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Cough, a hallmark of tuberculosis, transmits the disease. Ruhl et al. find that a Mycobacterium tuberculosis (Mtb)-specific lipid, SL-1, stimulates human nociceptive neurons and makes guinea pigs cough. Mtb extract, but not SL-1, also stimulates non-nociceptive neurons that participate in the cough reflex, suggesting additional cough-inducing mechanisms.
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Mycobacterium tuberculosis , Tuberculosis , Animales , Tos , Cobayas , Humanos , Lípidos , NociceptoresRESUMEN
A greater understanding of hematopoietic stem cell (HSC) regulation is required for dissecting protective versus detrimental immunity to pathogens that cause chronic infections such as Mycobacterium tuberculosis (Mtb). We have shown that systemic administration of Bacille Calmette-Guérin (BCG) or ß-glucan reprograms HSCs in the bone marrow (BM) via a type II interferon (IFN-II) or interleukin-1 (IL1) response, respectively, which confers protective trained immunity against Mtb. Here, we demonstrate that, unlike BCG or ß-glucan, Mtb reprograms HSCs via an IFN-I response that suppresses myelopoiesis and impairs development of protective trained immunity to Mtb. Mechanistically, IFN-I signaling dysregulates iron metabolism, depolarizes mitochondrial membrane potential, and induces cell death specifically in myeloid progenitors. Additionally, activation of the IFN-I/iron axis in HSCs impairs trained immunity to Mtb infection. These results identify an unanticipated immune evasion strategy of Mtb in the BM that controls the magnitude and intrinsic anti-microbial capacity of innate immunity to infection.
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Células Madre Hematopoyéticas/microbiología , Inmunidad , Mycobacterium tuberculosis/fisiología , Mielopoyesis , Animales , Células de la Médula Ósea/metabolismo , Proliferación Celular , Susceptibilidad a Enfermedades , Homeostasis , Interferón Tipo I/metabolismo , Hierro/metabolismo , Cinética , Pulmón/microbiología , Pulmón/patología , Macrófagos/inmunología , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Necrosis , Transducción de Señal , Transcripción Genética , Tuberculosis/inmunología , Tuberculosis/microbiología , Tuberculosis/patologíaRESUMEN
Tobin and colleagues show that both inhibition and excessive production of the inflammatory mediator TNFα impact the pathogenesis of tuberculosis (TB) and the response to therapy. Identifying a critical role for the genetically determined balance between pro- and anti-inflammatory eicosanoids in regulating TNFα levels provides a roadmap to tailored TB treatment based on host genotype.
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Mycobacteria are responsible for several human and animal diseases. NOD2 is a pattern recognition receptor that has an important role in mycobacterial recognition. However, the mechanisms by which mutations in NOD2 alter the course of mycobacterial infection remain unclear. Herein, we aimed to review the totality of studies directly addressing the relationship between NOD2 and mycobacteria as a foundation for moving the field forward. NOD2 was linked to mycobacterial infection at 3 levels: (1) genetic, through association with mycobacterial diseases of humans; (2) chemical, through the distinct NOD2 ligand in the mycobacterial cell wall; and (3) immunologic, through heightened NOD2 signaling caused by the unique modification of the NOD2 ligand. The immune response to mycobacteria is shaped by NOD2 signaling, responsible for NF-κB and MAPK activation, and the production of various immune effectors like cytokines and nitric oxide, with some evidence linking this to bacteriologic control. Absence of NOD2 during mycobacterial infection of mice can be detrimental, but the mechanism remains unknown. Conversely, the success of immunization with mycobacteria has been linked to NOD2 signaling and NOD2 has been targeted as an avenue of immunotherapy for diseases even beyond mycobacteria. The mycobacteria-NOD2 interaction remains an important area of study, which may shed light on immune mechanisms in disease.
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Infecciones por Mycobacterium , Mycobacterium , Humanos , Animales , Ratones , Ligandos , Transducción de Señal/fisiología , FN-kappa B/metabolismo , Citocinas/metabolismo , Mycobacterium/metabolismo , Proteína Adaptadora de Señalización NOD2/genéticaRESUMEN
Mycobacteria, the pathogens that cause tuberculosis and leprosy, establish long-term infections in host macrophages. Recent studies, including two genetic screens reported in this issue of Cell (Kumar et al., 2010; Tobin et al., 2010), reveal that virulent mycobacteria evade the host immune system by stimulating production of anti-inflammatory molecules and inhibiting autophagy.
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Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Autofagia , Predisposición Genética a la Enfermedad , Humanos , Evasión Inmune , Macrófagos/microbiología , Tuberculosis/genética , Tuberculosis/microbiologíaRESUMEN
BACKGROUND: Mycobacterium abscessus complex (MABC), an opportunistic nontuberculous mycobacteria (NTM), can lead to poor clinical outcomes in pulmonary infections. Conflicting data exist on person-to-person transmission of MABC within and across healthcare facilities. To investigate further, a comprehensive retrospective study across five healthcare institutions on the Island of Montréal was undertaken. METHODS: We analyzed the genomes of 221 MABC isolates obtained from 115 individuals (2010-2018) to identify possible links. Genetic similarity, defined as ≤25 single-nucleotide polymorphisms (SNPs), was investigated through a blinded epidemiological inquiry. RESULTS: Bioinformatics analyses identified 28 sequence types (STs), including globally observed dominant circulating clones (DCCs). Further analysis revealed 210 isolate pairs within the SNP threshold. Among these pairs, there was one possible lab contamination where isolates from different patients processed in the same lab differed by only 2 SNPs. There were 37 isolate pairs from patients who had provided specimens from the same hospital; however, epidemiological analysis found no evidence of healthcare-associated person-to-person transmission between these patients. Additionally, pan-genome analysis showed higher discriminatory power than core genome analysis for examining genomic similarity. CONCLUSIONS: Genomics alone is insufficient to establish MABC transmission, particularly considering the genetic similarity and wide distribution of DCCs, although pan-genome analysis has the potential to add further insight. Our findings indicate that MABC infections in Montréal are unlikely attributable to healthcare-associated person-to-person transmission.
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OBJECTIVES: Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). METHODS: Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. RESULTS: The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. CONCLUSION: The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.
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Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Humanos , Secuenciación Completa del Genoma , Mutación , Farmacorresistencia Bacteriana/genética , Rifampin/farmacología , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Antituberculosos/farmacología , Antituberculosos/uso terapéuticoRESUMEN
A new tuberculosis vaccine is a high priority. However, the classical development pathway is a major deterrent. Most tuberculosis cases arise within 2 years after Mycobacterium tuberculosis exposure, suggesting a 3-year trial period should be possible if sample size is large to maximize the number of early exposures. Increased sample size could be facilitated by working alongside optimized routine services for case ascertainment, with strategies for enhanced case detection and safety monitoring. Shortening enrolment could be achieved by simplifying screening criteria and procedures and strengthening site capacity. Together, these measures could enable radically shortened phase 3 tuberculosis vaccine trials.
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Mycobacterium tuberculosis , Vacunas contra la Tuberculosis , Tuberculosis , Humanos , Vacunas contra la Tuberculosis/inmunología , Nueces/inmunología , Tuberculosis/prevención & control , Tuberculosis/inmunología , Mycobacterium tuberculosis/inmunología , Método Doble CiegoRESUMEN
Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis.
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Antituberculosos/farmacología , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus/efectos de los fármacos , Valina/análogos & derivados , Animales , Quimioterapia Combinada/métodos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Valina/farmacología , Pez CebraRESUMEN
Developers of severe acute respiratory syndrome coronavirus 2 vaccines should consider some of the lessons from a "new" vaccine introduced in 1921, namely bacille Calmette-Guérin.
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Vacuna BCG/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , SARS-CoV-2/inmunología , Animales , Vacuna BCG/administración & dosificación , COVID-19/inmunología , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Humanos , Pandemias/prevención & control , Ensayos Clínicos Controlados Aleatorios como Asunto , Tuberculosis/inmunología , Tuberculosis/prevención & controlRESUMEN
BACKGROUND: In the last decade, tuberculosis (TB) incidence among Inuit in the Canadian Arctic has been rising. Our aim was to better understand the transmission dynamics of TB in this remote region of Canada using whole-genome sequencing. METHODS: Isolates from patients who had culture-positive pulmonary TB in Iqaluit, Nunavut, between 2009 and 2015 underwent whole-genome sequencing (WGS). The number of transmission events between cases within clusters was calculated using a threshold of aâ ≤3 single nucleotide polymorphism (SNP) difference between isolates and then combined with detailed epidemiological data using a reproducible novel algorithm. Social network analysis of epidemiological data was used to support the WGS data analysis. RESULTS: During the study period, 140 Mycobacterium tuberculosis isolates from 135 cases were sequenced. Four clusters were identified, all from Euro-American lineage. One cluster represented 62% of all cases that were sequenced over the entire study period. In this cluster, 2 large chains of transmission were associated with 3 superspreading events in a homeless shelter. One of the superspreading events was linked to a nonsanctioned gambling house that resulted in further transmission. Shelter to nonshelter transmission was also confirmed. An algorithm developed for the determination of transmission events demonstrated very good reproducibility (κ score .98, 95% confidence interval, .97-1.0). CONCLUSIONS: Our study suggests that socioeconomic factors, namely residing in a homeless shelter and spending time in a gambling house, combined with the superspreading event effect may have been significant factors explaining the rise in cases in this predominantly Inuit Arctic community.
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Mycobacterium tuberculosis , Canadá/epidemiología , Genoma Bacteriano , Humanos , Inuk , Epidemiología Molecular , Mycobacterium tuberculosis/genética , Nunavut/epidemiología , Polimorfismo de Nucleótido Simple , Reproducibilidad de los ResultadosRESUMEN
We compared clinical performance of p16/Ki-67 dual-stained cytology and human papillomavirus (HPV) genotyping, via different algorithms-alone, or in combination with cytology-to identify cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) in women referred to as colposcopy. We included 492 cervical specimens (134 normal, 130 CIN1, 99 CIN2, 121 CIN3, 8 cancers) randomly selected from 1158 specimens with valid conventional cytology, HPV (cobas 4800 HPV test) and biopsy results. Dual-stained cytology was retrospectively performed (CINtec PLUS assay) on PreservCyt material; slides were read by a cytologist and confirmed by two pathologists, blinded to cytology, biopsy and genotyping results. Sensitivity and specificity (95% confidence intervals in parentheses) of dual-stained cytology to detect CIN2+ and CIN3+ were compared to other screening tests available for the same women. Positivity rate for dual-stained cytology increased with histological severity: 30.6% in normal, 41.5% in CIN1, 72.7% in CIN2, 86.8% in CIN3 and 87.5% in cancer. Dual-stained cytology alone had lower sensitivity than HPV testing for CIN2+ [80.7% (75.0-85.6) vs 89.9% (85.3-93.5)] and CIN3+ [86.8% (79.7-92.1) vs 92.3% (86.2-96.2)]. However, corresponding specificity values were higher [64.0% (57.9-69.8) vs 56.1% (49.8-62.1) for CIN2+; 54.0% (48.7-59.2) vs 44.4% (39.2-49.6) for CIN3+]. Combining dual-stained cytology with an ASC-US abnormality threshold decreased specificity to 31.4% (25.9-37.4) for CIN2+ and 24.2% (19.9-29.0) for CIN3+. The corresponding values considering low squamous intraepithelial lesion threshold values were 42.8% (36.8-49.0) and 35.0% (30.1-40.1). Dual-stained cytology and HPV testing exhibited similar performance, although the former improved the specificity by 7.9% and 9.6% for CIN2+ and CIN3+, respectively.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Antígeno Ki-67/análisis , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/patología , Adolescente , Adulto , Anciano , Células Escamosas Atípicas del Cuello del Útero/metabolismo , Células Escamosas Atípicas del Cuello del Útero/patología , Células Escamosas Atípicas del Cuello del Útero/virología , Cuello del Útero/patología , Cuello del Útero/virología , Detección Precoz del Cáncer/métodos , Femenino , Humanos , Persona de Mediana Edad , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Coloración y Etiquetado/métodos , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virología , Adulto Joven , Displasia del Cuello del Útero/diagnóstico , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/virologíaRESUMEN
The production of itaconate by macrophages was only discovered in 2011. An increasing number of studies have since revealed essential biological functions for this small molecule, ranging from antimicrobial to immunomodulator. The antibacterial role of itaconate has however been questioned because the estimated concentration of itaconate in macrophages (low-millimolar) is lower than the minimum inhibitory concentration (MIC) of itaconate reported for several bacterial strains (low-to-mid-millimolar). We note that some of these investigations have tended to ignore the high acidity of this small diacid (pKas 3.85 and 5.45), thereby potentially biassing activity measurements. We measured the MIC of itaconate in Escherichia coli (not known to metabolize itaconate) and in Salmonella enterica serovar Typhimurium (known to metabolize itaconate) at varying pH values to probe the effect that pH has on itaconate toxicity. Herein, we demonstrate that the antimicrobial effect of itaconate is dependent upon the pH of the media and that itaconate does have antimicrobial activity at biologically relevant pH and concentrations. Under nutrient-poor conditions, the antimicrobial activity of itaconate in both E. coli and S. Typhimurium increased approximately 200-fold when the pH was dropped by one unit, whereas itaconate was not found to be toxic under nutrient rich conditions. Our results also reveal that the activity of itaconate is synergistic with acidity, yet is not a function of increased permeability with protonation. Similar experiments performed with succinate (a pKa-matched diacid) yielded drastically different results, consistent with a target-based mechanism of action for itaconate. Overall, our work shows the importance of controlling the pH when performing experiments with itaconic acid.
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Antibacterianos/química , Antibacterianos/farmacología , Macrófagos/química , Succinatos/química , Succinatos/farmacología , Antibacterianos/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Escherichia coli/efectos de los fármacos , Concentración de Iones de Hidrógeno , Macrófagos/metabolismo , Pruebas de Sensibilidad Microbiana , Salmonella typhimurium/efectos de los fármacos , Succinatos/metabolismoRESUMEN
Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.
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Antiácidos/metabolismo , Lípidos/biosíntesis , Lípidos/química , Mycobacterium tuberculosis/metabolismo , Fagosomas/metabolismo , Animales , Regulación Bacteriana de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Lisosomas , Macrófagos/metabolismo , Ratones , Estructura Molecular , Mycobacterium kansasii/genética , PrevalenciaRESUMEN
We used a genome-wide screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mice to identify genes in which recessive loss-of-function mutations protect against pathological neuroinflammation. We identified an R367Q mutation in the ZBTB7B (ThPOK) protein in which homozygosity causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Zbtb7bR367Q homozygous mice show a defect in the lymphoid compartment expressed as severe reduction in the number of single-positive CD4 T cells in the thymus and in the periphery, reduced brain infiltration of proinflammatory leukocytes in P. berghei ANKA-infected mice, and reduced production of proinflammatory cytokines by primary T cells ex vivo and in vivo Dampening of proinflammatory immune responses in Zbtb7bR367Q mice is concomitant to increased susceptibility to infection with avirulent (Mycobacterium bovis BCG) and virulent (Mycobacterium tuberculosis H37Rv) mycobacteria. The R367Q mutation maps to the first DNA-binding zinc finger domain of ThPOK and causes loss of base contact by R367 in the major groove of the DNA, which is predicted to impair DNA binding. Global immunoprecipitation of ThPOK-containing chromatin complexes coupled to DNA sequencing (ChIP-seq) identified transcriptional networks and candidate genes likely to play key roles in CD4+ CD8+ T cell development and in the expression of lineage-specific functions of these cells. This study highlights ThPOK as a global regulator of immune function in which alterations may affect normal responses to infectious and inflammatory stimuli.
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Proteínas de Unión al ADN/genética , Malaria Cerebral/genética , Factores de Transcripción/genética , Tuberculosis Pulmonar/genética , Animales , Encéfalo/microbiología , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD8-positivos/microbiología , Citocinas/genética , Femenino , Inflamación/genética , Inflamación/microbiología , Malaria Cerebral/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/patogenicidad , Plasmodium berghei/patogenicidad , Tuberculosis Pulmonar/microbiología , Virulencia/genéticaRESUMEN
DNA methylation analysis may improve risk stratification in cervical screening. We used a pan-epigenomic approach to identify new methylation markers along the continuum of cervical intraepithelial neoplasia (CIN) to cervical cancer. Physician-collected samples (54 normal, 50 CIN1, 40 CIN2 and 42 CIN3) were randomly selected from women at a single-center colposcopy clinic. Extracted DNA was subjected to Illumina Infinium EPIC array analysis, and methylation was assessed blinded to histopathological and clinical data. CpG sites whose state of methylation correlated with lesion grade were assessed (Spearman correlation), and a weighted methylation score was calculated comparing normal to CIN3. Validation of the top selected genes was performed in an independent cohort (100 normal, 50 CIN1, 50 CIN2, 50 CIN3 and 8 cervical cancers) of new patients, referred for colposcopic examination at three hospitals, using targeted DNA methylation Illumina amplicon sequencing. The relationship between a combined weighted marker score and progression from normal through precancerous lesions and cervical cancer was compared using one-way ANOVA. Our analyses revealed 7,715 CpGs whose methylation level correlated with progression (from normal to CIN1, CIN2 and CIN3), with a significant trend of increased methylation with lesion grade. We shortlisted a bigenic (hyaluronan synthase 1, HAS1 and ATPase phospholipid transporting 10A, ATP10A corresponding to cg03419058 and cg13944175 sites) marker set; r = 0.55, p < 0.0001. Validation of the four most discriminating genes (CA10, DPP10, FMN2 and HAS1) showed a significant correlation between methylation levels and disease progression (p-value < 2.2 × 10-16 , adjusted R2 = 0.952). Translational research of the identified genes to future clinical applications is warranted.
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Biomarcadores de Tumor/genética , Infecciones por Papillomavirus/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , Adulto , Cuello del Útero/metabolismo , Cuello del Útero/patología , Metilación de ADN , Progresión de la Enfermedad , Detección Precoz del Cáncer , Epigenómica , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Papillomaviridae/genética , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Reproducibilidad de los Resultados , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/diagnóstico , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
Isoniazid (INH) is a cornerstone of antitubercular therapy. Mycobacterium tuberculosis complex bacteria are the only mycobacteria sensitive to clinically relevant concentrations of INH. All other mycobacteria, including M. marinum and M. avium subsp. paratuberculosis are resistant. INH requires activation by bacterial KatG to inhibit mycobacterial growth. We tested the role of the differences between M. tuberculosis KatG and that of other mycobacteria in INH sensitivity. We cloned the M. boviskatG gene into M. marinum and M. avium subsp. paratuberculosis and measured the MIC of INH. We recombinantly expressed KatG of these mycobacteria and tested in vitro binding to, and activation of, INH. Introduction of katG from M. bovis into M. marinum and M. avium subsp. paratuberculosis rendered them 20 to 30 times more sensitive to INH. Analysis of different katG sequences across the genus found KatG evolution diverged from RNA polymerase-defined mycobacterial evolution. Biophysical and biochemical tests of M. bovis and nontuberculous mycobacteria (NTM) KatG proteins showed lower affinity to INH and substantially lower enzymatic capacity for the conversion of INH into the active form in NTM. The KatG proteins of M. marinum and M. avium subsp. paratuberculosis are substantially less effective in INH activation than that of M. tuberculosis, explaining the relative INH insensitivity of these microbes. These data indicate that the M. tuberculosis complex KatG is divergent from the KatG of NTM, with a reciprocal relationship between resistance to host defenses and INH resistance. Studies of bacteria where KatG is functionally active but does not activate INH may aid in understanding M. tuberculosis INH-resistance mechanisms, and suggest paths to overcome them.
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Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Catalasa/metabolismo , Isoniazida/farmacología , Mycobacterium/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Catalasa/genética , Activación Enzimática , Proteínas de Unión al Hemo/genética , Proteínas de Unión al Hemo/metabolismo , Mycobacterium/enzimología , Mycobacterium/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Micobacterias no Tuberculosas/efectos de los fármacos , Micobacterias no Tuberculosas/enzimología , Micobacterias no Tuberculosas/genética , Filogenia , Multimerización de Proteína , Alineación de Secuencia , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be partially restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation.