) with the transmission pattern and levels of exposure to M. leprae. However, the present study indicated that the population from highly endemic pockets will have exposure to M. leprae irrespective of season.
Topics: Adolescent; Adult; Aged; Carrier State; Child; Child, Preschool; Cohort Studies; DNA, Bacterial; Endemic Diseases; Female; Humans; Humidity; India; Leprosy; Male; Middle Aged; Mycobacterium leprae; Nasal Mucosa; Polymerase Chain Reaction; Seasons; Young Adult
PubMed: 23331372
DOI: 10.1111/1469-0691.12087 -
Immunological Reviews May 2021Mycobacterium leprae, the causative agent of leprosy, is still actively transmitted in endemic areas reflected by the fairly stable number of new cases detected each... (Review)
Review
Mycobacterium leprae, the causative agent of leprosy, is still actively transmitted in endemic areas reflected by the fairly stable number of new cases detected each year. Recognizing the signs and symptoms of leprosy is challenging, especially at an early stage. Improved diagnostic tools, based on sensitive and specific biomarkers, that facilitate diagnosis of leprosy are therefore urgently needed. In this review, we address the challenges that leprosy biomarker research is facing by reviewing cell types reported to be involved in host immunity to M leprae. These cell types can be associated with different possible fates of M leprae infection being either protective immunity, or pathogenic immune responses inducing nerve damage. Unraveling these responses will facilitate the search for biomarkers. Implications for further studies to disentangle the complex interplay between host responses that lead to leprosy disease are discussed, providing leads for the identification of new biomarkers to improve leprosy diagnostics.
Topics: Biomarkers; Humans; Immunity; Leprosy; Mycobacterium leprae
PubMed: 33709405
DOI: 10.1111/imr.12966 -
Journal of Clinical Microbiology Apr 2020Leprosy is caused by , and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent hybridization (FISH) for detecting in 27 skin samples...
Leprosy is caused by , and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent hybridization (FISH) for detecting in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d'Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified , including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.
Topics: Burkina Faso; DNA, Bacterial; Humans; In Situ Hybridization, Fluorescence; Leprosy; Mycobacterium leprae; Skin
PubMed: 32132193
DOI: 10.1128/JCM.02130-19 -
PLoS Neglected Tropical Diseases Mar 2015Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host...
Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of "HSP18-ATP" interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Bacterial Proteins; Biomarkers; Heat-Shock Proteins; Molecular Chaperones; Mycobacterium leprae; Protein Conformation; Protein Interaction Domains and Motifs; Sequence Alignment; Surface Plasmon Resonance; alpha-Crystallin B Chain
PubMed: 25811190
DOI: 10.1371/journal.pntd.0003661 -
Journal of Clinical Microbiology Sep 1990By using a set of four nested oligonucleotide primers, a two-step polymerase chain reaction assay for the detection and identification of Mycobacterium leprae that does...
By using a set of four nested oligonucleotide primers, a two-step polymerase chain reaction assay for the detection and identification of Mycobacterium leprae that does not require the use of radioactivity labeled hybridization probes was developed. The nested-primer procedure amplified a 347-base-pair product from M. leprae genomic DNA. No amplification products were produced from DNAs of 19 other Mycobacterium species, 19 non-Mycobacterium species, mouse cells, or human cells. Minor amplification products were observed with three additional Mycobacterium species, i.e., "M. lufu", M. simiae, and M. smegmatis. These products were easily distinguished from the M. leprae product by size and restriction enzyme cleavage patterns. The assay could amplify the 347-base-pair product from samples containing as little as 3 fg of M. leprae genomic DNA--the amount of DNA in a single bacillus. The assay also amplified target sequences in crude lysates of M. leprae bacilli isolated from tissue biopsy specimens from infected animals and humans. The entire assay, from sample preparation to data analysis, can be completed in less than 8 h.
Topics: Animals; Base Sequence; DNA Probes; DNA, Bacterial; Genes, Bacterial; Humans; Leprosy; Mice; Molecular Sequence Data; Mycobacterium leprae; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Species Specificity
PubMed: 2229372
DOI: 10.1128/jcm.28.9.1913-1917.1990 -
Journal of Clinical Microbiology Jun 2012Skin biopsy samples from 145 relapse leprosy cases and from five different regions in Brazil were submitted for sequence analysis of part of the genes associated with...
Skin biopsy samples from 145 relapse leprosy cases and from five different regions in Brazil were submitted for sequence analysis of part of the genes associated with Mycobacterium leprae drug resistance. Single nucleotide polymorphisms (SNPs) in these genes were observed in M. leprae from 4 out of 92 cases with positive amplification (4.3%) and included a case with a mutation in rpoB only, another sample with SNPs in both folP1 and rpoB, and two cases showing mutations in folP1, rpoB, and gyrA, suggesting the existence of multidrug resistance (MDR). The nature of the mutations was as reported in earlier studies, being CCC to CGC in codon 55 in folP (Pro to Arg), while in the case of rpoB, all mutations occurred at codon 531, with two being a transition of TCG to ATG (Ser to Met), one TCG to TTC (Ser to Phe), and one TCG to TTG (Ser to Leu). The two cases with mutations in gyrA changed from GCA to GTA (Ala to Val) in codon 91. The median time from cure to relapse diagnosis was 9.45 years but was significantly shorter in patients with mutations (3.26 years; P = 0.0038). More than 70% of the relapses were multibacillary, including three of the mutation-carrying cases; one MDR relapse patient was paucibacillary.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Anti-Bacterial Agents; Biopsy; Brazil; DNA, Bacterial; Drug Resistance, Bacterial; Female; Genes, Bacterial; Humans; Leprosy; Male; Middle Aged; Mutation, Missense; Mycobacterium leprae; Point Mutation; Polymorphism, Single Nucleotide; Prevalence; Prospective Studies; Recurrence; Sequence Analysis, DNA; Skin; Young Adult
PubMed: 22495562
DOI: 10.1128/JCM.06561-11 -
Infection and Immunity Nov 1983Soon after more than 10(6) Mycobacterium leprae, freshly harvested from armadillo liver or harvested and 60CO irradiated, were inoculated into the hind footpads of...
Soon after more than 10(6) Mycobacterium leprae, freshly harvested from armadillo liver or harvested and 60CO irradiated, were inoculated into the hind footpads of either normal or thymectomized and irradiated (T900R) mice, the organisms were found to reside within phagosomes of polymorphonuclear and mononuclear cells. On the other hand, 7 and 8 months after 10(4) freshly harvested M. leprae were inoculated into the footpads of normal or T900R mice and the organisms had multiplied to their maximum in the normal mice, many organisms, largely intact by electron-microscopic criteria, were found to reside free in the cytoplasm of the footpad macrophages, whereas damaged organisms were contained within phagosomes. After 11 months, many intact organisms were found to lie free in the cytoplasm of the macrophages of T900R mice, whereas only damaged intraphagosomal M. leprae cells were observed in the macrophages of normal mice. Finally, a remarkably large proportion of damaged extraphagosomal M. leprae was found in T900R mice administered rifampin for 2 days in a bactericidal dosage. It appears that M. leprae multiplies free in the cytoplasm of the footpad macrophages of infected mice, whereas the M. leprae cells resident within the phagosomes of the macrophages are dead. As the result of treatment with rifampin, the organisms appeared to have been killed in their extraphagosomal location, only afterwards being incorporated into phagosomes. However, the intracellular site in which M. leprae is killed in the course of an effective immune response remains unclear.
Topics: Animals; Female; Immunologic Deficiency Syndromes; Leprosy; Macrophages; Mice; Mice, Inbred Strains; Microscopy, Electron; Mycobacterium leprae; Phagocytosis; Rifampin; Thymectomy
PubMed: 6358034
DOI: 10.1128/iai.42.2.802-811.1983 -
FEMS Immunology and Medical Microbiology Nov 2008Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and...
Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and bacterial enumeration by microscopic examination is required for leprosy classification, choice in choosing and monitoring chemotherapy regimens, and diagnosis of relapse. However, detection and quantification using standard microscopy yields results of limited specificity and sensitivity. We describe an extremely sensitive and specific assay for the detection and quantification of M. leprae in skin biopsy specimens. Primers that amplified a specific 171-bp fragment of M. leprae 16S rRNA gene were chosen and specificity was verified by amplicon melting temperature. The method is sensitive enough to detect as low as 20 fg of M. leprae DNA, equivalent to four bacilli. The assay showed 100% concordance with clinical diagnosis in cases of multibacillary patients, and 50% of paucibacillary leprosy. The entire procedure of DNA extraction and PCR could be performed in c. 3 h. According to normalized quantitative real-time PCR, the patients in this study had bacilli numbers in the range of 1.07 x 10(2) -1.65 x 10(8) per 6-mm3 skin biopsy specimen. This simple real-time PCR assay is a facile tool with possible applications for rapid detection and simultaneous quantification of leprosy bacilli in clinical samples.
Topics: Humans; Leprosy; Mycobacterium leprae; Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; Sensitivity and Specificity; Skin
PubMed: 18783434
DOI: 10.1111/j.1574-695X.2008.00472.x -
Bulletin of the World Health... 1973Some criteria are presented to help evaluate papers appearing in the literature claiming successful cultivation of M. leprae either in the absence or in the presence of... (Review)
Review
Some criteria are presented to help evaluate papers appearing in the literature claiming successful cultivation of M. leprae either in the absence or in the presence of tissue-cultured cells. Recently, electron microscopic studies have definitely shown M. leprae to belong to the genus Mycobacterium and its division to occur through transverse section. A survey is given of the mycobacterial strains isolated in the last 10 years from leprosy lesions. These strains belong to taxonomically different species and cannot be considered to be M. leprae. No substantiated claim was made concerning the in vitro growth of M. leprae and the application of the tissue culture technique has been equally disappointing. The view is expressed that progress towards the in vitro cultivation of M. leprae can be made only as a result of increased knowledge about the intracellular environment and the metabolic activities of this organism, to be obtained by the application of modern biochemical and histochemical techniques.
Topics: Animals; Bacteriological Techniques; Cell Division; Cell Line; Cells, Cultured; Culture Media; Culture Techniques; Ganglia; Haplorhini; Humans; L Forms; Leprosy; Macrophages; Mice; Microscopy, Electron; Mycobacterium; Mycobacterium leprae; Mycobacterium lepraemurium; Rats
PubMed: 4212439
DOI: No ID Found