RESUMO
Drug-resistant tuberculosis (TB) is a global crisis and a threat to health security. Since conventional drug susceptibility testing (DST) takes several weeks, we herein described a molecular assay to rapidly identify multidrug-resistant (MDR) and extensively drug-resistant (XDR) and reveal transmission associated-mutations of Mycobacterium tuberculosis complex (MTBC) isolates in 6 to 7 hours. An array was designed with 12 pairs of primers and 60 single nucleotide polymorphisms of 9 genes: rpoB, katG, inhA, ahpC, embB, rpsL, gyrA, rrs and eis. We assessed the performance of the array using 176 clinical MTBC isolates. The results of culture-based DST were used as the gold standard, the GenoType MTBDRplus and MTBDRsl tests were used for parallel comparison, and gene sequencing was performed to resolve the discordance. The sensitivities and specificities of the array are comparable to those of the MTBDRplus test for resistance to isoniazid (INH) (100.0%, 96.7%) and rifampicin (RIF) (99.4%, 96.7%) and of the MTBDRsl test for resistance to fluoroquinolones (FQs) (100%, 100%) and second-line injectable drugs (SLIDs) (98.3%, 100%). The sensitivities of the array for detecting resistance to ethambutol and streptomycin were 79.3% and 64.9%, respectively. The array has potential as a powerful tool for clinical diagnosis and epidemiological investigations.
Assuntos
Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Mycobacterium tuberculosis/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , DNA Bacteriano/análise , DNA Bacteriano/genética , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are global challenges due to the limited number of effective drugs for treatment. Treatment with less than 4-5 effective drugs might lead to the further emergence of drug resistance and poor clinical outcomes. For better prediction of treatment outcomes, we compared drug-resistance profiles of consecutive clinical MDR Mycobacterium tuberculosis isolates from high- and low-burden settings. This was a retrospective cohort study. We analysed 225 and 229 MDR isolates from Moscow (Russia) and Taiwan, respectively, obtained between 2014 and 2015. Drug susceptibility testing was performed by the Bactec MGIT 960 automated system and the agar proportion method. Detection of resistance-associated mutations in the M. tuberculosis genome was carried out by an array and/or sequencing of selected loci. The principal differences between resistance profiles of MDR isolates in the two countries were the percentages of pre-XDR (40.9% vs. 14.8%) and XDR (34.7% vs. 1.7%) isolates, both of which were significantly higher in Moscow isolates. Forty-eight (33%) of 147 MDR and pre-XDR Russian isolates fall into a group with less than four effective drugs, which accounts for 40% (Nâ¯=â¯120) of these isolates. The other 60% in this group were XDR strains (Nâ¯=â¯72). Consequently, the average number of effective anti-tuberculosis drugs for MDR-TB treatment was lower for Russian isolates (3 vs. 7). Furthermore, a notable percentage (9%) of isolates resistant to kanamycin harboured mutations in the whiB7 locus, which was not detected by molecular tests targeting common mutations in the rrs and eis loci. We found that 98.2% and 45.9% of MDR isolates from Moscow and Taiwan, respectively, were resistant to streptomycin. Molecular tests for detecting resistance to drugs other than rifampicin, isoniazid, fluoroquinolones, and second-line injectable drugs are needed for individualized therapy. The conventional MDR treatment schemes most probably fail in these cases due to the limited number of effective drugs.
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/epidemiologia , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Genes MDR/genética , Genoma Bacteriano/genética , Humanos , Mutação , Estudos Retrospectivos , Federação Russa/epidemiologia , Taiwan/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Non-selenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx or GPx7) is an oxidative stress sensor that modulates the antioxidative activity of its target proteins through intermolecular disulfide bond formation. Given NPGPx's role in protecting cells from oxidative damage, identification of the oxidative stress-induced protein complexes, which forms with key stress factors, may offer novel insight into intracellular reactive oxygen species homeostasis. Here, we show that NPGPx forms a disulfide bond with the translational regulator cytoplasmic polyadenylation element-binding protein 2 (CPEB2) that results in negative regulation of hypoxia-inducible factor 1-alpha (HIF-1α) RNA translation. In NPGPx-proficient cells, high oxidative stress that disrupts this bonding compromises the association of CPEB2 with HIF-1α RNA, leading to elevated HIF-1α RNA translation. NPGPx-deficient cells, in contrast, demonstrate increased HIF-1α RNA translation under normoxia with both impaired induction of HIF-1α synthesis and blunted HIF-1α-programmed transcription following oxidative stress. Together, these results reveal a molecular mechanism for how NPGPx mediates CPEB2-controlled HIF-1α RNA translation in a redox-sensitive manner.
Assuntos
Proteínas de Transporte/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Estresse Oxidativo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Cisteína/análise , Dissulfetos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Ratos , Transcrição GênicaRESUMO
NPGPx is a member of the glutathione peroxidase (GPx) family; however, it lacks GPx enzymatic activity due to the absence of a critical selenocysteine residue, rendering its function an enigma. Here, we show that NPGPx is a newly identified stress sensor that transmits oxidative stress signals by forming the disulfide bond between its Cys57 and Cys86 residues. This oxidized form of NPGPx binds to glucose-regulated protein (GRP)78 and forms covalent bonding intermediates between Cys86 of NPGPx and Cys41/Cys420 of GRP78. Subsequently, the formation of the disulfide bond between Cys41 and Cys420 of GRP78 enhances its chaperone activity. NPGPx-deficient cells display increased reactive oxygen species, accumulated misfolded proteins, and impaired GRP78 chaperone activity. Complete loss of NPGPx in animals causes systemic oxidative stress, increases carcinogenesis, and shortens life span. These results suggest that NPGPx is essential for releasing excessive ER stress by enhancing GRP78 chaperone activity to maintain physiological homeostasis.