24 research outputs found
Comparison of Mycobacterium tuberculosis isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity, oligomeric state, pH tolerance and phylogenetic affiliation
Background: M.tb icd-1 and M.tb icd-2, have been identified in the Mycobacterium tuberculosis genome as probable isocitrate dehydrogenase (ICD) genes. Earlier we demonstrated that the two isoforms can elicit B cell response in TB patients and significantly differentiate TB infected population from healthy, BCG-vaccinated controls. Even though immunoassays suggest that these proteins are closely related in terms of antigenic determinants, we now show that M.tb icd-1 and M.tb icd-2 code for functional energy cycle enzymes and document the differences in their biochemical properties, oligomeric assembly and phylogenetic affiliation. Results: Functionally, both M.tb ICD-1 and ICD-2 proteins are dimers. Zn+2 can act as a cofactor for ICD-1 apart from Mg+2, but not for ICD-2. ICD-1 has higher affinity for metal substrate complex (Km (isocitrate) with Mg++:10 μM ± 5) than ICD-2 (Km (isocitrate) with Mg++:20 μM ± 1). ICD-1 is active across a wider pH range than ICD-2, retaining 33-35% activity in an acidic pH upto 5.5. Difference in thermal behaviour is also observed with ICD-2 being active across wider temperature range (20°C to 40°C) than ICD-1 (optimum temperature 40°C). The isozymes are NADP+ dependent with distinct phylogenetic affiliations; unlike M.tb ICD-2 that groups with bacterial ICDs, M.tb ICD-1 exhibits a closer lineage to eukaryotic NADP+ dependent ICDs. Conclusion: The data provide experimental evidence to show that the two open reading frames, Rv3339c (ICD-1) and Rv0066c (ICD-2), annotated as probable ICDs are functional TCA cycle enzymes with identical enzymatic function but different physio-chemical and kinetic properties. The differences in biochemical and kinetic properties suggest the possibility of differential expression of the two ICDs during different stages of growth, despite having identical metabolic function
Mutations in rpoB gene and their association with Rifampicin resistance levels in clinical isolates of Mycobacterium tuberculosis
Present study was aimed to identify most frequent mutations in rpoB gene region and to evaluate the association between mutations in rpoB gene and resistance levels to Rifampicin in clinical isolates of Mycobacterium tuberculosis of different geographical regions of India. A total of 100 clinical isolates of Mycobacterium tuberculosis were included in this study. Drug susceptibility testing against first line anti-tuberculosis drugs was performed on LJ medium by conventional minimal inhibitory concentration (MIC) method and the mutation(s) in rpoB gene of M. tuberculosis isolates were analyzed by sequencing method. Of the 100 M. tuberculosis isolates, 31 (31.0%) and 18 (18.0%) were found resistant and susceptible for all four first-line anti-tuberculosis drugs. The genetic mutations were observed in 96% (72/75) rifampicin-resistant M. tuberculosis isolates, while 4% (3/75) of rifampicin resistant isolates did not have any mutation in rpoB gene. The mutation TCG531TTG (Ser531Leu) was found as most common and frequent mutation in 69.3% (52/75) of rifampicin resistant isolates of M. tuberculosis with MIC level (≥ 512mg/l). The mutation at codon 511 was associated with low degree (128mg/l) of rifampicin resistance, deletions at codons 514-516 or substitution at codon 516 were found to be associated with moderate degree (256mg/l) of rifampicin resistance and mutations at codon 526, 531 were associated with the high degree (512mg/l) of rifampicin resistance in M. tuberculosis isolates of Indian origin. The findings of this study will be useful for the development of raid and more specific indigenous molecular tools for the early diagnosis of multidrug-resistant tuberculosis in the country.
Proteomic analysis of streptomycin resistant and sensitive clinical isolates of Mycobacterium tuberculosis
<p>Abstract</p> <p>Background</p> <p>Streptomycin (SM) is a broad spectrum antibiotic and is an important component of any anti-tuberculosis therapy regimen. Several mechanisms have been proposed to explain the emergence of resistance but still our knowledge is inadequate. Proteins form a very complex network and drugs are countered by their modification/efflux or over expression/modification of targets. As proteins manifest most of the biological processes, these are attractive targets for developing drugs, immunodiagnostics or therapeutics. The aim of present study was to analyze and compare the protein profile of whole cell extracts from <it>Mycobacterium tuberculosis </it>clinical isolates susceptible and resistant to SM.</p> <p>Results</p> <p>Two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was employed for analyzing the protein profiles. Homology and <it>in silico </it>characterization for identified proteins was assessed using BLAST, InterProScan and KEGG database searches. Computational studies on the possible interactions between SM and identified proteins were carried out by a battery of online servers and softwares, namely, CLUSTALW (KEGG), I-TASSER, VMD, PatchDock and FireDock. On comparing 2DE patterns, nine proteins were found consistently overexpressed in SM resistant isolates and were identified as Rv0350, Rv0440, Rv1240, Rv3075c, Rv2971, Rv3028c, Rv2145c, Rv2031c and Rv0569. <it>In silico </it>docking analysis showed significant interactions of SM with essential (Rv0350, Rv0440 and Rv2971) and non essential (Rv1240, Rv3075c and Rv2031c) genes.</p> <p>Conclusions</p> <p>The computational results suggest high protein binding affinity of SM and suggested many possible interactions between identified proteins and the drug. Bioinformatic analysis proves attributive for analysis of diversity of proteins identified by whole proteome analysis. In-depth study of the these proteins will give an insight into probable sites of drug action other than established primary sites and hence may help in search of novel chemotherapeutic agents at these new sites as inhibitors.</p
Detection of Mycobacterium leprae DNA for 36kDa protein in urine from leprosy patients: a preliminary report
We have searched for Mycobacterium leprae DNA for 36kDa protein in urine using a M. leprae specific PCR technique. A limited number of 16 patients (of which 11 belonged to lepromatous leprosy and five to tuberculoid leprosy) and eight healthy individuals were included for the present study. The number of urine samples positive by PCR were 36.4% (4/11) in lepromatous patients and 40% (2/5) in tuberculoid patients. None of the samples from healthy individuals was positive. To our knowledge, the results indicate, for the first time, the presence of M. leprae DNA in urine from leprosy patients. Another important finding obtained out of the study is that amongst treated patients 66.6% (4/6) were positive whereas amongst untreated only 20% (2/10) were positive. From the present indicative data it appears that treatment improves the PCR results with urine as a sample. Thus, the approach could prove to be useful for monitoring the treatment response of individual patients and needs to be further evaluated with a large number of patients.Pesquisamos o DNA do Mycobacterium leprae para proteína 36 kDa na urina usando a técnica do PCR específica para M. leprae. Um número limitado de 16 pacientes (dos quais 11 tinham hanseníase multibacilar e cinco hanseníase paucibacilar) e oito indivíduos saudáveis foram incluídos neste estudo. O número de amostras de urina positivas pelo PCR foi de 36,4% (4/11) em pacientes com hanseníase multibacilar e 40% (2/5) em pacientes com hanseníase paucibacilar. Nenhuma das amostras de indivíduos saudáveis foi positiva. Até onde chega o nosso conhecimento, os resultados indicam, pela primeira vez, a presença de DNA do M. leprae na urina de pacientes com hanseníase. Outro fato importante obtido através do exame é que entre os pacientes tratados 66.6% (4/6) eram positivos enquanto entre os não tratados somente 20% (2/10) foram positivos. Pelos presentes dados indicativos parece que o tratamento melhora os resultados do PCR em amostra de urina. Assim, o acesso a estes dados prova ser útil no monitoramento da resposta ao tratamento de pacientes individuais e precisa ser melhor avaliado com um grande número de pacientes
Applications of Molecular Methods in Human Medicine in India-Urgency to Bridge the Gap
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Current status of TB vaccines
During last 10 years, there has been extensive work for the development of potential tuberculosis vaccine candidates using the mice and guinea pig models. Though till date several promising candidates have been identified and at least eight vaccines have entered clinical evaluation. These recent advances in the clinical testing of new TB vaccines are very exciting and promising. However, there is a need to continue the search for additional vaccine candidates or vaccination strategies