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The impact of chronic exposure to type I interferons (IFN)-α2a, 2b, and ß on macrophage metabolism, intimately linked to macrophage function, is not well understood. This study assesses the nuanced host responses induced by type I IFN cytokines, offering insights into potential therapeutic approaches in diseases associated with these cytokines. Employing a combination of transcriptional profiling and real-time functional analysis, we delineated metabolic reprogramming in response to chronic IFN exposure. Our results reveal distinct transcriptional metabolic profiles between macrophages chronically exposed to IFN-α and IFN-ß. IFN-ß significantly diminishes the oxygen consumption rate and glycolytic proton extrusion rate in macrophages. Conversely, IFN-α2b decreased parameters of mitochondrial fitness and induced a shift toward glutamine oxidation. Assessing the ability of macrophages to induce glycolysis in response to antigenic stimuli (LPS and iH37Rv), we found that chronic exposure to all IFN subtypes limited glycolytic induction. This study addresses a critical oversight in the literature, where individual roles of IFN subtypes are frequently amalgamated and lack distinction. These findings not only provide novel insights into the divergent effects of IFN-α2a, α2b, and ß on macrophage metabolism but also highlight their potential implications for developing targeted therapeutic strategies.
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BACKGROUND: Flu-like reactions can occur after exposure to rifampin, rifapentine, or isoniazid. Prior studies have reported the presence of antibodies to rifampin, but associations with underlying pathogenesis are unclear. METHODS: We evaluated PREVENT TB study participants who received weekly isoniazid + rifapentine for 3 months (3HP) or daily isoniazid for 9 months (9H) as treatment for M. tuberculosis infection. Flu-like reaction was defined as a grade ≥2 of any of flu-like symptoms. Controls (3HP or 9H) did not report flu-like reactions. We developed a competitive enzyme-linked immunosorbent assays (ELISA) to detect antibodies against rifapentine, isoniazid, rifampin, and rifapentine metabolite. RESULTS: Among 128 participants, 69 received 3HP (22 with flu-like reactions; 47 controls) and 59 received 9H (12 with flu-like reactions; 47 controls). In participants receiving 3HP, anti-rifapentine IgG was identified in 2/22 (9%) participants with flu-like reactions and 6/47 (13%) controls (P = 0.7), anti-isoniazid IgG in 2/22 (9%) participants with flu-like reactions and 4/47 (9%) controls (P = 0.9), and anti-rifapentine metabolite IgG in 2/47 (4%) controls (P = 0.9). Among participants receiving 9H, IgG and IgM anti-isoniazid antibodies were each present in 4/47 (9%) controls, respectively, but none among participants with flu-like reactions; anti-rifapentine IgG antibodies were not present in any participants with flu-like reactions or controls. CONCLUSIONS: We detected anti-rifapentine, anti-isoniazid, and anti-rifapentine metabolite antibodies, but the proportions of participants with antibodies were low, and did not differ between participants with flu-like reactions and those without such reactions. This suggests that flu-like reactions associated with 3HP and 9H were not antibody-mediated.
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The study of genetic minority variants is fundamental to the understanding of complex processes such as evolution, fitness, transmission, virulence, heteroresistance and drug tolerance in Mycobacterium tuberculosis (Mtb). We evaluated the performance of the variant calling tool LoFreq to detect de novo as well as drug resistance conferring minor variants in both in silico and clinical Mtb next generation sequencing (NGS) data. The in silico simulations demonstrated that LoFreq is a conservative variant caller with very high precision (≥96.7%) over the entire range of depth of coverage tested (30x to1000x), independent of the type and frequency of the minor variant. Sensitivity increased with increasing depth of coverage and increasing frequency of the variant, and was higher for calling insertion and deletion (indel) variants than for single nucleotide polymorphisms (SNP). The variant frequency limit of detection was 0.5% and 3% for indel and SNP minor variants, respectively. For serial isolates from a patient with DR-TB; LoFreq successfully identified all minor Mtb variants in the Rv0678 gene (allele frequency as low as 3.22% according to targeted deep sequencing) in whole genome sequencing data (median coverage of 62X). In conclusion, LoFreq can successfully detect minor variant populations in Mtb NGS data, thus limiting the need for filtering of possible false positive variants due to sequencing error. The observed performance statistics can be used to determine the limit of detection in existing whole genome sequencing Mtb data and guide the required depth of future studies that aim to investigate the presence of minor variants.
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Mycobacterium tuberculosis/genética , Secuenciación Completa del Genoma , Proteínas Bacterianas , Frecuencia de los Genes , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación INDEL , Mutación , Polimorfismo de Nucleótido Simple , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.
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Proteínas Bacterianas , ARN Polimerasas Dirigidas por ADN , Mycobacterium tuberculosis , Multimerización de Proteína , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/química , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Holoenzimas/química , Holoenzimas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Microscopía Electrónica de Transmisión , Factor sigma/metabolismo , Factor sigma/química , Factor sigma/genética , Cromatografía en GelRESUMEN
BACKGROUND: Tuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates. METHODS: Nineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP). RESULTS: All 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ. CONCLUSIONS: Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.
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Antituberculosos , Diarilquinolinas , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efectos de los fármacos , Diarilquinolinas/farmacología , Humanos , Antituberculosos/farmacología , Irán , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Mutación , Proteínas de Transporte de Membrana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Verapamilo/farmacologíaRESUMEN
Mycobacterium tuberculosis (Mtb) employs various strategies to manipulate the host's cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis (M. bovis) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage infection.
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Infecciones por Mycobacterium , Mycobacterium bovis , Mycobacterium tuberculosis , Humanos , Mycobacterium bovis/fisiología , Cromatografía Liquida , Espectrometría de Masas en Tándem , Macrófagos/metabolismo , Autofagia , Infecciones por Mycobacterium/metabolismo , Proteína Quinasa C/metabolismoRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), is a major global health issue, with around 10 million new cases annually. Advances in TB immunology have improved our understanding of host signaling pathways, leading to innovative therapeutic strategies. Inflammasomes, protein complexes organized by cytosolic pattern recognition receptors (PRRs), play a crucial role in the immune response to M. tb by activating caspase 1, which matures proinflammatory cytokines IL1ß and IL18. While inflammation is necessary to fight infection, excessive or dysregulated inflammation can cause tissue damage, highlighting the need for precise inflammasome regulation. Drug-resistant TB strains have spurred research into adjunctive host-directed therapies (HDTs) that target inflammasome pathways to control inflammation. Canonical and non-canonical inflammasome pathways can trigger excessive inflammation, leading to immune system exhaustion and M. tb spread. Novel HDT interventions can leverage precision medicine by tailoring treatments to individual inflammasome responses. Studies show that medicinal plant derivatives like silybin, andrographolide, and micheliolide and small molecules such as OLT1177, INF39, CY-09, JJ002, Ac-YVAD-cmk, TAK-242, and MCC950 can modulate inflammasome activation. Molecular tools like gene silencing and knockouts may also be used for severe TB cases. This review explores these strategies as potential adjunctive HDTs in fighting TB.
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Inflamasomas , Mycobacterium tuberculosis , Tuberculosis , Humanos , Inflamasomas/metabolismo , Inflamasomas/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Animales , Antituberculosos/uso terapéutico , Antituberculosos/farmacología , Transducción de Señal , Interacciones Huésped-Patógeno/inmunologíaRESUMEN
A series of 13 new 3-substituted 5-(5-nitro-2-furyl)-1,2,4-oxadiazoles was synthesized from different aminonitriles. All compounds were screened in the disc diffusion test at a 100 µg/mL concentration to determine the bacterial growth inhibition zone presence and diameter, and then the minimum inhibitory concentrations (MICs) were determined for the most active compounds by serial dilution. The compounds showed antibacterial activity against ESKAPE bacteria, predominantly suppressing the growth of 5 species out of the panel. Some compounds had similar or lower MICs against ESKAPE pathogens compared to ciprofloxacin, nitrofurantoin, and furazidin. In particular, 3-azetidin-3-yl-5-(5-nitro-2-furyl)-1,2,4-oxadiazole (2h) inhibited S. aureus at a concentration lower than all comparators. Compound 2e (5-(5-nitro-2-furyl)-3-[4-(pyrrolidin-3-yloxy)phenyl]-1,2,4-oxadiazole) was active against Gram-positive ESKAPE pathogens as well as M. tuberculosis. Differences in the molecular periphery led to high selectivity for the compounds. The induced-fit docking (IFD) modeling technique was applied to in silico research. Molecular docking results indicated the targeting of compounds against various nitrofuran-associated biological targets.
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Antibacterianos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Nitrofuranos , Nitrofuranos/farmacología , Nitrofuranos/química , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Diseño de Fármacos , Relación Estructura-Actividad , Oxadiazoles/química , Oxadiazoles/farmacología , Estructura Molecular , Staphylococcus aureus/efectos de los fármacosRESUMEN
The study revealed no effects of pregnancy and childbirth on the course of tuberculosis in female BALB/c mice after aerosol infection with Mycobacterium tuberculosis. However, we demonstrated a negative effect of tuberculosis infection on the fertility of infected females, which manifested in a longer period from mating to pregnancy and in a smaller litter size. Impaired reproductive function in response to the effect of the systemic infectious process was accompanied by the development of immunosuppression confirmed by an immunological test (delayed-type hypersensitivity to tuberculin) and the formation of genital tract dysbiosis during pregnancy and postpartum period.
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Fertilidad , Ratones Endogámicos BALB C , Mycobacterium tuberculosis , Tuberculosis , Animales , Femenino , Ratones , Fertilidad/fisiología , Embarazo , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiología , Disbiosis/microbiología , Disbiosis/inmunología , Hipersensibilidad Tardía/inmunología , Tamaño de la CamadaRESUMEN
Many persons with immunological tests indicating Mycobacterium tuberculosis infection, such as tuberculin skin tests or interferon-γ release assays, are at risk of progression to tuberculosis disease. Persons whose tests revert to negative may no longer be at such risk. Therefore, identifying the rate of test reversion, potentially indicating cure of M. tuberculosis infection, is an important area of investigation. In their accompanying article (Am J Epidemiol. 2023;192(12):1937-1943), Schwalb et al. extract data on test reversion from prechemotherapy literature and construct a model to predict the rate of reversion, and thus the likely cure of infection. Unfortunately, the incompleteness of the historical data and the use of imprecise definitions of test positivity and reversion lead to substantial misclassification and limit the usefulness of the model. Better definitions and improved tests will be needed in order to develop a clear picture of this aspect of the natural history of tuberculosis.
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Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Humanos , Tuberculosis/diagnóstico , Tuberculosis/epidemiología , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/epidemiología , Ensayos de Liberación de Interferón gamma , Prueba de TuberculinaRESUMEN
Bacterial chitinases serve to hydrolyse chitin as food source or as defence mechanism. Given that chitin is not produced by mammals, it is intriguing that Mycobacterium tuberculosis, an exclusively human pathogen harbours Rv1987, a probable chitinase and secretes it. Interestingly genes annotated as chitinases are widely distributed among Mycobacterium tuberculosis complex species, clinical isolates and other human pathogens M. abscessus and M. ulcerans. However, Mycobacterial chitinases are not characterized and hence the functions remain unknown. In the present study, we show that Rv1987 is a chitin and cellulose binding protein lacking enzymatic activity in contrary to its current annotation. Further, we show Rv1987 has moon lighting functions in M. tuberculosis pathobiology signifying roles of bacterial cellulose binding clusters in infections.
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Quitinasas , Mycobacterium tuberculosis , Animales , Humanos , Quitinasas/genética , Quitina/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Portadoras , Celulosa/metabolismo , Mamíferos/metabolismoRESUMEN
Minority variants of Mycobacterium tuberculosis harboring mutations conferring resistance can become dominant populations during tuberculosis (TB) treatment, leading to treatment failure. Our understanding of drug-resistant within-host subpopulations and the frequency of resistance-conferring mutations in minority variants remains limited. M. tuberculosis sequences recovered from liquid cultures of culture-confirmed TB cases notified between January 2017 and December 2021 in New South Wales, Australia were examined. Potential drug resistance-conferring minority variants were identified using LoFreq, and mixed populations of different M. tuberculosis strains (≥100 SNPs apart) were examined using QuantTB. A total of 1831 routinely sequenced M. tuberculosis strains were included in the analysis. Drug resistance-conferring minority variants were detected in 3.5% (65/1831) of sequenced cultures; 84.6% (55/65) had majority strains that were drug susceptible and 15.4% (10/65) had majority strains that were drug resistant. Minority variants with high-confidence drug resistance-conferring mutations were 1.5 times more common when the majority strains were drug resistant. Mixed M. tuberculosis strain populations were documented in 10.0% (183/1831) of specimens. Minority variants with high-confidence drug resistance-conferring mutations were more frequently detected in mixed M. tuberculosis strain populations (2.7%, 5/183) than in single strain populations (0.6%, 10/1648; P = 0.01). Drug-resistant minority variants require monitoring in settings that implement routine M. tuberculosis sequencing. The frequency with which drug-resistant minority variants are detected is likely influenced by pre-culture requirement. Culture-independent sequencing methods should provide a more accurate reflection of drug-resistant subpopulations.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis , Humanos , Mycobacterium tuberculosis/genética , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Mutación , Tuberculosis/tratamiento farmacológico , Genómica , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
AIM: ß-defensins 2 and -3 (HBD-2 and HBD-3) and cathelicidin LL-37 are host defense peptides (HDPs) that play a crucial role in the immune response against mycobacteria. Given our former studies in tuberculosis patients wherein their plasma levels of such peptides correlated with steroid hormone concentrations, we now studied the reciprocal influence of cortisol and/or dehydroepiandrosterone (DHEA) on HDPs biosynthesis and LL-37 on adrenal steroidogenesis. MAIN METHODS: Cultures of macrophages derived from the THP-1 line were treated with cortisol (10-6M) and/or DHEA (10-6M and 10-7M) and stimulated with irradiated M. tuberculosis (Mi) or infected M. tuberculosis strain H37Rv to assess cytokine production, HDPs, reactive oxygen species (ROS) and colony forming units. Cultures of NCI-H295-R adrenal line were treated with LL37 (5, 10, and 15 µg/ml) for 24 h to further measure cortisol and DHEA levels together with steroidogenic enzyme transcripts. KEY FINDINGS: In macrophages, M. tuberculosis produced an increase of IL-1ß, TNFα, IL-6, IL-10, LL-37, HBD-2, and HBD-3 levels, irrespective of DHEA treatment. Adding cortisol to M. tuberculosis-stimulated cultures (with or without DHEA) decreased the amounts of these mediators, compared to only stimulated cultures. Although M. tuberculosis reduced ROS levels, DHEA increased these values in addition to diminishing intracellular mycobacterial growth (no matter cortisol treatment). In turn, studies on adrenal cells showed that LL-37 reduced the production of cortisol and DHEA besides modifying transcripts for some steroidogenic enzymes. SIGNIFICANCE: while adrenal steroids seem to influence the production of HDPs, the former compounds are also likely to modulate adrenal biogenesis.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Deshidroepiandrosterona , Hidrocortisona , Péptidos Catiónicos Antimicrobianos , Especies Reactivas de Oxígeno , EsteroidesRESUMEN
BACKGROUND: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown. METHODS: A tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG. RESULTS: Deletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death. CONCLUSIONS: Our findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy.
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Corismato Mutasa , Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculosis , Animales , Ratones , Apoptosis/genética , Corismato Mutasa/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Mitocondrias/genética , Mitocondrias/metabolismo , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Tuberculosis/genética , Tuberculosis/microbiologíaRESUMEN
Mammalian cell entry (mce) operons play a vital role in cell invasion and survival of M. tuberculosis. Of the mce genes, the function of Rv0590A is still unknown. The present study was performed to investigate the function and immunogenic properties of the protein Rv0590A. Human leukemia monocytic cell line (THP-1) derived macrophages were infected with M. tuberculosis H37Rv at 3, 6, and 24 h of infection. The maximum colony forming units (CFU) were observed at 6 h (p < 0.005), followed by 3 h after infection. M. tuberculosis H37Rv and clinical isolates representative of Delhi/CAS, EAI, Beijing, Haarlem and Euro-American-superlineage were included in the study for expression analysis of mce1A, mce2A, mce3A, mce4A, and Rv0590A genes. Maximum upregulation of all mce genes was observed at 3 h of infection. All the five clinical isolates and H37Rv upregulated Rv0590A at various time points. Macrophage infection with M. tuberculosis H37Rv-overexpressing Rv0590A gene showed higher intracellular CFU as compared to that of wild-type H37Rv. Further, purified Rv0590A protein stimulated the production of TNFα, IFNγ, and IL-10 in macrophages. Thus, Rv0590A was found to be involved in cell invasion and showed good immunological response.
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Mycobacterium tuberculosis , Tuberculosis , Animales , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Internalización del Virus , Mycobacterium tuberculosis/genética , Antígenos Bacterianos/genética , MamíferosRESUMEN
The EccC enzyme of Mycobacterium tuberculosis ESX-1 secretion system is involved in EsxAB virulence factor secretion and offers an attractive target for antivirulence inhibitors development against M. tuberculosis. The EccCb1 polypeptide of the EccC enzyme contains two Ftsk/SpoIIIE type ATPase domains (D2 and D3) and binds to the EsxAB factor at the C-terminal region of the D3 domain. In the current study, we have determined a low-resolution structure of EccCb1, and its mechanism involved in ATPase activity and EsxAB factor binding. Small-angle X-ray scattering data yielded a double hexameric ring structure of EccCb1 in solution and was further confirmed by SEC-MALS and dynamic light scattering. ATPase activity of wild-type, D2, and D3 mutants showed that D2-K90A and D3-K382A mutations led to a complete loss of enzyme activity. The full-length EccCb1 showed â¼3.7-fold lower catalytic efficiency than D2 domain and â¼1.7 fold lower than D3 domain. The EsxAB factor binds EccCb1 with Kd â¼ 11.3 ± 0.6â nM and its affinity is enhanced â¼2 fold in presence of ATP + Mg2+. These data indicate the involvement of ATPase activity in EsxAB factor translocation. Molecular dynamics simulation on wild-type, ATP + Mg2+, and EsxAB + ATP + Mg2+ bound EccCb1 double-ring structure showed enhanced stability of enzyme upon ATP + Mg2+ and EsxAB binding. Overall, our study showed a low-resolution structure of EccCb1, and the mechanism involved in ATPase activity and EsxAB factor recognition, which can be targeted for the development of antivirulence drugs against M. tuberculosis.
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Mycobacterium tuberculosis , Sistemas de Secreción Tipo VII , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Humanos , Magnesio/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculosis/microbiología , Sistemas de Secreción Tipo VII/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Cerebrovascular complications of central nervous system tuberculosis (TB) are predictors of poor prognosis and adverse outcomes. These complications are mainly intracranial arterial involvement, with occasional venous involvement. Here, we present a 67-year-old woman with concurrent cerebral infarction and intracranial tuberculoma induced by the carotid plaque complicated by miliary tuberculosis. Mycobacterium tuberculosis was observed on the luminal side of the carotid plaques in pathological specimens. Treatment with anti-TB drugs alone would likely not cure the patient, as M. tuberculosis would continue to disseminate. Endarterectomy could directly remove the embolic source, and a complete cure was achieved.
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Tuberculoma Intracraneal , Tuberculoma , Tuberculosis Miliar , Femenino , Humanos , Anciano , Tuberculoma Intracraneal/complicaciones , Tuberculoma Intracraneal/tratamiento farmacológico , Tuberculosis Miliar/complicaciones , Tuberculosis Miliar/tratamiento farmacológico , Antituberculosos/uso terapéutico , Infarto Cerebral , Tuberculoma/complicaciones , Tuberculoma/tratamiento farmacológicoRESUMEN
Tuberculosis (TB) of the central nervous system (CNS) presents high mortality due to brain damage and inflammation events. The formation and deposition of immune complexes (ICs) in the brain microvasculature during Mycobacterium tuberculosis (Mtb) infection are crucial for its pathobiology. The relevance of ICs to Mtb antigens in the pathogenesis of CNS-TB has been poorly explored. Here, we aimed to establish a murine experimental model of ICs-mediated brain vasculitis induced by cell wall antigens of Mtb. We administered a cell wall extract of the prototype pathogenic Mtb strain H37Rv to male BALB/c mice by subcutaneous and intravenous routes. Serum concentration and deposition of ICs onto blood vessels were determined by polyethylene glycol precipitation, ELISA, and immunofluorescence. Histopathological changes in the brain, lung, spleen, liver, and kidney were evaluated by hematoxylin and eosin staining. Our results evidenced that vasculitis developed in the studied tissues. High serum levels of ICs and vascular deposition were evident in the brain, lung, and kidneys early after the last cell wall antigen administration. Cell wall Mtb antigens induce strong type III hypersensitivity reactions and the development of systemic vasculitis with brain vascular changes and meningitis, supporting a role for ICs in the pathogenesis of TB.
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Mycobacterium tuberculosis , Tuberculosis , Vasculitis , Masculino , Animales , Ratones , Complejo Antígeno-Anticuerpo , Modelos Animales de Enfermedad , Tuberculosis/microbiología , Antígenos Bacterianos , Pared CelularRESUMEN
Tuberculosis is a major global threat to human health. Since the widely used BCG vaccine is poorly effective in adults, there is a demand for the development of a new type of boost tuberculosis vaccine. We designed a novel intranasal tuberculosis vaccine candidate, TB/FLU-04L, which is based on an attenuated influenza A virus vector encoding two mycobacterium antigens, Ag85A and ESAT-6. As tuberculosis is an airborne disease, the ability to induce mucosal immunity is one of the potential advantages of influenza vectors. Sequences of ESAT-6 and Ag85A antigens were inserted into the NS1 open reading frame of the influenza A virus to replace the deleted carboxyl part of the NS1 protein. The vector expressing chimeric NS1 protein appeared to be genetically stable and replication-deficient in mice and non-human primates. Intranasal immunization of C57BL/6 mice or cynomolgus macaques with the TB/FLU-04L vaccine candidate induced Mtb-specific Th1 immune response. Single TB/FLU-04L immunization in mice showed commensurate levels of protection in comparison to BCG and significantly increased the protective effect of BCG when applied in a "prime-boost" scheme. Our findings show that intranasal immunization with the TB/FLU-04L vaccine, which carries two mycobacterium antigens, is safe, and induces a protective immune response against virulent M. tuberculosis.
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Vacunas contra la Influenza , Gripe Humana , Mycobacterium tuberculosis , Vacunas contra la Tuberculosis , Tuberculosis , Adulto , Ratones , Humanos , Animales , Vacuna BCG , Antígenos Bacterianos/genética , Ratones Endogámicos C57BL , Tuberculosis/prevención & control , Proteínas Bacterianas/genética , Aciltransferasas/genéticaRESUMEN
The human stress hormones catecholamines play a critical role in communication between human microbiota and their hosts and influence the outcomes of bacterial infections. However, it is unclear how M. tuberculosis senses and responds to certain types of human stress hormones. In this study, we screened several human catecholamine stress hormones (epinephrine, norepinephrine, and dopamine) for their effects on Mycobacterium growth. Our results showed that epinephrine significantly stimulated the growth of M. tuberculosis in the serum-based medium as well as macrophages. In silico analysis and molecular docking suggested that the extra-cytoplasmic domain of the MprB might be the putative adrenergic sensor. Furthermore, we showed that epinephrine significantly enhances M. tuberculosis biofilm formation, which has distinct texture composition, antibiotic resistance, and stress tolerance. Together, our data revealed the effect and mechanism of epinephrine on the growth and biofilm formation of M. tuberculosis, which contributes to the understanding of the environmental perception and antibiotic resistance of M. tuberculosis and provides important clues for the understanding of bacterial pathogenesis and the development of novel antibacterial therapeutics.