RESUMEN
The length and complexity of tuberculosis (TB) therapy, as well as the propensity of Mycobacterium tuberculosis to develop drug resistance, are major barriers to global TB control efforts. M. tuberculosis is known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a mechanism of genetically encoded but rapidly reversible drug tolerance in M. tuberculosis caused by transient frameshift mutations in a homopolymeric tract (HT) of 7 cytosines (7C) in the glpK gene. Inactivating frameshift mutations associated with the 7C HT in glpK produce small colonies that exhibit heritable multidrug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the same glpK HT region. These reversible frameshift mutations in the 7C HT of M. tuberculosis glpK occur in clinical isolates, accumulate in M. tuberculosis-infected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction of dosR and sigH and repression of kstR regulons, similar to that observed in other in vitro models of M. tuberculosis tolerance. These results suggest that GlpK phase variation may contribute to drug tolerance, treatment failure, and relapse in human TB. Drugs effective against phase-variant M. tuberculosis may hasten TB treatment and improve cure rates.
Asunto(s)
Tolerancia a Medicamentos/genética , Glicerol Quinasa/genética , Mycobacterium tuberculosis/genética , Animales , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Femenino , Glicerol Quinasa/metabolismo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/metabolismo , Regiones Promotoras Genéticas/genética , Tuberculosis/microbiologíaRESUMEN
We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosisorn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 µg/ml) produces an SCV with an EMB MIC of 32 µg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 µg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.
Asunto(s)
Farmacorresistencia Bacteriana , Etambutol , Mutación del Sistema de Lectura , Mycobacterium tuberculosis , Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Etambutol/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Pentosiltransferasa/genéticaRESUMEN
The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the iniBAC promoter. In this study, we set out to identify the regulator of the iniBAC operon in Mycobacterium marinum using an unbiased transposon mutagenesis screen in a constitutively iniBAC-expressing mutant background. We obtained multiple mutants in the mce1 locus as well as mutants in an uncharacterized putative transcriptional regulator (MMAR_0612). This latter gene was shown to function as the iniBAC regulator, as overexpression resulted in constitutive iniBAC induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the M. tuberculosis homologue (Rv0339c) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the iniBAC operon. We therefore propose to name this dedicated regulator iniBAC Regulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for iniBAC activation, which could also explain the effect of the mce1 mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose.
Asunto(s)
Adenosina Trifosfatasas/genética , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Trehalosa/metabolismo , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Pared Celular/genética , Pared Celular/metabolismo , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Mutagénesis Insercional , Operón , Regiones Promotoras Genéticas , Transducción de Señal , Transcripción GenéticaRESUMEN
We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.
Asunto(s)
Redes Reguladoras de Genes , Hipoxia/genética , Redes y Vías Metabólicas/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Adaptación Fisiológica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Genómica , Hipoxia/metabolismo , Metabolismo de los Lípidos/genética , Modelos Biológicos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/fisiología , Oxígeno/farmacología , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
The majority of Mycobacterium tuberculosis (Mtb) infections are clinically latent, characterized by drug tolerance and little or no bacterial replication. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Here, we tested the role of serine/threonine phosphorylation in the Mtb response to altered oxygen status, using an in vitro model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of Mtb in reaeration. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. Mtb replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability and gross morphological defects in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the Mtb life cycle-active disease, latency, and reactivation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Oxígeno/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Anaerobiosis , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Carbazoles/farmacología , Alcaloides Indólicos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Oxígeno/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Serina/metabolismo , Transducción de Señal , Treonina/metabolismoRESUMEN
Mycobacterium tuberculosis (MTB) is the causative bacterium of tuberculosis, a disease responsible for over a million deaths worldwide annually with a growing number of strains resistant to antibiotics. The development of better therapeutics would greatly benefit from improved understanding of the mechanisms associated with MTB responses to different genetic and environmental perturbations. Therefore, we expanded a genome-scale regulatory-metabolic model for MTB using the Probabilistic Regulation of Metabolism (PROM) framework. Our model, MTBPROM2.0, represents a substantial knowledge base update and extension of simulation capability. We incorporated a recent ChIP-seq based binding network of 2555 interactions linking to 104 transcription factors (TFs) (representing a 3.5-fold expansion of TF coverage). We integrated this expanded regulatory network with a refined genome-scale metabolic model that can correctly predict growth viability over 69 source metabolite conditions and predict metabolic gene essentiality more accurately than the original model. We used MTBPROM2.0 to simulate the metabolic consequences of knocking out and overexpressing each of the 104 TFs in the model. MTBPROM2.0 improves performance of knockout growth defect predictions compared to the original PROM MTB model, and it can successfully predict growth defects associated with TF overexpression. Moreover, condition-specific models of MTBPROM2.0 successfully predicted synergistic growth consequences of overexpressing the TF whiB4 in the presence of two standard anti-TB drugs. MTBPROM2.0 can screen in silico condition-specific transcription factor perturbations to generate putative targets of interest that can help prioritize future experiments for therapeutic development efforts.
Asunto(s)
Redes Reguladoras de Genes/genética , Genoma Bacteriano/genética , Redes y Vías Metabólicas/genética , Modelos Biológicos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Biología de SistemasRESUMEN
The resilience of Mycobacterium tuberculosis (MTB) is largely due to its ability to effectively counteract and even take advantage of the hostile environments of a host. In order to accelerate the discovery and characterization of these adaptive mechanisms, we have mined a compendium of 2325 publicly available transcriptome profiles of MTB to decipher a predictive, systems-scale gene regulatory network model. The resulting modular organization of 98% of all MTB genes within this regulatory network was rigorously tested using two independently generated datasets: a genome-wide map of 7248 DNA-binding locations for 143 transcription factors (TFs) and global transcriptional consequences of overexpressing 206 TFs. This analysis has discovered specific TFs that mediate conditional co-regulation of genes within 240 modules across 14 distinct environmental contexts. In addition to recapitulating previously characterized regulons, we discovered 454 novel mechanisms for gene regulation during stress, cholesterol utilization and dormancy. Significantly, 183 of these mechanisms act uniquely under conditions experienced during the infection cycle to regulate diverse functions including 23 genes that are essential to host-pathogen interactions. These and other insights underscore the power of a rational, model-driven approach to unearth novel MTB biology that operates under some but not all phases of infection.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Mycobacterium tuberculosis/genética , Colesterol/metabolismo , Perfilación de la Expresión Génica , Genoma Bacteriano , Modelos Genéticos , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative σ-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.
Asunto(s)
Adaptación Biológica/fisiología , Regulación Bacteriana de la Expresión Génica/genética , Mycobacterium tuberculosis/fisiología , Presión Osmótica/fisiología , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Western Blotting , Proteínas Fluorescentes Verdes , Análisis por Micromatrices , Mycobacterium tuberculosis/enzimología , Concentración Osmolar , FosforilaciónRESUMEN
Mycobacterium tuberculosis (MTB) is a highly successful pathogen that infects over a billion people. As with most organisms, MTB adapts to stress by modifying its transcriptional profile. Remodeling of the transcriptome requires both altering the transcription rate and clearing away the existing mRNA through degradation, a process that can be directly regulated in response to stress. To understand better how MTB adapts to the harsh environs of the human host, we performed a global survey of the decay rates of MTB mRNA transcripts. Decay rates were measured for 2139 of the ~4000 MTB genes, which displayed an average half-life of 9.5 min. This is nearly twice the average mRNA half-life of other prokaryotic organisms where these measurements have been made. The transcriptome was further stabilized in response to lowered temperature and hypoxic stress. The generally stable transcriptome described here, and the additional stabilization in response to physiologically relevant stresses, has far-ranging implications for how this pathogen is able to adapt in its human host.
Asunto(s)
Mycobacterium tuberculosis/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , Frío , Semivida , Mycobacterium tuberculosis/metabolismo , Estrés Fisiológico/genética , TranscriptomaRESUMEN
We analyzed whole genome-based transcriptional profiles of Mycobacterium tuberculosis subjected to prolonged hypoxia to guide the discovery of novel potential Ags, by a combined bioinformatic and empirical approach. We analyzed the fold induction of the 100 most highly induced genes at 7 d of hypoxia, as well as transcript abundance, peptide-binding prediction (ProPred) adjusted for population-specific MHC class II allele frequency, and by literature search. Twenty-six candidate genes were selected by this bioinformatic approach and evaluated empirically using IFN-γ and IL-2 ELISPOT using immunodominant Ags (Acr-1, CFP-10, ESAT-6) as references. Twenty-three of twenty-six proteins induced an IFN-γ response in PBMCs of persons with active or latent tuberculosis. Five novel immunodominant proteins-Rv1957, Rv1954c, Rv1955, Rv2022c, and Rv1471-were identified that induced responses similar to CFP-10 and ESAT-6 in both magnitude and frequency. IL-2 responses were of lower magnitude than were those of IFN-γ. Only moderate evidence of infection stage-specific recognition of Ags was observed. Reconciliation of bioinformatic and empirical hierarchies of immunodominance revealed that Ags could be predicted, providing transcriptomic data were combined with peptide-binding prediction adjusted by population-specific MHC class II allele frequency.
Asunto(s)
Biología Computacional/métodos , Hipoxia/genética , Hipoxia/inmunología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/microbiología , Tuberculosis Pulmonar/prevención & control , Adulto , Anciano , Anciano de 80 o más Años , Marcación de Gen , Genoma Bacteriano/genética , Genoma Bacteriano/inmunología , Humanos , Hipoxia/microbiología , Persona de Mediana Edad , Mycobacterium tuberculosis/crecimiento & desarrollo , Valor Predictivo de las Pruebas , Subgrupos de Linfocitos T/metabolismo , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Adulto JovenRESUMEN
M. tuberculosis (MTB) species-specific antigenic determinants of the human T cell response are important for immunodiagnosis and vaccination. As hypoxia is a stimulus in chronic tuberculosis infection, we analyzed transcriptional profiles of MTB subject to 168 hours of hypoxia to test the hypothesis that upregulation by hypoxia might result in gene products being recognized as antigens. We identified upregulation of two region of difference (RD) 11 (Rv2658C and Rv2659c), and one RD2 (Rv1986) absent from commonly used BCG strains. In MTB infected persons, the IL-2 ELISpot response to Rv1986 peptides was several times greater than the corresponding IFN-γ response to the reference immunodominant ESAT-6 or CFP-10 antigens. The IL-2 response was confined to two epitopic regions containing residues 61-80 and 161-180. The biggest population of IL-2 secreting T cells was single cytokine positive central memory T cells. The IL-2 response to live MTB bacilli lacking Rv1986 was significantly lower than the response to wild type or mutant complemented with Rv1986. In addition, the IL-2 response to Rv1986 was significantly lower in HIV-TB co-infected persons than in HIV uninfected persons, and significantly increased during antiretroviral therapy. These findings demonstrate that Rv1986 is an immunodominant target of memory T cells and is therefore of relevance when considering the partial efficacy of currently used BCG vaccines and provide evidence for a clinical trial comparing BCG strains.
Asunto(s)
Hipoxia/inmunología , Epítopos Inmunodominantes/genética , Linfocitos T/inmunología , Activación Transcripcional , Tuberculosis/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Perfilación de la Expresión Génica , Humanos , Epítopos Inmunodominantes/biosíntesis , Memoria Inmunológica , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
Transposon-based strategies provide a powerful and unbiased way to study the bacterial stress response1-8, but these approaches cannot fully capture the complexities of network-based behaviour. Here, we present a network-based genetic screening approach: the transcriptional regulator-induced phenotype (TRIP) screen, which we used to identify previously uncharacterized network adaptations of Mycobacterium tuberculosis to the first-line anti-tuberculosis drug isoniazid (INH). We found regulators that alter INH susceptibility when induced, several of which could not be identified by standard gene disruption approaches. We then focused on a specific regulator, mce3R, which potentiated INH activity when induced. We compared mce3R-regulated genes with baseline INH transcriptional responses and implicated the gene ctpD (Rv1469) as a putative INH effector. Evaluating a ctpD disruption mutant demonstrated a previously unknown role for this gene in INH susceptibility. Integrating TRIP screening with network information can uncover sophisticated molecular response programs.
Asunto(s)
Antituberculosos/farmacología , Redes Reguladoras de Genes/genética , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Transcripción Genética/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Mycobacterium tuberculosis/genética , Estrés Fisiológico/fisiologíaRESUMEN
Tuberculosis (TB) is a heterogeneous disease manifesting in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). Unlike human TB, murine infection results in uniformly high lung bacterial burdens and poorly organized granulomas. To develop a TB model that more closely resembles human disease, we infected mice with an ultra-low dose (ULD) of between 1-3 founding bacteria, reflecting a physiologic inoculum. ULD-infected mice exhibited highly heterogeneous bacterial burdens, well-circumscribed granulomas that shared features with human granulomas, and prolonged Mtb containment with unilateral pulmonary infection in some mice. We identified blood RNA signatures in mice infected with an ULD or a conventional Mtb dose (50-100 CFU) that correlated with lung bacterial burdens and predicted Mtb infection outcomes across species, including risk of progression to active TB in humans. Overall, these findings highlight the potential of the murine TB model and show that ULD infection recapitulates key features of human TB.
Asunto(s)
Modelos Animales de Enfermedad , Mycobacterium tuberculosis/patogenicidad , Tuberculosis Pulmonar , Animales , Carga Bacteriana , Biomarcadores/sangre , Progresión de la Enfermedad , Femenino , Granuloma/patología , Humanos , Pulmón/microbiología , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/crecimiento & desarrollo , RNA-Seq , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
Tuberculosis is a massive public health problem on a global scale and the success of Mycobacterium tuberculosis is linked to its ability to persist within humans for long periods without causing any overt disease symptoms. Hypoxia is predicted to be a key host-induced stress limiting growth of the pathogen in vivo. However, multiple studies in vitro and in vivo indicate that M. tuberculosis adapts to oxygen limitation by entering into a metabolically altered state, while awaiting the opportunity to reactivate. Molecular signatures of bacteria adapted to hypoxia in vitro are accumulating, although correlations to human disease are only now being established. Similarly, defining the mechanisms that control this adaptation is an active area of research. In this review we discuss the historical precedents linking hypoxia and latency, and the gathering knowledge of M. tuberculosis hypoxic responses. We also examine the role of these responses in tuberculosis latency, and identify promising avenues for future studies.
Asunto(s)
Mycobacterium tuberculosis/metabolismo , Oxígeno/metabolismo , Tuberculosis/microbiología , Anaerobiosis , Animales , Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica , Humanos , Hipoxia/metabolismo , Hipoxia/microbiología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Proteínas Quinasas/fisiología , Tuberculosis/metabolismo , VirulenciaRESUMEN
Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths each year. Current assays of antimicrobial resistance need knowledge of mutations that confer drug resistance, or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. ODELAM enables rapid quantitative measures of growth kinetics in as little as 30 hrs under a wide variety of environmental conditions. We demonstrate ODELAM's utility by identifying ofloxacin resistance in cultured clinical isolates of Mtb and benchmark its performance with standard minimum inhibitory concentration (MIC) assays. ODELAM identified ofloxacin heteroresistance and the presence of drug resistant colony forming units (CFUs) at 1 per 1000 CFUs in as little as 48 hrs. ODELAM is a powerful new tool that can rapidly evaluate Mtb drug resistance in a laboratory setting.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad Microbiana , Microscopía por Video , Mycobacterium tuberculosis/efectos de los fármacos , Ofloxacino/farmacología , Imagen de Lapso de Tiempo , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Recuento de Colonia Microbiana , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Cinética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Flujo de TrabajoRESUMEN
The 60 kDa heat shock proteins, also known as Cpn60s (GroELs) are components of the essential protein folding machinery of the cell, but are also dominant antigens in many infectious diseases. Although generally essential for cellular survival, in some organisms such as Mycobacterium tuberculosis, one or more paralogous Cpn60s are known to be dispensable. In M. tuberculosis, Cpn60.2 (GroEL2) is essential for cell survival, but the biological role of the non-essential Cpn60.1 (GroEL1) is still elusive. To understand the relevance of Cpn60.1 (GroEL1) in M. tuberculosis physiology, detailed transcriptomic analyses for the wild type H37Rv and cpn60.1 knockout (groEL1-KO) were performed under in vitro stress conditions: stationary phase, cold shock, low aeration, mild cold shock and low pH. Additionally, the survival of the groEL1-KO was assessed in macrophages at multiplicity of infection (MOI) of 1:1 and 1:5. We observed that survival under low aeration was significantly compromised in the groEL1-KO. Further, the gene expression analyses under low aeration showed change in expression of several key virulence factors like two component system PhoP/R and MprA/B, sigma factors SigM and C and adversely affected known hypoxia response regulators Rv0081, Rv0023 and DosR. Our work is therefore suggestive of an important role of Cpn60.1 (GroEL1) for survival under low aeration by affecting the expression of genes known for hypoxia response.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chaperonina 60/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/genética , Chaperonina 60/genética , Frío , Respuesta al Choque por Frío , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Técnicas de Silenciamiento del Gen , Concentración de Iones de Hidrógeno , Viabilidad Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/patogenicidad , Oxígeno/metabolismo , Transcripción Genética , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Mycobacterium tuberculosis (MTB) is a pathogenic bacterium responsible for 12 million active cases of tuberculosis (TB) worldwide. The complexity and critical regulatory components of MTB pathogenicity are still poorly understood despite extensive research efforts. In this study, we constructed the first systems-scale map of transcription factor (TF) binding sites and their regulatory target proteins in MTB. We constructed FLAG-tagged overexpression constructs for 206 TFs in MTB, used ChIP-seq to identify genome-wide binding events and surveyed global transcriptomic changes for each overexpressed TF. Here we present data for the most comprehensive map of MTB gene regulation to date. We also define elaborate quality control measures, extensive filtering steps, and the gene-level overlap between ChIP-seq and microarray datasets. Further, we describe the use of TF overexpression datasets to validate a global gene regulatory network model of MTB and describe an online source to explore the datasets.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Perfilación de la Expresión Génica , Modelos Genéticos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20-30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 'dormant' DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression.
Asunto(s)
Proteínas Bacterianas/química , ADN Bacteriano/química , Proteínas de Unión al ADN/química , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/química , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Inmunoprecipitación de Cromatina , Biología Computacional , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Vectores Genéticos , Estudio de Asociación del Genoma Completo , Mycobacterium tuberculosis/genética , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Curva ROC , Proteínas Recombinantes/química , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
BACKGROUND: Mycobacterium tuberculosis senses and responds to the shifting and hostile landscape of the host. To characterize the underlying intertwined gene regulatory network governed by approximately 200 transcription factors of M. tuberculosis, we have assayed the global transcriptional consequences of overexpressing each transcription factor from an inducible promoter. RESULTS: We cloned and overexpressed 206 transcription factors in M. tuberculosis to identify the regulatory signature of each. We identified 9,335 regulatory consequences of overexpressing each of 183 transcription factors, providing evidence of regulation for 70% of the M. tuberculosis genome. These transcriptional signatures agree well with previously described M. tuberculosis regulons. The number of genes differentially regulated by transcription factor overexpression varied from hundreds of genes to none, with the majority of expression changes repressing basal transcription. Exploring the global transcriptional maps of transcription factor overexpressing (TFOE) strains, we predicted and validated the phenotype of a regulator that reduces susceptibility to a first line anti-tubercular drug, isoniazid. We also combined the TFOE data with an existing model of M. tuberculosis metabolism to predict the growth rates of individual TFOE strains with high fidelity. CONCLUSION: This work has led to a systems-level framework describing the transcriptome of a devastating bacterial pathogen, characterized the transcriptional influence of nearly all individual transcription factors in M. tuberculosis, and demonstrated the utility of this resource. These results will stimulate additional systems-level and hypothesis-driven efforts to understand M. tuberculosis adaptations that promote disease.