RESUMEN
The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M. tuberculosis genome associated with resistance to anti-tuberculosis drugs. We ascertained consensus on the use of the results of molecular DST for clinical treatment decisions in TB patients. This document has been developed by TBNET and RESIST-TB groups to reach a consensus about reporting standards in the clinical use of molecular DST results. Review of the available literature and the search for evidence included hand-searching journals and searching electronic databases. The panel identified single nucleotide mutations in genomic regions of M. tuberculosis coding for katG, inhA, rpoB, embB, rrs, rpsL and gyrA that are likely related to drug resistance in vivo. Identification of any of these mutations in clinical isolates of M. tuberculosis has implications for the management of TB patients, pending the results of in vitro DST. However, false-positive and false-negative results in detecting resistance-associated mutations in drugs for which there is poor or unproven correlation between phenotypic and clinical drug resistance complicate the interpretation. Reports of molecular DST results should therefore include specific information on the mutations identified and provide guidance for clinicians on interpretation and on the choice of the appropriate initial drug regimen.
Asunto(s)
Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/genética , Antituberculosos/farmacología , Conferencias de Consenso como Asunto , Humanos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/epidemiologíaRESUMEN
Here we study links between aminoglycoside-induced mistranslation, protein misfolding and neuropathy. We demonstrate that aminoglycosides induce misreading in mammalian cells and assess endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways. Genome-wide transcriptome and proteome analyses revealed upregulation of genes related to protein folding and degradation. Quantitative PCR confirmed induction of UPR markers including C/EBP homologous protein, glucose-regulated protein 94, binding immunoglobulin protein and X-box binding protein-1 (XBP1) mRNA splicing, which is crucial for UPR activation. We studied the effect of a compromised UPR on aminoglycoside ototoxicity in haploinsufficient XBP1 (XBP1(+/-)) mice. Intra-tympanic aminoglycoside treatment caused high-frequency hearing loss in XBP1(+/-) mice but not in wild-type littermates. Densities of spiral ganglion cells and synaptic ribbons were decreased in gentamicin-treated XBP1(+/-) mice, while sensory cells were preserved. Co-injection of the chemical chaperone tauroursodeoxycholic acid attenuated hearing loss. These results suggest that aminoglycoside-induced ER stress and cell death in spiral ganglion neurons is mitigated by XBP1, masking aminoglycoside neurotoxicity at the organismal level.
Asunto(s)
Proteínas de Unión al ADN/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Gentamicinas/farmacología , Pérdida Auditiva de Alta Frecuencia , Ácido Tauroquenodesoxicólico/farmacología , Factores de Transcripción/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/patología , Femenino , Células HEK293 , Células Ciliadas Auditivas/patología , Pérdida Auditiva de Alta Frecuencia/inducido químicamente , Pérdida Auditiva de Alta Frecuencia/genética , Pérdida Auditiva de Alta Frecuencia/patología , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos CBA , Neuronas/patología , Biosíntesis de Proteínas/efectos de los fármacos , Pliegue de Proteína , Deficiencias en la Proteostasis , Empalme del ARN/genética , Factores de Transcripción del Factor Regulador X , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/efectos de los fármacos , Ganglio Espiral de la Cóclea/patología , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiología , Proteína 1 de Unión a la X-BoxRESUMEN
The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.
Asunto(s)
Respuesta al Choque Térmico , Macrófagos/microbiología , Mycobacterium tuberculosis/efectos de los fármacos , Estrés Oxidativo , Peroxidasas/genética , Ácido Peroxinitroso/farmacología , Línea Celular , Farmacorresistencia Bacteriana , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/patogenicidad , PeroxirredoxinasRESUMEN
Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain M. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulator oxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulent M. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome of M. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the ahpC::Km(r) mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.
Asunto(s)
Antioxidantes/metabolismo , Mycobacterium tuberculosis/enzimología , Estrés Oxidativo , Peroxidasas/metabolismo , Animales , Femenino , Perfilación de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/patogenicidad , Peroxidasas/genética , Peroxirredoxinas , Recombinación Genética , VirulenciaRESUMEN
Stimulation of the human epithelial-like cell line, HeLa, with interferon gamma (IFN-gamma) induces steady-state levels of HLA-DR alpha mRNA. Using a sensitive RNase-mapping procedure, we detect induced HLA-DR alpha mRNA as early as 8 hr after IFN-gamma treatment; maximal accumulation occurs by 48 hr. Treatment with the protein synthesis inhibitor, cycloheximide, abolishes the IFN-gamma-induced accumulation of HLA-DR alpha mRNA, indicating that de novo synthesis of a trans-acting protein factor is required for induction of this major histocompatibility complex class II gene. Nuclear run-off transcription assays revealed that IFN-gamma acts by directly stimulating the transcription rate of HLA-DR alpha. Similarly, IFN-gamma increased the transcription rate of the class I HLA-A2-encoding gene as well as that of the human invariant chain gene. IFN-gamma-induced transcription of HLA-DR alpha and of the invariant chain gene was blocked by treatment with cycloheximide, but IFN-gamma-induced transcription of HLA-A2 was unaffected. Our findings show that transcriptional induction of HLA-DR alpha and the invariant chain gene by IFN-gamma requires the action of an unidentified trans-acting protein.