Cloning ofmce1locus ofMycobacterium lepraeinMycobacterium smegmatismc2155 SMR5 and evaluation of expression ofmce1genes inM. smegmatisandM. leprae

Santhosh, Ramachandran Sarojini; Pandian, Shunmugiah Karutha; Nirmala, Lini; Shabaana, A. K.; Nagavardhini, Avuthu; Dharmalingam, Kuppamuthu

doi:10.1016/j.femsim.2005.05.004

Cited by 8 publications

(15 citation statements)

References 34 publications

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“…In addition, we used paraffin-embedded leprosy biopsies for these studies, which reduce the chances of the direct handling of infectious material. The use of PET biopsy samples for the analysis of host-as well as pathogen-specific mRNA by RT-PCR has been reported in previous publications from our laboratory (Santhosh et al, 2005;Shabaana et al, 2001Shabaana et al, , 2003. Recombinant plasmid carrying the cloned amplicon, prepared using the same primer set used for real-time PCR analysis, was constructed, and used for the preparation of a standard curve.…”

Section: Resultsmentioning

confidence: 78%

“…Formalin-fixed and paraffin-embedded tissue (PET) is less vulnerable to contamination for molecular pathology studies and diagnostic applications (Santhosh et al, 2005;Shabaana et al, 2001). The use of PET in bacterial load determination and gene expression studies by real-time PCR has been reported to be very sensitive and highly reproducible (Godfrey et al, 2000;Lehmann & Kreipe, 2001).…”

Section: Introductionmentioning

confidence: 99%

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Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases

Nirmala

Dharmalingam

2009

Journal of Medical Microbiology

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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.

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Section: Resultsmentioning

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases

Nirmala

Dharmalingam

2009

Journal of Medical Microbiology

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“…Mammalian cell entry 1A (Mce1A) (ML2589; mce1A) protein on the surface of M. leprae ( Santhosh et al 2005 ) was first identified in Mycobacterium tuberculosis by Arruda et al (1993) . Recombinant Escherichia coli expressing the M. tuberculosis Mce1A protein could invade and survive in HeLa cells and macrophages ( Arruda et al 1993 ).…”

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confidence: 99%

ELISA-based assay of immunoglobulin G antibodies against mammalian cell entry 1A (Mce1A) protein: a novel diagnostic approach for leprosy

Lima

Takenami

Cavalcanti

et al. 2017

Mem. Inst. Oswaldo Cruz

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BACKGROUNDLeprosy is a chronic infectious disease caused by the obligate intracellular bacillus Mycobacterium leprae. Because leprosy diagnosis is complex and requires professional expertise, new tools and methodologies are needed to detect cases in early stages and prevent transmission. The M. leprae genome contains mce1A, which encodes a putative mammalian cell entry protein (Mce1A). We hypothesised that the presence of Mce1A on the cell surface could be detected by the host's immune system.OBJECTIVEThe aim of this study was to evaluate antibody responses against the Mce1A protein in leprosy patients, household contacts of patients, and the general population to present an addition tool for leprosy diagnosis.METHODSA cross-sectional study involving 89 volunteers [55 leprosy cases, 12 household contacts (HHC) and 22 endemic controls (EC)] was conducted at Couto Maia Hospital, in Salvador, Bahia (BA), Brazil.RESULTSThe median anti-Mce1A IgA was significantly higher in multibacillary (MB) and paucibacillary (PB) cases than in EC (p < 0.0001). A similar trend was observed in IgM levels, which were significantly higher in both MB (p < 0.0001) and PB (p = 0.0006) groups compared to in EC individuals. The greatest differences were observed for IgG class-specific antibodies against Mce1A. The median levels of MB and PB were significantly higher compared to both controls HHC and EC (MB or PB vs EC, MB vs HHC p < 0.0001; PB vs HHC, p = 0.0013). Among leprosy cases, IgG enzyme-linked immunosorbent assay sensitivity and specificity were 92.7% and 97.1%, respectively. IgG positivity was confirmed in 92.1% and 94.1% of MB and PB patients, respectively.CONCLUSIONThis novel diagnostic approach presents an easy, non-invasive, and inexpensive method for leprosy screening, which may be applicable in endemic areas.

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“…Although considered nonpathogenic, M. smegmatis provides a popular model for studying virulence mechanisms of slow-growing, pathogenic relatives such as Mycobacterium tuberculosis (9,16,37,41) and Mycobacterium leprae (35,42). An important aspect of mycobacterial pathogenesis is the ability of the pathogen to establish latent infections in hosts lasting for several years.…”

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Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis

et al. 2008

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Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of Mycobacterium smegmatis, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (M. smegmatis ⌬rel Msm strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the lsr2 gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated rel Msm and lsr2 genes, and so a single lsr2 mutant was created to analyze the gene's function. The lsr2 gene was inactivated in the wild-type M. smegmatis mc 2 155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and ⌬rel Msm strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as "hypermotility"), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing lsr2 (pLSR2) reverts the strain to the mc 2 155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.Mycobacterium smegmatis is a fast-growing, saprophytic mycobacterial species. Although considered nonpathogenic, M. smegmatis provides a popular model for studying virulence mechanisms of slow-growing, pathogenic relatives such as Mycobacterium tuberculosis (9,16,37,41) and Mycobacterium leprae (35,42). An important aspect of mycobacterial pathogenesis is the ability of the pathogen to establish latent infections in hosts lasting for several years. Persistent M. tuberculosis bacilli in the host manifest drastic changes in gene expression that set the cells apart from actively growing tubercle bacilli (36,40). One bacterial regulatory network that coordinates nutrient deprivation with adaptive metabolism is the stringent response. In mycobacteria this global regulatory system is controlled by a single gene called rel, and deletion of this gene in M. tuberculosis results in a severe defect in both long-term in vitro and in vivo survival (10, 30). We recently reported that the rel gene of M. smegmatis (rel Msm ) is involved in the regulation of cellular and colony morphologies (9). As seen with M. tuberculosis, the stringent response is required for long-term survival of M. smegmatis in culture, since the rel Msm mutant readily dies over a month-long period while in stationary phase.Here we report the appearance of a mutant strain, called DL1215, that arose spontaneously from the parental M. smegmatis ⌬rel Msm strain. We...

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Cloning ofmce1locus ofMycobacterium lepraeinMycobacterium smegmatismc²155 SMR5 and evaluation of expression ofmce1genes inM. smegmatisandM. leprae

Cited by 8 publications

References 34 publications

Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases

Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases

ELISA-based assay of immunoglobulin G antibodies against mammalian cell entry 1A (Mce1A) protein: a novel diagnostic approach for leprosy

Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis

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