RESUMO
Background: Extrapulmonary tuberculosis (EPTB), accounting for 10%-20% of all cases of tuberculosis (TB), is known to be determined by host immunity. However, the contribution of bacterial factors to the development of EPTB has not been studied extensively. Mycolic acids are predominant lipids constituting the cell wall of Mycobacterium tuberculosis, and keto-mycolic acid is involved in the synthesis of foamy macrophages that facilitate persistence of mycobacteria. Hence, the present study was performed to gain an insight into variable expression of mycolic acids in clinical isolates of M. tuberculosis under stress. Methods: Pansusceptible clinical isolates of M. tuberculosis from patients with lymph node TB (LNTB) (n = 10) and pulmonary TB (PTB) (n = 10) were subjected to sodium dodecyl sulfate (SDS) stress, and the expression of mycolic acid and its biosynthetic genes was compared. Any bias arising due to the genotype of the clinical isolates was ruled out by performing single-nucleotide polymorphism cluster grouping (SCG), wherein no significant difference was observed between the SCG of LNTB or PTB isolates. Results: The expression of α-mycolic acid during the exposure to SDS was high in 7/10 (70%) LNTB and 6/10 (60%) PTB isolates. Methoxy mycolic acid showed an increased expression in 7/10 (70%) LNTB isolates and 4/10 (40%) PTB isolates. Increased expression of keto-mycolic acid on exposure with SDS was observed in 8/10 (80%) M. tuberculosis LNTB and 3/10 (30%) PTB isolates. Similarly, the mycolic acid synthesis gene, fas, was upregulated more in LNTB isolates than PTB isolates in vitro and ex vivo. SCG 3a was the most common SCG observed in 40% (8/20) of the isolates, followed by SCG 3b in 30% (6/20) of the isolates. There was no significant difference between the SCG of LNTB or PTB isolates. Conclusion: The higher expression of keto-mycolic acid in LNTB as against PTB isolates may indicate better survival in LNTB isolates in the presence of stress.
Assuntos
Expressão Gênica/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Proteínas de Bactérias/genética , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Dodecilsulfato de Sódio/farmacologia , Estresse Fisiológico , Células THP-1 , Tuberculose dos Linfonodos/microbiologia , Tuberculose Pulmonar/microbiologiaRESUMO
Despite the consideration of chromosomal mutations as the major cause of rifampicin (RIF) resistance in M. tuberculosis, the role of other mechanisms such as efflux pumps cannot be ruled out. We evaluated the role of four efflux pumps viz., MmpL2 (Rv0507), MmpL5 (Rv0676c), Rv0194 and Rv1250 in providing RIF resistance in M. tuberculosis. The real time expression of the efflux pumps was analyzed in 16 RIF resistant and 11 RIF susceptible clinical isolates of M. tuberculosis after exposure to RIF. Expression of efflux pumps in these isolates was also correlated with mutations in the rpoB gene and MICs of RIF in the presence and absence of efflux pump inhibitors. Under RIF stress, Rv0194 was induced in 8/16 (50%) RIF resistant and 2/11 (18%) RIF susceptible isolates; mmpL5 in 7/16 (44%) RIF resistant and 1/11 (9%) RIF susceptible isolates; Rv1250 in 4/16 (25%) RIF resistant and 2/11 (18%) RIF susceptible isolates; and mmpL2 was upregulated in 2/16 (12.5%) RIF resistant and 1/11 (9%) RIF susceptible isolates. This preliminary study did not find any association between Rv0194, MmpL2, MmpL5 and Rv1250 and RIF resistance. However, the overexpression of Rv0194 and mmpL5 in greater number of RIF resistant isolates as compared to RIF susceptible isolates and expression of Rv0194 in wild type (WT) resistant isolates suggests a need for further investigations.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Tuberculose/tratamento farmacológico , Antituberculosos/uso terapêutico , Análise Mutacional de DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Índia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/uso terapêutico , Tuberculose/microbiologiaRESUMO
Cholesterol, an essential cellular component in macrophages, is exploited for entry and long-term survival of Mycobacterium inside the host. Cholesterol-deficient macrophages can restrict the cholesterol-dependent entry of Mycobacterium. Rv3499c protein in Mycobacterium has high binding affinity for cholesterol. Rv3499c gene is a part of mce4 operon which is reported to act as cholesterol transport system in mycobacteria. Earlier we reported Rv3499c protein to localise on cell wall and facilitate entry of Mycobacterium inside macrophages. Here we performed fold recognition and multiple sequence alignment to find similarity with methyl-accepting chemotaxis protein (MCP). MCP allows detection of level of nutrient in the medium, which in this case is cholesterol. We showed Rv3499c protein expression is important for host cholesterol utilization by Mycobacterium for its survival. Infected female balb/c mice presented increased CFU of Rv3499c overexpressing M. tuberculosis H37Rv marked with early disease conditions and increased lung pathology. Thus, findings suggest specific domain of MCP of Rv3499c help in regulation of downstream PDIM synthesis pathways for ligand utilization by M. tuberculosis H37Rv.
Assuntos
Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Pulmão/microbiologia , Macrófagos/microbiologia , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose Pulmonar/microbiologia , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Humanos , Lipídeos , Proteínas Quimiotáticas Aceptoras de Metil/genética , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células THP-1RESUMO
BACKGROUND: Isoniazid (INH) resistance in Mycobacterium tuberculosis has been mainly attributed to mutations in katG (64%) and inhA (19%). However, 20%-30% resistance to INH cannot be explained by mutations alone. Hence, other mechanisms besides mutations may play a significant role in providing drug resistance. Here, we explored the role of 24 putative efflux pump genes conferring INH-resistance in M. tuberculosis. MATERIALS AND METHODS: Real-time expression profiling of the efflux pump genes was performed in five INH-susceptible and six high-level INH-resistant clinical isolates of M. tuberculosis exposed to the drug. Isolates were also analyzed for mutations in katG and inhA. RESULTS: Four high-level INH-resistant isolates (minimum inhibitory concentration [MIC] ≥2.5 mg/L) with mutations at codon 315 (AGC-ACC) of katG showed upregulation of one of the efflux genes Rv1634, Rv0849, efpA, or p55. Another high-level INH-resistant isolate (MIC 1.5 mg/L), with no mutations at katG or inhA overexpressed 8/24 efflux genes, namely, Rv1273c, Rv0194, Rv1634, Rv1250, Rv3823c, Rv0507, jefA, and p55. Five of these, namely, Rv0194, Rv1634, Rv1250, Rv0507, and p55 were induced only in resistant isolates. CONCLUSION: The high number of efflux genes overexpressed in an INH-resistant isolate with no known INH resistance associated mutations, suggests a role for efflux pumps in resistance to this antituberculous agent, with the role of Rv0194 and Rv0507 in INH resistance being reported for the first time.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Isoniazida/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catalase/genética , Catalase/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
PURPOSE: We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. METHODOLOGY: A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. RESULTS: The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100â% specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. CONCLUSION: The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , Chaperonina 60/genética , Marcadores Genéticos , Humanos , Mycobacterium/classificação , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/isolamento & purificação , Reação em Cadeia da Polimerase/economia , Polimorfismo de Fragmento de Restrição/genética , Sensibilidade e Especificidade , Tuberculose/microbiologia , Tuberculose Pulmonar/microbiologiaRESUMO
BACKGROUND: The mce4 operon is one of the four mce operons with eight genes (yrbE4A, yrbE4B, mce4A, mce4B, mce4C, mce4D, mce4E and mce4F) of Mycobacterium tuberculosis. It expresses in the later phase of infection and imports cholesterol for long term survival of the bacilli. To cause latent infection, M. tuberculosis undergoes metabolic reprogramming of its genes to survive in the hostile environment like low availability of oxygen and nutrition depletion inside the host. OBJECTIVE: To analyze real time expression profile of mce4 operon under various stress conditions. METHODS: M. tuberculosis H37Rv was exposed to surface stress (0.1% SDS for 30min and 90min in late log and stationary phase of culture), hypoxia (5, 10, 15 and 20days) and grown in the presence of either glycerol or cholesterol as sole source of carbon. The expression profile of genes of mce4 operon was analyzed by real time PCR. RESULTS: Surface stress induced expression of mce4C and yrbE4B in late log phase on 30min and 90min exposure respectively. The SDS exposure for 30min induced mce4C, mce4D and mce4F in stationary phase. All eight genes were induced significantly on 10th and 15th days of hypoxia and in the presence of cholesterol. CONCLUSION: Hypoxia and cholesterol are potent factors for the expression of mce4 operon of M. tuberculosis.
Assuntos
Proteínas de Bactérias/biossíntese , Perfilação da Expressão Gênica , Mycobacterium tuberculosis/fisiologia , Óperon , Estresse Fisiológico , Proteínas de Bactérias/genética , Colesterol/metabolismo , Detergentes/toxicidade , Glicerol/metabolismo , Humanos , Hipóxia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase em Tempo Real , Dodecilsulfato de Sódio/toxicidadeRESUMO
Several attempts have been made to associate phylogenetic differences among Mycobacterium tuberculosis strains to variations in the clinical outcome of the disease and to drug resistance. We genotyped 139 clinical isolates of M. tuberculosis obtained from patients of pulmonary tuberculosis in North Delhi region. The isolates were analyzed using nine Single nucleotide polymorphism (SNP) markers, spoligotyping and MIRU-VNTRs; and the results were correlated with their drug susceptibility profile. Results of SNP cluster group (SCG) analysis (available for 138 isolates) showed that the most predominant cluster was SCG 3a, observed in 58.7% (81/138) of the isolates with 44.4% (36/81) of these being drug susceptible, while 16% (13/81) were multidrug resistant (MDR). Of the ancestral cluster SCG 1 observed in 19.5% (27/138) of the isolates, 14.8% (4/27) were MDR while 44.4% (12/27) were drug susceptible. SCG 2 formed 5.79% (8/138) of the isolates and 50% (4/8) of these were multidrug resistant (MDR). Spoligotyping subdivided the strains into 45 shared types (n = 125) and 14 orphan strains. The orphan strains were mostly associated with SCG 3a or SCG 1, reflecting the principal SCGs found in the Indian population. SCG 1 and SCG 2 genotypes were concordant with the East African Indian (EAI) and Beijing families respectively. Central Asian (CAS) clade and its sublineages were predominantly associated with SCG 3a. No consistent association was seen between the SCGs and Harlem, T or X clades. The 15 loci MIRU-VNTR typing revealed 123/136 isolates to be unclustered, while 13 isolates were present in 6 clusters of 2-3 isolates each. However, correlating the cluster analysis with patient details did not suggest any evidence of recent transmission. In conclusion, though our study revealed the preponderance of SCG 1 and 3a in the M. tuberculosis population circulating in the region, the diversity of strains highlights the changes occurring within lineages and reemphasizes the importance of cluster investigations in extended studies.
Assuntos
Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Análise por Conglomerados , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Filogenia , Adulto JovemRESUMO
Several reports have elaborated on the role of efflux pumps in drug resistance in Mycobacterium tuberculosis by analysing the mRNA expression profiles. However, there is no uniformity in the subinhibitory concentrations of drugs chosen in these studies. Some investigators studied the expression of efflux pumps under a drug concentration of 1/2 minimum inhibitory concentration (MIC), while others used 1/3, 1/4 or 1/8MIC. The present study was planned to understand the effect of different concentrations of antituberculosis drugs on the expression of efflux pump genes. Log phase culture of the laboratory strain M. tuberculosis H37Rv was exposed to rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) at different drug concentrations (1/2MIC, 1/3MIC and 1/4MIC). The expression of 10 putative efflux pump genes was studied using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). We observed an optimal expression of efflux pumps at higher concentrations of INH; and at lower concentrations of RIF and EMB. However, in the presence of SM, a decreased expression of efflux genes with increasing concentrations of the drug was confounded by a significant reduction in Colony Forming Units (CFU).
Assuntos
Antituberculosos/administração & dosagem , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Contagem de Colônia Microbiana , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana/genética , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Humanos , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Especificidade por Substrato , Regulação para Cima/efeitos dos fármacosRESUMO
Lipoprotein signal peptidase, lspA (Rv1539), is the only known gene in mycobacterial genome for cleaving the signal sequence from prolipoprotein to form mature lipoprotein. It has been implicated in maintaining the virulence of Mycobacterium tuberculosis. The regulation of lspA had not been studied so far. Here, we identify a novel operon lspA-Rv1540 in M. tuberculosis. We detected co-transcription of the open reading frames of lspA-Rv1540 in in-vitro as well as in ex-vivo conditions. Analysis of the sequence upstream to lspA revealed a strong promoter activity that was shown to be induced significantly by surface stress and acidic environment.
Assuntos
Ácidos/farmacologia , Ácido Aspártico Endopeptidases/genética , Proteínas de Bactérias/genética , Transferases Intramoleculares/genética , Mycobacterium tuberculosis/genética , Estresse Fisiológico , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Óperon/efeitos dos fármacos , Óperon/genética , Regiões Promotoras Genéticas , Estresse Mecânico , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Propriedades de Superfície , Transcrição Gênica/efeitos dos fármacosRESUMO
The mce4 operon is reported to be involved in cholesterol utilization and intracellular survival of Mycobacterium tuberculosis (M. tuberculosis). The regulatory mechanism of this important operon was unknown so far. Here we report detection of the promoter region and regulatory factors of the mce4 operon. The in silico analyzed putative promoter region was cloned in promoter selection vector and promoter strength was measured by O-Nitrophenyl-ß-D-galactopyranosidase (ONPG) assay. The transcription start site was determined by 5' Rapid amplification of C terminal end (5'RACE). Surface stress, hypoxia and presence of cholesterol, were found to be stimulatory for mce4 operon promoter induction. Pull down assay coupled with 2D gel electrophoresis resolved many proteins; few prominent spots were processed for identification. MALDI TOF-TOF identified proteins of M. tuberculosis which supported the regulatory function of the identified promoter region and cholesterol utilization of mce4 operon. Since mce4 operon is involved in cholesterol utilization and intracellular survival of M. tuberculosis in the later phase of infection, identification of the promoter sequence as reported in the present communication may facilitate development of effective inhibitors to regulate expression of mce4 operon which may prove to be a good drug target to prevent latency in tuberculosis.
Assuntos
Proteínas de Bactérias/genética , Mycobacterium tuberculosis/genética , Óperon/genética , Proteínas de Bactérias/fisiologia , Sequência de Bases , Colesterol/metabolismo , Colesterol/farmacologia , Simulação por Computador , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estresse Fisiológico/genética , Sítio de Iniciação de Transcrição , Virulência/genética , beta-Galactosidase/metabolismoRESUMO
The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.
Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Técnicas de Diagnóstico Molecular/métodos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Tuberculose/diagnóstico , Adolescente , Adulto , Análise Custo-Benefício , Humanos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Fatores de Tempo , Adulto JovemRESUMO
A large number of potentially pathogenic non-tuberculous mycobacteria (NTM) encountered in the clinical laboratory makes it necessary to identify their species to ensure appropriate treatment. However, labor-intensive conventional methods of speciation are not used in every laboratory, and hence NTM infections are often ignored. Polymerase chain reaction (PCR) restriction analysis (PRA) was applied in this study for early identification and speciation of mycobacterial species on 306 cultures of acid-fast bacilli isolated from patients suspected of suffering from tuberculosis. Mycobacterium tuberculosis was identified in 85.6% of the isolates. The NTM isolated most commonly was Mycobacterium kansasii/gastri group (3.5%), followed by Mycobacterium fortuitum (3.2%). Four of the M. fortuitum were grown from cultures obtained on the same day, but from samples from different patients and were probably laboratory contaminants. Mycobacterium intracellulare and Mycobacterium avium were identified in 2.94% and 2.28% of the isolates, respectively. Three isolates of M. avium and two isolates of M. intracellulare were obtained in repeated cultures from sputum samples of the same patients and were thus pathogenic. A single isolate of Mycobacterium abscessus was obtained from a breast abscess. A rare pathogen Mycobacterium phocaicum was isolated from one patient with epididymitis. However, whether it was the causative agent of epididymitis in this patient remains doubtful. The results of this study highlight the importance of speciation of mycobacteria for appropriate diagnosis and the importance of including molecular assays to augment conventional methods of diagnosis of mycobacterial diseases for rapid identification of NTM so that these potential pathogens are not overlooked in routine diagnostic procedures.
RESUMO
We attempted to apply the PCR-restriction fragment length polymorphism (PCR-RFLP) technique for the early detection and identification of Mycobacterium tuberculosis directly from clinical samples. PCR-RFLP of hsp65 was applied to the DNA extracted directly from sputum samples (n = 226) collected from 226 patients. We could detect and identify M. tuberculosis in 84.5% of the acid-fast bacillus (AFB) smear-positive samples (n = 149) and 11% of the AFB smear-negative samples (n = 18) obtained from patients with clinical and radiological evidence of tuberculosis. Sputum samples (n = 59) obtained from patients suffering from respiratory diseases other than tuberculosis were included as negative controls. To test the sensitivity of the assay, we spiked a smear-negative sample with serial dilutions of H37Rv. The protocol could detect down to 10 organisms/microl. PCR-RFLP was found to be a simple and reproducible method for early detection of M. tuberculosis from sputum samples. The present assay could be used to augment conventional methods of diagnosis of mycobacterial diseases and thus help clinicians to differentiate between M. tuberculosis complex and non-tuberculous mycobacteria directly in clinical samples. The assay could help clinicians to select appropriate chemotherapeutic agents early, which could considerably reduce the morbidity due to mycobacterial diseases.