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For many diseases there are delays in diagnosis due to a lack of objective biomarkers for disease onset. Here, in 41,931 individuals from the United Kingdom Biobank Pharma Proteomics Project, we integrated measurements of ~3,000 plasma proteins with clinical information to derive sparse prediction models for the 10-year incidence of 218 common and rare diseases (81-6,038 cases). We then compared prediction models developed using proteomic data with models developed using either basic clinical information alone or clinical information combined with data from 37 clinical assays. The predictive performance of sparse models including as few as 5 to 20 proteins was superior to the performance of models developed using basic clinical information for 67 pathologically diverse diseases (median delta C-index = 0.07; range = 0.02-0.31). Sparse protein models further outperformed models developed using basic information combined with clinical assay data for 52 diseases, including multiple myeloma, non-Hodgkin lymphoma, motor neuron disease, pulmonary fibrosis and dilated cardiomyopathy. For multiple myeloma, single-cell RNA sequencing from bone marrow in newly diagnosed patients showed that four of the five predictor proteins were expressed specifically in plasma cells, consistent with the strong predictive power of these proteins. External replication of sparse protein models in the EPIC-Norfolk study showed good generalizability for prediction of the six diseases tested. These findings show that sparse plasma protein signatures, including both disease-specific proteins and protein predictors shared across several diseases, offer clinically useful prediction of common and rare diseases.
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SNPs in the FAM13A locus are amongst the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases, however the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: 'long' and 'short', but their functions remain unknown, partly due to a lack of isoform conservation in mice. We performed in-depth characterisation of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate RhoGAP activity of this domain. In Xenopus laevis, which conserve the long isoform, Fam13a-deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long isoform deficiency did not affect multiciliogenesis but reduced cilia co-ordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform co-ordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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BACKGROUND: Chronic sputum production impacts on quality of life and is a feature of many respiratory diseases. Identification of the genetic variants associated with chronic sputum production in a disease agnostic sample could improve understanding of its causes and identify new molecular targets for treatment. METHODS: We conducted a genome-wide association study (GWAS) of chronic sputum production in UK Biobank. Signals meeting genome-wide significance (p<5×10-8) were investigated in additional independent studies, were fine-mapped and putative causal genes identified by gene expression analysis. GWASs of respiratory traits were interrogated to identify whether the signals were driven by existing respiratory disease among the cases and variants were further investigated for wider pleiotropic effects using phenome-wide association studies (PheWASs). RESULTS: From a GWAS of 9714 cases and 48 471 controls, we identified six novel genome-wide significant signals for chronic sputum production including signals in the human leukocyte antigen (HLA) locus, chromosome 11 mucin locus (containing MUC2, MUC5AC and MUC5B) and FUT2 locus. The four common variant associations were supported by independent studies with a combined sample size of up to 2203 cases and 17 627 controls. The mucin locus signal had previously been reported for association with moderate-to-severe asthma. The HLA signal was fine-mapped to an amino acid change of threonine to arginine (frequency 36.8%) in HLA-DRB1 (HLA-DRB1*03:147). The signal near FUT2 was associated with expression of several genes including FUT2, for which the direction of effect was tissue dependent. Our PheWAS identified a wide range of associations including blood cell traits, liver biomarkers, infections, gastrointestinal and thyroid-associated diseases, and respiratory disease. CONCLUSIONS: Novel signals at the FUT2 and mucin loci suggest that mucin fucosylation may be a driver of chronic sputum production even in the absence of diagnosed respiratory disease and provide genetic support for this pathway as a target for therapeutic intervention.
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Estudio de Asociación del Genoma Completo , Esputo , Humanos , Esputo/metabolismo , Cadenas HLA-DRB1 , Calidad de Vida , Proteínas , Mucinas , Moco/metabolismo , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido SimpleRESUMEN
Geographic atrophy (GA) is an advanced form of dry age-related macular degeneration (AMD), in which local inflammation and hyperactivity of the complement pathway have been implicated in its pathophysiology. This study explores whether any surrogate biomarkers are specifically associated with GA. Plasma from subjects with GA, intermediate dry AMD and non-AMD control were evaluated in 2 cohorts. Cohort 1 was assayed in a 320-analyte Luminex library. Statistical analysis was performed using non-parametric and parametric methods (Kruskal-Wallis, principal component analysis, partial least squares and multivariate analysis of variance (MANOVA) and univariate ANCOVAs). Bioinformatic analysis was conducted and identified connections to the amyloid pathway. Statistically significant biomarkers identified in Cohort 1 were then re-evaluated in Cohort 2 using individual ELISA and multiplexing. Of 320 analytes in Cohort 1, 273 were rendered measurable, of which 56 were identified as changing. Among these markers, 40 were identified in univariate ANCOVAs. Serum amyloid precursor protein (sAPP) was analyzed by a separate ELISA and included in further analyses. The 40 biomarkers, sAPP and amyloid-ß (Aß) (1-42) (included for comparison) were evaluated in Cohort 2. This resulted in 11 statistically significant biomarkers, including sAPP and Aß(1-40), but not Aß(1-42). Other biomarkers identified included serum proteases- tissue plasminogen activator, tumor-associated trypsinogen inhibitor, matrix metalloproteinases 7 and 9, and non-proteases- insulin-like growth factor binding protein 6, AXL receptor tyrosine kinase, omentin, pentraxin-3 and osteopontin. Findings suggest that there is a preferential processing of APP to Aß(1-40) over Aß(1-42), and a potential role for the carboxylase activity of the γ-secretase protein, which preferentially splices sAPPß to Aß(1-40). Other markers are associated with the breakdown and remodeling of the extracellular matrix, and loss of homeostasis, possibly within the photoreceptor-retinal pigment epithelium-choriocapillaris complex. These data suggest novel disease pathways associated with GA pathogenesis and could provide potential novel targets for treatment of GA.
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Péptidos beta-Amiloides/metabolismo , Atrofia Geográfica/sangre , Degeneración Macular/complicaciones , Epitelio Pigmentado de la Retina/patología , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/metabolismo , Estudios de Cohortes , Biología Computacional , Femenino , Fondo de Ojo , Atrofia Geográfica/diagnóstico , Atrofia Geográfica/etiología , Atrofia Geográfica/patología , Humanos , Degeneración Macular/sangre , Degeneración Macular/patología , Masculino , Persona de Mediana Edad , Imagen Óptica , Transducción de Señal , Activador de Tejido PlasminógenoRESUMEN
Genome editing is a tool that has many applications, including the validation of potential drug targets. However, performing genome editing in low-passage primary human cells with the greatest physiological relevance is notoriously difficult. High editing efficiency is desired because it enables gene knockouts (KO) to be generated in bulk cellular populations and circumvents the problem of having to generate clonal cell isolates. Here, we describe a single-step workflow enabling >90% KO generation in primary human lung fibroblasts via CRISPR ribonucleoprotein delivery in the absence of antibiotic selection or clonal expansion. As proof of concept, we edited two SMAD family members and demonstrated that in response to transforming growth factor beta, SMAD3, but not SMAD2, is critical for deposition of type I collagen in the fibrotic response. The optimization of this workflow can be readily transferred to other primary cell types.
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Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Cultivo Primario de Células/métodos , Sistemas CRISPR-Cas/genética , Técnicas de Cultivo de Célula/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Fibroblastos/metabolismo , Ingeniería Genética/métodos , Vectores Genéticos , Humanos , Pulmón/patología , Proteínas Smad/genética , Proteínas Smad/metabolismoRESUMEN
BACKGROUND: The Centre for Therapeutic Target Validation (CTTV - https://www.targetvalidation.org/) was established to generate therapeutic target evidence from genome-scale experiments and analyses. CTTV aims to support the validity of therapeutic targets by integrating existing and newly-generated data. Data integration has been achieved in some resources by mapping metadata such as disease and phenotypes to the Experimental Factor Ontology (EFO). Additionally, the relationship between ontology descriptions of rare and common diseases and their phenotypes can offer insights into shared biological mechanisms and potential drug targets. Ontologies are not ideal for representing the sometimes associated type relationship required. This work addresses two challenges; annotation of diverse big data, and representation of complex, sometimes associated relationships between concepts. METHODS: Semantic mapping uses a combination of custom scripting, our annotation tool 'Zooma', and expert curation. Disease-phenotype associations were generated using literature mining on Europe PubMed Central abstracts, which were manually verified by experts for validity. Representation of the disease-phenotype association was achieved by the Ontology of Biomedical AssociatioN (OBAN), a generic association representation model. OBAN represents associations between a subject and object i.e., disease and its associated phenotypes and the source of evidence for that association. The indirect disease-to-disease associations are exposed through shared phenotypes. This was applied to the use case of linking rare to common diseases at the CTTV. RESULTS: EFO yields an average of over 80% of mapping coverage in all data sources. A 42% precision is obtained from the manual verification of the text-mined disease-phenotype associations. This results in 1452 and 2810 disease-phenotype pairs for IBD and autoimmune disease and contributes towards 11,338 rare diseases associations (merged with existing published work [Am J Hum Genet 97:111-24, 2015]). An OBAN result file is downloadable at http://sourceforge.net/p/efo/code/HEAD/tree/trunk/src/efoassociations/. Twenty common diseases are linked to 85 rare diseases by shared phenotypes. A generalizable OBAN model for association representation is presented in this study. CONCLUSIONS: Here we present solutions to large-scale annotation-ontology mapping in the CTTV knowledge base, a process for disease-phenotype mining, and propose a generic association model, 'OBAN', as a means to integrate disease using shared phenotypes. AVAILABILITY: EFO is released monthly and available for download at http://www.ebi.ac.uk/efo/.
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Ontologías Biológicas , Terapia Molecular Dirigida , Fenotipo , Enfermedades Raras/tratamiento farmacológico , Minería de Datos , Bases de Datos Factuales , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Reproducibilidad de los ResultadosRESUMEN
The p38 mitogen-activated protein kinase (MAPK) intracellular signaling pathway responds to a variety of extracellular stimuli, including cytokines, Toll-like receptor agonists, and components of cigarette smoke to influence the expression of proinflammatory mediators. Activation of p38 MAPK is increased within the lungs of chronic obstructive pulmonary disease (COPD) patients. In clinical trials, treatment of COPD patients with p38 MAPK inhibitors has been shown to reduce systemic inflammation plasma biomarkers C-reactive protein (CRP) and fibrinogen. As CRP and fibrinogen have been associated with poor clinical outcomes in COPD patients, such as mortality, exacerbation, and hospitalization, we analyzed gene expression data from COPD subjects treated with dilmapimod with the aim of understanding the effects of p38 MAPK inhibition on the inflammatory genome of immune cells within the systemic circulation. Whole blood and induced sputum samples were used to measure mRNA levels by gene array and PCR. Pathway and network analysis showed STAT1, MMP-9, CAV1, and IL-1ß as genes regulated by dilmapimod that could also influence fibrinogen levels, while only IL-1ß was identified as a gene regulated by dilmapimod that could influence CRP levels. This suggests that p38 MAPK inhibits specific inflammatory pathways, leading to to differential effects on CRP and fibrinogen levels in COPD patients.
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Wide variation in glucocorticoid (Gc) sensitivity exists between individuals which may influence susceptibility to, and treatment response of, inflammatory diseases. To determine a genetic fingerprint of Gc sensitivity 100 healthy human volunteers were polarized into the 10% most Gc-sensitive and 10% most Gc-resistant following a low dose dexamethasone (0.25 mg) suppression test. Gene expression profiling of primary lymphocytes identified the 98 most significantly Gc regulated genes. These genes were used to build a subnetwork of Gc signaling, with 54 genes mapping as nodes, and 6 non-Gc regulated genes inferred as signaling nodes. Twenty four of the 98 genes showed a difference in Gc response in vitro dependent on the Gc sensitivity of their donor individuals in vivo. A predictive model was built using both partial least squares discriminate analysis and support vector machines that predicted donor glucocorticoid sensitivity with 87% accuracy. Discriminating genes included bone morphogenetic protein receptor, type II (BMPRII). Transfection studies showed that BMPRII modulated Gc action. These studies reveal a broad base of gene expression that predicts Gc sensitivity and determine a Gc signaling network in human primary T lymphocytes. Furthermore, this combined gene profiling, and functional analysis approach has identified BMPRII as a modulator of Gc signaling.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Adulto , Análisis por Conglomerados , Dexametasona/farmacología , Femenino , Humanos , Análisis de los Mínimos Cuadrados , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The Beijing strain family has often been associated with tuberculosis (TB) outbreaks and drug resistance worldwide. In this study the authors have compared the protein expression and antigen recognition profiles of a local Beijing strain with a less prevalent clinical isolate belonging to the family 23 strain lineage, and the laboratory strain Mycobacterium tuberculosis H37Rv. Using two-dimensional electrophoresis, liquid chromatography tandem mass spectrometry and Western blot analysis several proteins were identified as quantitatively increased or decreased in both clinical strains compared to H37Rv. Remarkably, the Beijing strain showed increased expression of alpha-crystallin and decreased expression of Hsp65, PstS1, and the 47 kDa protein compared to the other clinical strain and H37Rv. One- and two-dimensional Western blot analysis of antigens expressed by the three strains, using plasma from TB patients, confirmed differential antigen expression by strains and patient-to-patient variation in humoral immunity. These observed protein differences could aid the elucidation of mechanisms underlying the success of the Beijing strain family, measured by global dissemination, compared to other M. tuberculosis strains.
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Proteínas Bacterianas/análisis , Mycobacterium tuberculosis/química , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Monoclonales , Antígenos Bacterianos/análisis , Proteínas Bacterianas/inmunología , China , Electroforesis en Gel Bidimensional , Humanos , Ratones , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/aislamiento & purificación , Proteoma/análisis , Proteoma/inmunología , Especificidad de la Especie , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiologíaRESUMEN
The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.
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Regulación Bacteriana de la Expresión Génica , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Membrana Celular/química , Clonación Molecular , Citoplasma/química , Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Genes Bacterianos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Mutagénesis Insercional , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Tuberculosis/mortalidad , Virulencia/genéticaRESUMEN
Genomic technologies have the potential to greatly increase the efficiency of the drug development process. As part of our tuberculosis drug discovery program, we used DNA microarray technology to profile drug-induced effects in Mycobacterium tuberculosis. Expression profiles of M. tuberculosis treated with compounds that inhibit key metabolic pathways are required as references for the assessment of novel antimycobacterial agents. We have studied the response of M. tuberculosis to treatment with the mycolic acid biosynthesis inhibitors isoniazid, thiolactomycin, and triclosan. Thiolactomycin targets the beta-ketoacyl-acyl carrier protein (ACP) synthases KasA and KasB, while triclosan inhibits the enoyl-ACP reductase InhA. However, controversy surrounds the precise mode of action of isoniazid, with both InhA and KasA having been proposed as the primary target. We have shown that although the global response profiles of isoniazid and thiolactomycin are more closely related to each other than to that of triclosan, there are differences that distinguish the mode of action of these two drugs. In addition, we have identified two groups of genes, possibly forming efflux and detoxification systems, through which M. tuberculosis may limit the effects of triclosan. We have developed a mathematical model, based on the expression of 21 genes, which is able to perfectly discriminate between isoniazid-, thiolactomycin-, or triclosan-treated M. tuberculosis. This model is likely to prove invaluable as a tool to improve the efficiency of our drug development programs by providing a means to rapidly confirm the mode of action of thiolactomycin analogues or novel InhA inhibitors as well as helping to translate enzyme activity into whole-cell activity.
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Antibacterianos/farmacología , Antiinfecciosos Locales/farmacología , Antituberculosos/farmacología , Regulación Bacteriana de la Expresión Génica , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tiofenos/farmacología , Triclosán/farmacología , ADN Complementario/biosíntesis , ADN Complementario/genética , Regulación hacia Abajo , Análisis de Secuencia por Matrices de Oligonucleótidos , Operón , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3-regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3-regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.
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Proteínas Bacterianas/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Fosfotransferasas/genética , Animales , Línea Celular , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genes Reguladores , Humanos , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Mycobacterium tuberculosis/crecimiento & desarrollo , Regiones Promotoras Genéticas , Regulón , Tuberculosis/microbiología , Virulencia/genéticaRESUMEN
Different phenotypes are displayed by Mycobacterium tuberculosis (M. tuberculosis) strains, fuelling speculation that certain strains are "hypervirulent" and able to evade host defenses better than others. Furthermore, differential antigen expression by M. tuberculosis strains may explain why certain patients are susceptible to a repeat episode of tuberculosis. The objective of this study was to compare protein expression by M. tuberculosis H37Rv and clinical isolates in order to determine whether differential protein expression contributes to the different phenotypes expressed by these strains. Expression of alpha-crystallin, the antigen 85 complex, PstS-1, L-alanine dehydrogenase and the 65 kDa antigen was analysed by Western blotting and enzyme-linked immunosorbent assays, using mouse monoclonal antibodies. We found no significant difference in the growth rate of the M. tuberculosis strains in vitro, and although M. tuberculosis protein expression showed phase variation during growth, expression seemed to be qualitatively, but not quantitatively, conserved in the strains investigated. These results have potentially important implications for vaccine development and serodiagnosis.
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Proteínas Bacterianas/biosíntesis , Mycobacterium tuberculosis/metabolismo , Animales , Anticuerpos Monoclonales/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Western Blotting , Dermatoglifia del ADN/métodos , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Regulación Bacteriana de la Expresión Génica , Genotipo , Humanos , Ratones , Peso Molecular , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Esputo/microbiologíaRESUMEN
We have used RNA-RNA in situ hybridization to detect the expression of several Mycobacterium tuberculosis genes in tuberculous granulomas in lung tissue sections from tuberculosis patients. The M. tuberculosis genes chosen fall into two classes. Four genes (icl, narX, and Rv2557 and Rv2558) have been implicated in the persistence of the bacterium in the host, and two genes (iniB and kasA) are upregulated in response to isoniazid exposure. Both necrotic and nonnecrotic granulomas were identified in all of the patients. Necrotic granulomas were divided into three zones: an outer lymphocyte cuff containing lymphocytes and macrophages, a transition zone consisting of necrotic material interspersed with macrophages, and a central acellular necrotic region. Transcripts of all of the genes studied were found in nonnecrotic granulomas and in the lymphocyte cuff of necrotic granulomas. Mycobacterial gene expression was associated with CD68-positive myeloid cells. Rv2557 and/or its homologue Rv2558, kasA, and iniB were expressed within the transition zone of necrotic granulomas, whereas icl and narX transcripts were absent from this area. There was no evidence of transcription of any of the genes examined in the central necrotic region, although mycobacterial DNA was present. The differential expression of genes within granulomas demonstrates that M. tuberculosis exists in a variety of metabolic states and may be indicative of the response to different microenvironments. These observations confirm that genes identified in models of persistence or in response to drug treatment in vitro are expressed in the human host.
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Perfilación de la Expresión Génica , Granuloma del Sistema Respiratorio/microbiología , Mycobacterium tuberculosis/genética , ARN Mensajero/análisis , Tuberculosis Pulmonar/microbiología , Adolescente , Adulto , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Femenino , Granuloma del Sistema Respiratorio/metabolismo , Granuloma del Sistema Respiratorio/patología , Humanos , Hibridación in Situ , Masculino , Necrosis , Tuberculosis Pulmonar/metabolismo , Tuberculosis Pulmonar/patologíaRESUMEN
A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.
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Elementos Transponibles de ADN/genética , Biblioteca de Genes , Mutagénesis Insercional , Mycobacterium tuberculosis/patogenicidad , Tuberculosis Pulmonar/microbiología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones SCID , Mutación , Mycobacterium tuberculosis/genética , Sistemas de Lectura Abierta/genética , VirulenciaRESUMEN
The availability of the complete genome sequence of Mycobacterium tuberculosis has revolutionised many areas of tuberculosis research and facilitated functional genomics studies. Most notably, transcriptomics and proteomics have become important tools in gaining increased understanding of the biology of M. tuberculosis and offer the promise of being able to deliver novel drug targets and vaccine candidates.
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Genoma , Proteoma , Tuberculosis/tratamiento farmacológico , Tuberculosis/genética , Electroforesis en Gel Bidimensional , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Programas Informáticos , VacunasRESUMEN
The search for new TB drugs that rapidly and effectively sterilize the tissues and are thus able to shorten the duration of chemotherapy from the current 6 months has been hampered by a lack of understanding of the metabolism of the bacterium when in a 'persistent' or latent form. Little is known about the condition in which the bacilli survive, although laboratory models have shown that Mycobacterium tuberculosis can exist in a non-growing, drug-resistant state that may mimic persistence in vivo. Using nutrient starvation, we have established a model in which M. tuberculosis arrests growth, decreases its respiration rate and is resistant to isoniazid, rifampicin and metronidazole. We have used microarray and proteome analysis to investigate the response of M. tuberculosis to nutrient starvation. Proteome analysis of 6-week-starved cultures revealed the induction of several proteins. Microarray analysis enabled us to monitor gene expression during adaptation to nutrient starvation and confirmed the changes seen at the protein level. This has provided evidence for slowdown of the transcription apparatus, energy metabolism, lipid biosynthesis and cell division in addition to induction of the stringent response and several other genes that may play a role in maintaining long-term survival within the host. Thus, we have generated a model with which we can search for agents active against persistent M. tuberculosis and revealed a number of potential targets expressed under these conditions.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/fisiología , Adaptación Fisiológica , Membrana Celular/metabolismo , Electroforesis en Gel Bidimensional , Metabolismo Energético , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Lípidos/biosíntesis , Modelos Biológicos , Mycobacterium tuberculosis/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxígeno/metabolismo , Biosíntesis de Proteínas , ARN Bacteriano/biosíntesis , Ribosomas/metabolismo , Rifampin/farmacología , Transcripción GenéticaRESUMEN
Mycobacterium tuberculosis resides within the macrophages of the host, but the molecular and cellular mechanisms of survival are poorly understood. Recent evidence suggests that the attenuated vaccine strain Mycobacterium bovis BCG is both a deletion and regulatory mutant, yet retains both its immunoprotective and intra-macrophage survival potential. In an attempt to define M. bovis BCG genes expressed during interaction with macrophages, the patterns of protein synthesis were examined by both one- and two-dimensional gel electrophoresis of BCG while inside the human leukaemic macrophage cell line THP-1. This study demonstrated that BCG expresses proteins while resident inside macrophages that are not expressed during in vitro growth in culture media or under conditions of heat shock. Western blotting analysis revealed that some of the differentially expressed proteins are specifically recognized by human M. tuberculosis-infected sera. Proteome analysis by two-dimensional electrophoresis and MS identified six abundant proteins that showed increased expression inside macrophages: 16 kDa alpha-crystallin (HspX), GroEL-1 and GroEL-2, a 31.7 kDa hypothetical protein (Rv2623), InhA and elongation factor Tu (Tuf). Identification of proteins by such a strategy will help elucidate the molecular basis of the attenuation and the vaccine potential of BCG, and may provide antigens that distinguish infection with M. tuberculosis from vaccination with BCG.
Asunto(s)
Proteínas Bacterianas/metabolismo , Macrófagos/microbiología , Mycobacterium bovis/metabolismo , Fagocitosis , Proteoma , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Western Blotting , Electroforesis en Gel Bidimensional , Humanos , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiologíaRESUMEN
The genome sequences of two virulent strains of Mycobacterium tuberculosis (H37Rv and CDC 1551) are now available. CDC 1551 is a recent clinical isolate and H37Rv is a commonly used lab strain which has been subject to in vitro passage. The two strains have been shown to display differing phenotypes both in vivo and in vitro. The proteome of the two strains grown in liquid culture were examined over time to determine whether there are any major differences between them at the protein level and the differences were compared to the genome data. Total cell lysates of the two strains were analysed by two-dimensional electrophoresis. Approximately 1750 protein spots were visualized by silver staining and the protein profiles of the two strains were found to be highly similar. Out of a total of 17 protein spot differences, seven were unique to CDC 1551 and three to H37Rv. Two further spots showed increased intensity in H37Rv, one spot showed differing vertical mobility between the strains and four showed differing spot intensities with time. Twelve of the spot differences were identified using mass spectrometry; however, no obvious association with phenotype could be deduced. When genome differences were analysed and related to the proteome differences, a mobility shift identified in the MoxR protein could be explained by a point mutation at the gene level. This proteome analysis reveals that, despite having been maintained under vastly different conditions, namely in vitro passage and in vivo transmission, these two strains have remained highly similar.