Genomic Reduction at TTC Repeats in the Bacterial Genome of Treated Cases of Hansen's Disease: A Possible Survival Mechanism of Mycobacterium leprae.

INTRODUCTION: Mycobacterium leprae has a small genome and a tendency of persisting as a very low-grade infection. The authors have shown earlier, that the changes in TTC repeats, in M. leprae genome may contribute to the restriction of the pathogenicity of the bacterium and its survival strategy in case of pure neural Hansen's disease. We suspect, that a similar genomic reduction if happens in treated cases of Hansen's disease, can be a determining factor for developing persisters and relapse. AIM: The present study aimed to find out if there was any evidence of genomic reduction in treated cases of Hansen's disease that showed microbiological nonresponse. METHODS: Skin biopsies were taken from treated cases of Hansen's disease at tertiary centers in Kolkata and at Raipur who had bacterial index (BI) unchanged or increased compared to their pretreatment BI. Analysis for the mutation in rpoB gene and folP1 gene were done to rule out rifampicin and dapsone resistance, respectively. The entire TTC repeat region of the bacteria was amplified by polymerase chain reaction and was subjected to sequencing. The obtained sequences were then analyzed by CLUSTALW. RESULTS: A total of 127 patients were included in the study of which in 52 the BI remained same and 75 had an increase in BI, even after 6 months of completion of multidrug therapy. Among the samples, 2 had positive rpoB gene mutation. No mutation was found in the folP1 gene. The TTC repeat of both the rpoB-resistant samples was found to have 17 copies, which matched their pretreatment copy number. In other 125 cases, 60 cases showed no change from their pretreatment TTC number. Of those 65 samples that showed evidence of genomic reduction, 11 samples showed one copy, 41 showed 2 copies, and 13 showed 3 copies deletion. We also observed a significant regional variation. CONCLUSION: We concluded that there was evidence of genomic reduction, which might lead to microbiological nonresponse in treated cases of Hansen's disease. This indicated a possibility of future persistence and relapse.

Mutation at codon 442 in the rpoB gene of Mycobacterium leprae does not confer resistance to rifampicin.

BACKGROUND: Rifampicin is the major drug in the treatment of leprosy. The rifampicin resistance of Mycobacterium leprae results from a mutation in the rpoB gene, encoding the ß subunit of RNA polymerase. As M. leprae is a non-cultivable organism observation of its growth using mouse food-pad (MFP) is the only Gold Standard assay used for confirmation of "in-vivo" drug resistance. OBJECTIVE: Any mutation at molecular level has to be verified by MFP assay for final confirmation of drug resistance in leprosy. MATERIAL AND METHODS: In the present study, M. leprae strains showing a mutation only at codon 442 Gln-His and along with mutation either at codon 424 Val-Gly or at 438 Gln-Val within the Rifampicin Resistance Determining Region (RRDR) confirmed by DNA sequencing and by high resolution melting (HRM) analysis were subjected for its growth in MFP. RESULT AND CONCLUSION: The M. leprae strain having the new mutation at codon 442 Gln-His was found to be sensitive to all the three drugs and strains having additional mutations at 424 Val-Gly and 438 Gln-Val were conferring resistance with Multi drug therapy (MDT) in MFP. These results indicate that MFP is the gold standard method for confirming the mutations detected by molecular techniques.

Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: a study from Eastern India.

BACKGROUND: The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very efficient opportunity for the diagnosis of drug resistance by in vitro method. AIM: We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. METHODS: A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplified by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI - BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. RESULTS: A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp - 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specific for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). LIMITATIONS: The major limitations of multiple-primer PCR amplification refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. CONCLUSION: The study indicates the existence of rifampicin drug resistance in Eastern India.

Genetics of fetal hemoglobin in tribal Indian patients with sickle cell anemia.

India tops the list of countries with sickle cell disease (SCD) with an estimated 44,000 live births in 2010 and a prevalence of 10%-33%. In the present study, the first from India, we have investigated the effect of genetic variants in the BCL11A, the HMIP (HBS1L-MYB intergenic polymorphism) locus, in addition to the HBB locus, which are known to be associated with fetal hemoglobin (HbF) levels, a major modulator of the disease phenotype. The present study was conducted on 240 individuals with SCD and 60 with sickle cell trait. Genotyping was performed for the BCL11A rs11886868 and rs34211119; HMIP rs9399137, rs189600565, rs7776196, rs34778774, and rs53293029; HBG2 Xmn1 polymorphism rs7482144; and -68C > T HBD promoter polymorphism. All the 3 quantitative trait loci were associated with HbF levels in Indian patients with SCD. The highest difference was seen in the Xmn1 single-nucleotide polymorphism, which accounted for 11% of the trait variance, the BCL11A rs11886868 for 3.65%, whereas the HMIP rs9399137 for 3.8%. The present study indicates the BCL11A, HMIP, and ß-globin region to be associated with increased HbF levels in Indian patient. Further interrogation of these genotypes with respect to pain crisis is warranted in this population, which may help in prognostication, as also a genome-wide association study, which may help uncover new loci controlling HbF levels.

Structural studies of an immunoenhancing glucan of an ectomycorrhizal fungus Ramaria botrytis.

A water-soluble glucan was isolated from the alkaline extract of an ectomycorrhizal fungus, Ramaria botrytis. On the basis of sugar analysis, methylation analysis, Smith degradation, partial hydrolysis, and 1D/2D NMR studies, the structure of the repeating unit of the glucan was established as: [structure: see text]. This glucan showed immunostimulating activity by NO production on RAW 264.7, a murine macrophage cell line. Splenocyte and thymocyte proliferation were measured using, respectively, single cell suspensions of spleen and thymus obtained from normal mice.

Characterization and lectin microarray of an immunomodulatory heteroglucan from Pleurotus ostreatus mycelia.

Glucans isolated from various mushroom and mycelia sources are interestingly being studied nowadays as a potent therapeutic agent. The present work was focused on the isolation, characterization and immunomodulatory study of a novel water soluble glucan from the pure mycelia of Pleurotus ostreatus. The extracted glucan was found to have a high molecular weight of ∼2.7 × 10(6)Da and mainly comprised of glucose, mannose and fucose in a ratio of 3:2:1 with both ß and α linkages. Presence of terminal or interior glucose, mannose and fucose residues was also revealed using a high throughput miniaturized platform of lectin microarray. The heteroglucan folded into a triple helical conformation and exhibited enhanced immune cell activation and anti-tumor potential in tumor bearing mice model. Thus, potential biological functions incorporated in these glucan molecules acts in accord with its structural property and exploration of such structure-function relationship will unveil its diverse mechanism of action.

A heteroglycan from the mycelia of Pleurotus ostreatus: structure determination and study of antioxidant properties.

A heteroglycan (PS) isolated from the mycelia of Pleurotus ostreatus was found to consist of l-fucose, d-mannose, and d-glucose in a molar ratio of nearly 1:2:3. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR studies (1H, 13C, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, HMBC, and DEPT), the structure of the repeating unit of the PS was determined. The repeating unit had a branched backbone composed of (1→6)-linked α and ßd-glucose and (1→2)-linked α-l-fucose. Branching occurred at C-4 position of (1→6)-linked α-d-glucopyranosyl residue with terminal ß-d-mannose and C-3 position of (1→6)-linked ß-d-glucopyranosyl residue with (1→6)-α-d-mannopyranosyl moiety terminated by α-d-glucose. The structure of the repeating unit of the PS was proposed as: [formula: see text]. The antioxidant properties of the PS were studied. The scavenging activity of hydroxyl free radicals whose EC50 value of PS at 943 µg/mL was determined. Further, EC50 value of the PS at 53 µg/mL was observed for scavenging activity of superoxide free radicals. Chelating effects (54.82%) of ferrous ions were observed at 1mg/mL of the PS.

Pectic polysaccharide from immature onion stick (Allium cepa): structural and immunological investigation.

The structure of a water-soluble pectic polysaccharide (PS) isolated from immature onion stick (Allium cepa) was investigated using acid hydrolysis, methylation analysis, periodate oxidation study, and NMR studies ((1)H, (13)C, DQF-COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC). The results of the above experiments indicated that the PS contained d-galactose, 6-O-Me-D-galactose, 3-O-acetyl-D-methyl galacturonate and D-methyl galacturonate in a molar ratio of nearly 1:1:1:1 and possesses a backbone of [→4)-α-D-GalpA6Me-(1→4)-α-D-GalpA6Me-(1→] in which one methyl galacturonate was substituted at O-3 position by an acetyl group and the neighboring methyl galacturonate being substituted at O-2 with a side chain, α-D-Galp-(1→4)-6-O-Me-ß-D-Galp-(1→. The probable structure of repeating unit of the pectic polysaccharide was established as: [formula in text] The pectic polysaccharide showed in vitro splenocyte, thymocyte as well as macrophage activations.

Structural and immunological studies of hetero polysaccharide isolated from the alkaline extract of Tricholoma crassum (Berk.) Sacc.

An immunoenhancing water-soluble hetero polysaccharide was isolated from an alkaline extract of the fruit bodies of an ectomycorrhizal edible mushroom, Tricholoma crassum (Berk.) Sacc. The structure of the molecule was investigated using acid hydrolysis, methylation analysis; periodate oxidation study, and NMR spectroscopy techniques ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC). On the basis of the above-mentioned experiments the structure of the repeating unit of the polysaccharide was established as: This polysaccharide exhibited splenocyte, thymocyte as well as macrophage activations.

Structural characterization of a heteropolysaccharide isolated from hot water extract of the stems of Amaranthus tricolor Linn. (Amaranthus gangeticus L.).

A water-soluble polysaccharide (PS-I), isolated from the aqueous extract of the stems of Amaranthus tricolor Linn. (Amaranthus gangeticus L.), was found to consist of L-arabinose, methyl-D-galacturonate, D-galactose, and 3-O-Ac-L-rhamnose in a molar ratio of nearly 1:1:1:1. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of the PS-I is determined as: -->5)-alpha-L-Araf-(1-->4)-beta-D-Galp-(1-->2)-3-O-Ac-beta-L-Rhap-(1-->4)-alpha-D-Gal-A6Me-(1-->

Structural studies of a heteropolysaccharide (PS-I) isolated from hot water extract of fruits of Psidium guajava (Guava).

A water-soluble polysaccharide was isolated from hot aqueous extracts of fruits of Psidium guajava. The polysaccharide was found to contain 2-O-methyl-l-arabinose, 2-O-acetyl-D-galactose, and D-methyl galacturonate in a molar ratio of approximately 1:1:1. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of the polysaccharide was established as [carbohydrate structure: see text].

Structural characterization of a water-soluble beta-(1-->6)-linked D-glucan isolated from the hot water extract of an edible mushroom, Agaricus bitorquis.

A water-soluble polysaccharide, isolated from the hot aqueous extract of an edible mushroom, Agaricus bitorquis, was found to consist of d-glucose only. On the basis of total hydrolysis, methylation analysis, and NMR studies ((1)H, (13)C, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit was established as -->6)-beta-D-Glcp-(1-->.