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The world has seen unprecedented gains in the global genomic surveillance capacities for pathogens with pandemic and epidemic potential within the last 4 years. To strengthen and sustain the gains made, WHO is working with countries and partners to implement the Global Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 2022-2032. A key technical product developed through these multi-agency collaborative efforts is a genomics costing tool (GCT), as sought by many countries. This tool was developed by five institutions - Association of Public Health Laboratories, FIND, The Global Fund to Fight AIDS, Tuberculosis and Malaria, UK Health Security Agency, and the World Health Organization. These institutions developed the GCT to support financial planning and budgeting for SARS-CoV-2 next-generation sequencing activities, including bioinformatic analysis. The tool costs infrastructure, consumables and reagents, human resources, facility and quality management. It is being used by countries to (1) obtain costs of routine sequencing and bioinformatics activities, (2) optimize available resources, and (3) build an investment case for the scale-up or establishment of sequencing and bioinformatics activities. The tool has been validated and is available in English and Russian at https://www.who.int/publications/i/item/9789240090866. This paper aims to highlight the rationale for developing the tool, describe the process of the collaborative effort in developing the tool, and describe the utility of the tool to countries.
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COVID-19 , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , SARS-CoV-2 , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/economía , COVID-19/economía , COVID-19/prevención & control , SARS-CoV-2/genética , Biología Computacional , Defensa Civil/economía , Pandemias/economía , Salud GlobalRESUMEN
The increasing availability of high-throughput sequencing (frequently termed next-generation sequencing (NGS)) data has created opportunities to gain deeper insights into the mechanisms of a number of diseases and is already impacting many areas of medicine and public health. The area of infectious diseases stands somewhat apart from other human diseases insofar as the relevant genomic data comes from the microbes rather than their human hosts. A particular concern about the threat of antimicrobial resistance (AMR) has driven the collection and reporting of large-scale datasets containing information from microbial genomes together with antimicrobial susceptibility test (AST) results. Unfortunately, the lack of clear standards or guiding principles for the reporting of such data is hampering the field's advancement. We therefore present our recommendations for the publication and sharing of genotype and phenotype data on AMR, in the form of 10 simple rules. The adoption of these recommendations will enhance AMR data interoperability and help enable its large-scale analyses using computational biology tools, including mathematical modelling and machine learning. We hope that these rules can shed light on often overlooked but nonetheless very necessary aspects of AMR data sharing and enhance the field's ability to address the problems of understanding AMR mechanisms, tracking their emergence and spread in populations, and predicting microbial susceptibility to antimicrobials for diagnostic purposes.
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Antibacterianos , Antiinfecciosos , Humanos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Bacterias/genética , Genoma Microbiano , Genotipo , FenotipoRESUMEN
Background: Tracking the emergence and spread of antimalarial drug resistance has become critical to sustaining progress towards the control and eventual elimination of malaria in South Asia, especially India. Methods: An amplicon sequencing protocol was used for high-throughput molecular surveillance of antimalarial drug resistance in a total of 158 isolates at three sites in India: Chennai, Nadiad and Rourkela. Five genes of the Plasmodium falciparum implicated in antimalarial resistance were investigated here; Pfcrt for chloroquine resistance, Pfdhfr for pyrimethamine resistance, Pfdhps for sulfadoxine resistance, Pfk13 for artemisinin resistance and Pfmdr1 for resistance to multiple antimalarials. Results: Mutations in the propeller domain of PfK13 were observed in two samples only, however these mutations are not validated for artemisinin resistance. A high proportion of parasites from the P. falciparum dominant site Rourkela showed wild-type Pfcrt and Pfdhfr haplotypes, while mutant Pfcrt and Pfdhfr haplotypes were fixed at the P. vivax dominant sites Chennai and Nadiad. The wild-type PfDHPS haplotype was predominant across all study sites. Finally, we observed the largest proportion of suspected multi-clonal infections at Rourkela, which has the highest transmission of P. falciparum among our study sites. Conclusion: This is the first simultaneous high-throughput next generation sequencing of five complete P. falciparum genes from infected patients in India.
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Universal drug susceptibility testing (DST) for tuberculosis is a major goal of the END TB strategy. PCR-based molecular diagnostic tests have been instrumental in increasing DST globally and several assays have now been endorsed by the World Health Organization (WHO) for use in the diagnosis of drug resistance. These endorsed assays, however, each interrogate a limited number of mutations associated with resistance, potentially limiting their sensitivity compared to sequencing-based methods. We applied an in silico method to compare the sensitivity and specificity of WHO-endorsed molecular based diagnostics to the mutation set identified by the WHO mutations catalogue using phenotypic DST as the reference. We found that, in silico, the mutation sets used by probe-based molecular diagnostic tests to identify rifampicin, isoniazid, pyrazinamide, levofloxacin, moxifloxacin, amikacin, capreomycin and kanamycin resistance produced similar sensitivities and specificities to the WHO mutation catalogue. PCR-based diagnostic tests were most sensitive for drugs where mechanisms of resistance are well established and localised to small genetic regions or a few prevalent mutations. Approaches using sequencing technologies can provide advantages for drugs where our knowledge of resistance is limited, or where complex resistance signatures exist.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Isoniazida , Pirazinamida , Rifampin , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Pruebas de Sensibilidad Microbiana , Capreomicina , Mycobacterium tuberculosis/genética , Amicacina , Levofloxacino , Moxifloxacino , Genotipo , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/genética , Organización Mundial de la SaludRESUMEN
Background: Molecular diagnostics are considered the most promising route to achieving rapid, universal drug susceptibility testing for Mycobacterium tuberculosiscomplex (MTBC). We aimed to generate a WHO endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: A candidate gene approach was used to identify mutations as associated with resistance, or consistent with susceptibility, for 13 WHO endorsed anti-tuberculosis drugs. 38,215 MTBC isolates with paired whole-genome sequencing and phenotypic drug susceptibility testing data were amassed from 45 countries. For each mutation, a contingency table of binary phenotypes and presence or absence of the mutation computed positive predictive value, and Fisher's exact tests generated odds ratios and Benjamini-Hochberg corrected p-values. Mutations were graded as Associated with Resistance if present in at least 5 isolates, if the odds ratio was >1 with a statistically significant corrected p-value, and if the lower bound of the 95% confidence interval on the positive predictive value for phenotypic resistance was >25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: 15,667 associations were computed for 13,211 unique mutations linked to one or more drugs. 1,149/15,667 (7·3%) mutations were classified as associated with phenotypic resistance and 107/15,667 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was >80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were classified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: This first WHO endorsed catalogue of molecular targets for MTBC drug susceptibility testing provides a global standard for resistance interpretation. Its existence should encourage the implementation of molecular diagnostics by National Tuberculosis Programmes. Funding: UNITAID, Wellcome, MRC, BMGF.
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Etambutol , Mycobacterium tuberculosis , Antituberculosos/farmacología , Resistencia a Medicamentos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Organización Mundial de la SaludRESUMEN
Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. One hundred four DNA samples extracted from smear-positive sputum sediments, previously sequenced using the Deeplex assay on an Illumina MiniSeq, were resequenced on MinION after applying a custom library preparation. MinION read quality, mapping statistics, and variant calling were computed using an in-house pipeline and compared to the reference MiniSeq data. The average percentage of MinION reads mapped to an H37RV reference genome was 90.8%, versus 99.5% on MiniSeq. The mean depths of coverage were 4,151× and 4,177× on MinION and MiniSeq, respectively, with heterogeneous distribution across targeted genes. Composite reference coverage breadth was >99% for both platforms. We observed full concordance between technologies in reporting the clinically relevant drug-resistant markers, including full gene deletions. In conclusion, we demonstrated that the workflow and sequencing data obtained from Deeplex on MinION are comparable to those for the MiniSeq, despite the higher raw error rates on MinION, with the added advantage of MinION's portability, versatility, and low capital costs. Targeted NGS on MinION is a promising DST solution for rapidly providing clinically relevant data to manage complex DR-TB cases.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Análisis de Secuencia de ADN , Tuberculosis Resistente a Múltiples Medicamentos/diagnósticoRESUMEN
Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world.
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Anticuerpos Antiprotozoarios/sangre , Formación de Anticuerpos , Malaria Falciparum/sangre , Malaria Vivax/sangre , Adolescente , Adulto , Anciano , Antígenos de Protozoos/inmunología , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , India , Malaria Falciparum/epidemiología , Malaria Falciparum/inmunología , Malaria Vivax/epidemiología , Malaria Vivax/inmunología , Masculino , Persona de Mediana Edad , Proyectos Piloto , Plasmodium falciparum/inmunología , Plasmodium vivax/inmunología , Análisis por Matrices de Proteínas , Adulto JovenRESUMEN
A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.
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Antimaláricos/farmacología , Resistencia a Medicamentos , Técnicas de Genotipaje/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Alelos , Biología Computacional/métodos , Humanos , India , Plasmodium falciparum/aislamiento & purificación , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodosRESUMEN
BACKGROUND: The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models. RESULTS: The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses. CONCLUSION: The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.
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Genoma Bacteriano , Mycobacterium tuberculosis/genética , Estreptomicina/farmacología , Transcriptoma , Hibridación Genómica Comparativa , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación INDEL , Modelos Moleculares , Anotación de Secuencia Molecular , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/efectos de los fármacos , Filogenia , Polimorfismo de Nucleótido Simple , Estructura Terciaria de Proteína , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Análisis de Secuencia de ARNRESUMEN
The study of the three protagonists in malaria-the Plasmodium parasite, the Anopheles mosquito, and the human host-is key to developing methods to control and eventually eliminate the disease. Genomic technologies, including the recent development of next-generation sequencing, enable interrogation of this triangle to an unprecedented level of scrutiny, and promise exciting progress toward real-time epidemiology studies and the study of evolutionary adaptation. We discuss the use of genomics by the International Centers of Excellence for Malaria Research, a network of field sites and laboratories in malaria-endemic countries that undertake cutting-edge research, training, and technology transfer in malarious countries of the world.
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Anopheles/genética , Genética de Población , Malaria/genética , Plasmodium/genética , Animales , Marcadores Genéticos/genética , Genética de Población/métodos , Estudio de Asociación del Genoma Completo , Técnicas de Genotipaje , Humanos , Cooperación Internacional , Malaria/epidemiología , Malaria/parasitología , Malaria/prevención & control , Repeticiones de Microsatélite/genética , Plasmodium falciparum/genética , Plasmodium vivax/genética , Análisis de Secuencia de ADNRESUMEN
The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.
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Proteínas Bacterianas/genética , Proteínas de Transporte de Membrana/biosíntesis , Mycobacterium tuberculosis/metabolismo , ARN no Traducido/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Productos del Gen tat/metabolismo , Humanos , Ratones Endogámicos C57BL , Mutación/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Operón/genética , Proteómica/métodos , ARN no Traducido/genética , Virulencia , beta-Lactamasas/metabolismoRESUMEN
The antileprosy drug clofazimine is also of interest for the treatment of multidrug-resistant tuberculosis. To understand possible resistance mechanisms, clofazimine-resistant Mycobacterium tuberculosis mutants were isolated in vitro, and, unexpectedly, found to be cross-resistant to bedaquiline. Mutations in the transcriptional regulator Rv0678, with concomitant upregulation of the multisubstrate efflux pump, MmpL5, accounted for this cross-resistance. Mutation in Rv0678 should therefore be considered a confounding factor for the treatment of tuberculosis with clofazimine or bedaquiline.
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Antituberculosos/farmacología , Clofazimina/farmacología , Diarilquinolinas/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Farmacorresistencia Bacteriana MúltipleRESUMEN
The transcriptional regulator GlxR has been characterized as a global hub within the gene-regulatory network of Corynebacterium glutamicum. Chromatin immunoprecipitation with a specific anti-GlxR antibody and subsequent high-throughput sequencing (ChIP-seq) was applied to C. glutamicum to get new in vivo insights into the gene composition of the GlxR regulon. In a comparative approach, C. glutamicum cells were grown with either glucose or acetate as the sole carbon source prior to immunoprecipitation. High-throughput sequencing resulted in 69 million reads and 2.6 Gb of genomic information. After mapping of these data on the genome sequence of C. glutamicum, 107 enriched DNA fragments were detected from cells grown with glucose as carbon source. GlxR binding sites were identified in the sequence of 79 enriched DNA fragments, of which 21 sites were not previously reported. Electrophoretic mobility shift assays with 40-mer oligomers covering the GlxR binding sites were performed for validation of the in vivo results. The detection of new binding sites confirmed the role of GlxR as a regulator of carbon source metabolism and energy conversion, but additionally revealed binding of GlxR in front of the 6C non-coding RNA gene and to non-canonical DNA binding sites within protein-coding regions. The present study underlines the dynamics within the GlxR regulon by identifying in vivo targets during growth on glucose and contributes to the expansion of knowledge of this important transcriptional regulator.
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Corynebacterium glutamicum/genética , ADN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/metabolismo , Ácido Acético/metabolismo , Sitios de Unión , Carbono/metabolismo , Inmunoprecipitación de Cromatina , Corynebacterium glutamicum/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Glucosa/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Unión Proteica , RegulónRESUMEN
To construct a regulatory map of the genome of the human pathogen, Mycobacterium tuberculosis, we applied two complementary high-resolution approaches: strand-specific RNA-seq, to survey the global transcriptome, and ChIP-seq, to monitor the genome-wide dynamics of RNA polymerase (RNAP) and the anti-terminator NusA. Although NusA does not bind directly to DNA, but rather to RNAP and/or to the nascent transcript, we demonstrate that NusA interacts with RNAP ubiquitously throughout the chromosome, and that its profile mirrors RNAP distribution in both the exponential and stationary phases of growth. Generally, promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease in signal strength reflecting transcriptional polarity. Differential binding of RNAP and NusA in the two growth conditions correlated with transcriptional activity as reflected by RNA abundance. Indeed, a significant association between expression levels and the presence of NusA throughout the gene body was detected, confirming the peculiar transcription-promoting role of NusA. Integration of the data sets pinpointed transcriptional units, mapped promoters and uncovered new anti-sense and non-coding transcripts. Highly expressed transcriptional units were situated mainly on the leading strand, despite the relatively unbiased distribution of genes throughout the genome, thus helping the replicative and transcriptional complexes to align.
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Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Genoma Fúngico , Mycobacterium tuberculosis/genética , Proteínas de Unión al ARN/metabolismo , Transcriptoma , Inmunoprecipitación de Cromatina , Codón , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , ARN sin Sentido/biosíntesis , ARN de Transferencia/metabolismo , Análisis de Secuencia de ARN , Elongación de la Transcripción Genética , Iniciación de la Transcripción GenéticaRESUMEN
Tuberculosis, a global threat to public health, is becoming untreatable due to widespread drug resistance to frontline drugs such as the InhA-inhibitor isoniazid. Historically, by inhibiting highly vulnerable targets, natural products have been an important source of antibiotics including potent anti-tuberculosis agents. Here, we describe pyridomycin, a compound produced by Dactylosporangium fulvum with specific cidal activity against mycobacteria. By selecting pyridomycin-resistant mutants of Mycobacterium tuberculosis, whole-genome sequencing and genetic validation, we identified the NADH-dependent enoyl- (Acyl-Carrier-Protein) reductase InhA as the principal target and demonstrate that pyridomycin inhibits mycolic acid synthesis in M. tuberculosis. Furthermore, biochemical and structural studies show that pyridomycin inhibits InhA directly as a competitive inhibitor of the NADH-binding site, thereby identifying a new, druggable pocket in InhA. Importantly, the most frequently encountered isoniazid-resistant clinical isolates remain fully susceptible to pyridomycin, thus opening new avenues for drug development. âSee accompanying article http://dx.doi.org/10.1002/emmm.201201811.
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Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Productos Biológicos/farmacología , Micromonosporaceae/química , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Antituberculosos/aislamiento & purificación , Productos Biológicos/aislamiento & purificación , Vías Biosintéticas/efectos de los fármacos , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Mutación , Ácidos Micólicos/metabolismo , Oligopéptidos/aislamiento & purificación , Selección GenéticaRESUMEN
In mycobacteria, CycA a D-serine, L- and D-alanine, and glycine transporter also functions in the uptake of D-cycloserine, an important second-line anti-tubercular drug. A single nucleotide polymorphism identified in the cycA gene of BCG was hypothesized to contribute to the increased resistance of Mycobacterium bovis bacillus Calmette-Guérin (BCG) to D-cycloserine compared to wild-type Mycobacterium tuberculosis or Mycobacterium bovis. Working along these lines, a merodiploid strain of BCG expressing Mycobacterium tuberculosis CycA was generated and found to exhibit increased susceptibility to D-cycloserine albeit not to the same extent as wild-type Mycobacterium tuberculosis or Mycobacterium bovis. In addition, recombinant Mycobacterium smegmatis strains expressing either Mycobacterium tuberculosis or Mycobacterium bovis CycA but not BCG CycA were rendered more susceptible to D-cycloserine. These findings support the notion that CycA-mediated uptake in BCG is impaired as a result of a single nucleotide polymorphism; however, the partial contribution of this impairment to D-cycloserine resistance suggests the involvement of additional genetic lesions in this phenotype.
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Sistemas de Transporte de Aminoácidos Neutros/genética , Proteínas Bacterianas/genética , Cicloserina/farmacología , Farmacorresistencia Bacteriana/genética , Mycobacterium bovis/genética , Mutación Puntual , Simportadores/genética , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Antibióticos Antituberculosos/farmacología , Vacuna BCG/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cicloserina/química , Técnicas de Sustitución del Gen , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium bovis/inmunología , Mycobacterium bovis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/metabolismo , Polimorfismo de Nucleótido Simple , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Simportadores/química , Simportadores/metabolismoRESUMEN
The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes.
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Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Mycobacterium tuberculosis/genética , Proteínas Represoras/genética , Factores de Virulencia/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano , Proteínas de Unión al ADN/metabolismo , Genes Reguladores , Proteínas de Homeodominio/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Proteínas Represoras/metabolismo , Virulencia , Factores de Virulencia/metabolismoRESUMEN
In Mycobacterium tuberculosis the alternative sigma factor SigF controls the expression of a particular subset of genes by altering RNA polymerase specificity. Here, we utilize two genome-wide approaches to identify SigF-binding sites: chromatin immunoprecipitation (ChIP-on-chip) and microarray analysis of SigF-mediated transcripts. Since SigF is not an abundant protein in the logarithmic phase of growth, a pristinamyin IA-inducible system was used to control its expression. We identified 67 high-affinity SigF-binding sites and 16 loci where a SigF promoter directs the expression of a transcript. These loci include sigF itself, genes involved in lipid and intermediary metabolism and virulence, and at least one transcriptional regulator (Rv2884), possibly acting downstream of SigF. In addition, SigF was also found to direct the transcription of the gene for small RNA F6. Many loci were also found where SigF may be involved in antisense transcription, and in two cases (Rv1358 and Rv1870c) the SigF-dependent promoter was located within the predicted coding sequence. Quantitative PCR confirmed the microarray findings and 5'-rapid amplification of cDNA ends was used to map the SigF-specific transcriptional start points. A canonical SigF consensus promoter sequence GGTTT-N((15-17))-GGGTA was found prior to 11 genes. Together, these data help to define the SigF regulon and show that SigF not only governs expression of proteins such as the virulence factor, HbhA, but also impacts novel functions, such as noncoding RNAs and antisense transcripts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Genoma Bacteriano , Mycobacterium tuberculosis/metabolismo , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Composición de Base , ADN sin Sentido , Perfilación de la Expresión Génica , Dispositivos Laboratorio en un Chip , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mycobacterium tuberculosis/genética , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Análisis por Matrices de Proteínas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN no Traducido/metabolismo , Factor sigma/genéticaRESUMEN
The 23-membered Esx protein family is involved in the host-pathogen interactions of Mycobacterium tuberculosis. These secreted proteins are among the most immunodominant antigens recognized by the human immune system and have thus been used to develop vaccines and immunodiagnostic tests for tuberculosis (TB). Gene pairs for 10 Esx proteins are contained in the ESX-1 to ESX-5 loci, encoding type VII secretion systems. A subset of Esx proteins can be further classified into the Mtb9.9, QILSS, and TB10.4 subfamilies. To survey genetic diversity in the Esx family and its potential for antigenic variation, we sequenced all esx genes from 108 clinical isolates of M. tuberculosis from different clades by using a targeted approach. A total of 109 unique single nucleotide polymorphisms (SNPs) were observed, and 59 of these were nonsynonymous. Some of the resultant amino acid substitutions affect known Esx epitopes, including two in the EsxB (CFP-10) and EsxH (TB10.4) antigens. Assessment of the SNP distribution across the Esx proteins revealed high genetic variability, especially in the Mtb9.9 and QILSS subfamilies, and more conservation in the ESX-1 to ESX-4 loci. Comparison of the DNA sequences of variable esx genes provided clear evidence for recombination events between different genes in the same strain, some of which are predicted to truncate the corresponding protein. Many of these polymorphisms escape detection by ultrahigh-throughput sequencing using short sequence reads, as such approaches cannot distinguish between closely related genes. The esx gene family is dynamic, and sequence changes likely lead to immune variation.
Asunto(s)
Proteínas Bacterianas/genética , Epítopos/genética , Conversión Génica , Variación Genética , Genómica , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/química , Secuencia de Bases , Epítopos/química , Humanos , Datos de Secuencia Molecular , Complejos Multiproteicos/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo de Nucleótido Simple/genética , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Tuberculosis/microbiologíaRESUMEN
Onset of the adaptive immune response in mice infected with Mycobacterium tuberculosis is accompanied by slowing of bacterial replication and establishment of a chronic infection. Stabilization of bacterial numbers during the chronic phase of infection is dependent on the activity of the gamma interferon (IFN-γ)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identify M. tuberculosis "counterimmune" mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in the rv0072, rv0405, and rv2958c genes. These mutants were impaired for replication and virulence in NOS2(-/-) mice but were growth-proficient and virulent in IFN-γ(-/-) mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-γ-dependent immune mechanism other than NOS2. Here, we report that the attenuation of these strains is attributable to an underlying transposon-independent deficiency in biosynthesis of phthiocerol dimycocerosate (PDIM), a cell wall lipid that is required for full virulence in mice. We performed whole-genome resequencing of a PDIM-deficient clone and identified a spontaneous point mutation in the putative polyketide synthase PpsD that results in a G44C amino acid substitution. We demonstrate by complementation with the wild-type ppsD gene and reversion of the ppsD gene to the wild-type sequence that the ppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2(-/-) and wild-type C57BL/6 mice, and a growth advantage in vitro in liquid culture. We conclude that PDIM biosynthesis is required for M. tuberculosis resistance to an IFN-γ-mediated immune response that is independent of NOS2.