RESUMEN
The covalent modification of bacterial (lipo)polysaccharides with discrete substituents may impact their biosynthesis, export and/or biological activity. Whether mycobacteria use a similar strategy to control the biogenesis of its cell envelope polysaccharides and modulate their interaction with the host during infection is unknown despite the report of a number of tailoring substituents modifying the structure of these glycans. Here, we show that discrete succinyl substituents strategically positioned on Mycobacterium tuberculosis (Mtb) lipoarabinomannan govern the mannose-capping of this lipoglycan and, thus, much of the biological activity of the entire molecule. We further show that the absence of succinyl substituents on the two main cell envelope glycans of Mtb, arabinogalactan and lipoarabinomannan, leads to a significant increase of pro-inflammatory cytokines and chemokines in infected murine and human macrophages. Collectively, our results validate polysaccharide succinylation as a critical mechanism by which Mtb controls inflammation.
Asunto(s)
Lipopolisacáridos , Tuberculosis , Humanos , Animales , Ratones , Manosa , InflamaciónRESUMEN
Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100-fold enhancement in M.â tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M.â tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Sondas Moleculares , Trehalosa , Colorantes FluorescentesRESUMEN
Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis. Despite intensive TB control campaigns, there are sporadic outbreaks of bovine TB in regions declared TB free. It is unclear how M. bovis is able to survive in the environment for long periods of time. We hypothesized that Free-living amoebae (FLA), as ubiquitous inhabitants of soil and water, may act as long-term reservoirs of M. bovis in the environment. In our model, M. bovis would be taken up by amoebal trophozoites, which are the actively feeding, replicating and mobile form of FLA. Upon exposure to hostile environmental conditions, infected FLA will encyst and provide an intracellular niche allowing their M. bovis cargo to persist for extended periods of time. Here, we show that five FLA species (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba lenticulata, Vermamoeba vermiformis and Dictyostellium discoideum) are permissive to M. bovis infection and that the M. bovis bacilli may survive within the cysts of four of these species for over 60 days. We further show that exposure of M. bovis-infected trophozoites and cysts to Balb/c mice leads to pulmonary TB. This work describes for the first time that FLA carrying M. bovis can transmit TB.
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Amebozoos/microbiología , Reservorios de Enfermedades/microbiología , Mycobacterium bovis/crecimiento & desarrollo , Acanthamoeba/microbiología , Animales , Bovinos , Dictyostelium/microbiología , Ratones , Ratones Endogámicos BALB C , Mycobacterium bovis/patogenicidad , Tuberculosis Bovina/microbiología , Tuberculosis Bovina/transmisiónRESUMEN
A library of 2-aminobenzimidazole derivatives was screened for the ability to suppress ß-lactam resistance in Mycobacterium smegmatis. Several non-bactericidal compounds were identified that reversed intrinsic resistance to ß-lactam antibiotics in a manner distinct from ß-lactamase inhibitors. Activity also translates to M.â tuberculosis, with a lead compound from this study potently suppressing carbenicillin resistance in multiple M.â tuberculosis strains (including multidrug-resistant strains). Preliminary mechanistic studies revealed that the lead compounds act through a mechanism distinct from that of traditional ß-lactamase inhibitors.
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Antibacterianos/farmacología , Bencimidazoles/farmacología , Lactamas/farmacología , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Bencimidazoles/química , Descubrimiento de Drogas , Lactamas/síntesis química , Lactamas/química , Estructura Molecular , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
In recent years, whole-cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further rendered M. tuberculosis hypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.
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Antituberculosos/farmacología , Proteínas Bacterianas/genética , Pulmón/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Carga Bacteriana/efectos de los fármacos , Transporte Biológico , Ciprofloxacina/farmacología , Modelos Animales de Enfermedad , Doxiciclina/farmacología , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Isoniazida/farmacología , Pulmón/microbiología , Pulmón/patología , Proteínas de Transporte de Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/patología , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
Over the last 10 years, Mycobacterium abscessus group strains have emerged as important human pathogens, which are associated with significantly higher fatality rates than any other rapidly growing mycobacteria. These opportunistic pathogens are widespread in the environment and can cause a wide range of clinical diseases, including skin, soft tissue, central nervous system, and disseminated infections; by far, the most difficult to treat is the pulmonary form. Infections with M. abscessus are often multidrug-resistant (MDR) and require prolonged treatment with various regimens and, many times, result in high mortality despite maximal therapy. We report here the evaluation of diverse mouse infection models for their ability to produce a progressive high level of infection with M. abscessus. The nude (nu/nu), SCID (severe combined immunodeficiency), gamma interferon knockout (GKO), and granulocyte-macrophage colony-stimulating factor (GMCSF) knockout mice fulfilled the criteria for an optimal model for compound screening. Thus, we set out to assess the antimycobacterial activity of clarithromycin, clofazimine, bedaquiline, and clofazimine-bedaquiline combinations against M. abscessus-infected GKO and SCID murine infection models. Treatment of GKO and SCID mice with a combination of clofazimine and bedaquiline was the most effective in decreasing the M. abscessus organ burden.
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Antibacterianos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Mycobacterium/efectos de los fármacos , Animales , Claritromicina/farmacología , Clofazimina/farmacología , Diarilquinolinas/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Ratones , Ratones Noqueados , Ratones SCID , Pruebas de Sensibilidad Microbiana , Infecciones por MycobacteriumRESUMEN
Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.
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Proteínas Bacterianas/metabolismo , Inmunidad Innata , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Neuropéptidos/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Tuberculosis Pulmonar/inmunología , Proteína de Unión al GTP rac1/metabolismo , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/microbiología , Línea Celular Transformada , Células Cultivadas , Femenino , Humanos , Macrófagos/enzimología , Macrófagos/microbiología , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/fisiología , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/genética , Nucleósido-Difosfato Quinasa/antagonistas & inhibidores , Nucleósido-Difosfato Quinasa/genética , Oligorribonucleótidos Antisentido , Fagosomas/enzimología , Fagosomas/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tuberculosis Pulmonar/enzimología , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patología , Virulencia , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genéticaRESUMEN
Impaired glucose tolerance and type 2 diabetes were induced in guinea pigs to model the emerging comorbidity of Mycobacterium tuberculosis infection in diabetic patients. Type 2 diabetes mellitus was induced by low-dose streptozotocin in guinea pigs rendered glucose intolerant by first feeding a high-fat, high-carbohydrate diet before M. tuberculosis exposure. M. tuberculosis infection of diabetic guinea pigs resulted in severe and rapidly progressive tuberculosis (TB) with a shortened survival interval, more severe pulmonary and extrapulmonary pathology, and a higher bacterial burden compared with glucose-intolerant and nondiabetic controls. Compared with nondiabetics, diabetic guinea pigs with TB had an exacerbated proinflammatory response with more severe granulocytic inflammation and higher gene expression for the cytokines/chemokines interferon-γ, IL-17A, IL-8, and IL-10 in the lung and for interferon-γ, tumor necrosis factor-α, IL-8, and monocyte chemoattractant protein-1 in the spleen. TB disease progression in guinea pigs with impaired glucose tolerance was similar to that of nondiabetic controls in the early stages of infection but was more severe by day 90. The guinea pig model of type 2 diabetes-TB comorbidity mimics important features of the naturally occurring disease in humans. This model will be beneficial in understanding the complex pathogenesis of TB in diabetic patients and to test new strategies to improve TB and diabetes control when the two diseases occur together.
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Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/inmunología , Tuberculosis/complicaciones , Tuberculosis/inmunología , Animales , Comorbilidad , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Citometría de Flujo , Cobayas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tuberculosis/patologíaRESUMEN
Two lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), play various, albeit incompletely defined, roles in the interactions of mycobacteria with the host. Growing evidence points to the modification of LM and LAM with discrete covalent substituents as a strategy used by these bacteria to modulate their biological activities. One such substituent, originally identified in Mycobacterium tuberculosis (Mtb), is a 5-methylthio-d-xylose (MTX) sugar, which accounts for the antioxidative properties of LAM. The widespread distribution of this motif across Mtb isolates from several epidemiologically important lineages have stimulated interest in MTX-modified LAM as a biomarker of tuberculosis infection. Yet, several lines of evidence indicate that MTX may not be restricted to Mtb and that this motif may substitute more acceptors than originally thought. Using a highly specific monoclonal antibody to the MTX capping motif of Mtb LAM, we here show that MTX motifs not only substitute the mannoside caps of LAM but also the mannan core of LM in Mtb. MTX substituents were also found on the LM and LAM of pathogenic, slow-growing nontuberculous mycobacteria. The presence of MTX substituents on the LM and LAM from Mtb enhances the pro-apoptotic properties of both lipoglycans on LPS-stimulated THP-1 macrophages. A comparison of the cytokines and chemokines produced by resting and LPS-activated THP-1 cells upon exposure to MTX-proficient versus MTX-deficient LM further indicates that MTX substituents confer anti-inflammatory properties upon LM. These findings add to our understanding of the glycan-based strategies employed by slow-growing pathogenic mycobacteria to alter the host immune response to infection.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Lipopolisacáridos , Tuberculosis/microbiologíaRESUMEN
Bacille Calmette-Guerin (BCG) is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease. However, BCG has limited efficacy, necessitating the development of better vaccines. Non-tuberculous mycobacteria (NTMs) are opportunistic pathogens present ubiquitously in the environment. TB endemic countries experience higher exposure to NTMs, but previous studies have not elucidated the relationship between NTM exposure and BCG efficacy against TB. Therefore, we develop a mouse model (BCG + NTM) to simulate human BCG immunization regime and continuous NTM exposure. BCG + NTM mice exhibit superior and prolonged protection against pulmonary TB, with increased B cell influx and anti-Mtb antibodies in serum and airways, compared with BCG alone. Notably, spatial transcriptomics and immunohistochemistry reveal that BCG + NTM mice formed B cell aggregates with features of germinal center development, which correlate with reduced Mtb burden. Our studies suggest a direct relationship between NTM exposure and TB protection, with B cells playing a crucial role.
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Mycobacterium tuberculosis , Tuberculosis Pulmonar , Tuberculosis , Ratones , Humanos , Animales , Vacuna BCG , Micobacterias no Tuberculosas , Inmunidad CelularRESUMEN
Biosynthesis of phosphatidylinositol (PI)-containing lipoarabinomannan (LAM) and lipomannan (LM) of Mycobacterium spp. follows a conserved pathway involving multiple membrane-associated, substrate-specific mannosyltransferases (ManTs) responsible for the sequential addition of alpha-mannopyranosyl (Manp) units donated by decaprenyl-P-Manp on the periplasmic side of the plasma membrane. Because of their receptor-binding and immunomodulatory properties, the alpha(1-->2)-linked di- and tri-Manp motifs that functionalize the nonreducing arabinan termini of LAM (ManLAM) in Mycobacterium tuberculosis are of crucial importance. We now show that the M. tuberculosis ManT, Rv2181, is required for the addition of these alpha(1-->2)-linked Manp residues but also at other locations of the LAM molecule. Structural analyses of the LM and LAM variants produced by a M. tuberculosis Rv2181 knockout mutant revealed the presence of but a single Manp residue on the nonreducing arabinan termini of LAM and also a complete absence of alpha(1-->2)-linked Man branching on the mannan backbones of LM and LAM. A recombinant strain was constructed in ManLAM-deficient Mycobacterium smegmatis that coexpressed Rv2181 and Rv1635c-the ManT responsible for the addition of the first Manp capping residue of ManLAM. Analysis revealed LAM termini fully capped with di- and tri-Manp motifs in addition to alpha(1-->2)Man branching on the mannan backbones of LM and LAM, confirming the involvement of the alpha(1-->2)ManT Rv2181 in the dual role of Man capping and mannan-core branching, and in the process generated a rapidly growing, ManLAM-containing strain, a tool for the study of the role of ManLAM in the pathogenesis of tuberculosis.
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Lipopolisacáridos/metabolismo , Manosa/metabolismo , Manosiltransferasas/metabolismo , Mycobacterium tuberculosis/enzimología , Alelos , Electroforesis en Gel de Poliacrilamida , Lipopolisacáridos/análisis , Lipopolisacáridos/química , Metilación , Mutación/genética , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Fenotipo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system.
RESUMEN
The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this preliminary evaluation provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results of this preliminary evaluation suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have potential utility in the preparation of one such vaccine candidate.
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Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel flow cytometry analysis pipeline to identify a plethora of cell populations efficiently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population differences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a filtering step refines the data from all identified cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The pipeline's modularity allows customization of statistical testing, adoption of alternative initial gating steps, and incorporation of other datasets. Validation of this pipeline through manual gating of two datasets (murine splenocytes and human whole blood) confirmed its accuracy in identifying even rare subsets. Lastly, this pipeline can be applied in all disciplines utilizing flow cytometry regardless of cytometer or panel design. The code is available at https://github.com/aef1004/cyto-feature_engineering.
Asunto(s)
Citodiagnóstico/métodos , Susceptibilidad a Enfermedades/inmunología , Citometría de Flujo , Animales , Biomarcadores , Células Sanguíneas/metabolismo , Citometría de Flujo/métodos , Humanos , Inmunofenotipificación , Ratones , Mycobacterium tuberculosis/inmunología , Fenotipo , Tuberculosis/diagnóstico , Tuberculosis/inmunología , Tuberculosis/microbiologíaRESUMEN
Flow cytometry allows the visualization of physical, functional, and/or biological properties of cells including antigens, cytokines, size, and complexity. With increasingly large flow cytometry panels able to analyze up to 50 parameters, there is a need to standardize flow cytometry protocols to achieve high-quality data that can be input into analysis algorithms. Without this clean data, algorithms may incorrectly categorize the cell populations present in the samples. In this protocol, we outline a comprehensive methodology to prepare samples for polychromatic flow cytometry. The use of multiple washing steps and rigorous controls creates high-quality data with good separation between cell populations. Experimental data acquired using this protocol can be analyzed via computational algorithms that perform end-to-end analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of single-cell suspension for flow cytometry Support Protocol 1: Lung preparation Support Protocol 2: Counting cells on a flow cytometer Basic Protocol 2: Surface and intracellular flow cytometry staining Support Protocol 3: Single-color bead controls.
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Citometría de Flujo/métodos , Citometría de Flujo/normas , Animales , Recuento de Células , Espacio Intracelular/metabolismo , Pulmón/citología , Ratones Endogámicos C57BL , Análisis de la Célula Individual , Bazo/citología , Linfocitos T/inmunologíaRESUMEN
Tuberculosis (TB) is a chronic inflammatory disease that is often associated with alterations in systemic and cellular metabolism that resolves following successful antimicrobial drug treatment. We hypothesized that altered systemic glucose metabolism as a consequence of Mycobacterium tuberculosis (Mtb) infection, contributes to TB pathogenesis, and when normalized with anti-glycemic drugs would improve clinical outcomes. To test this hypothesis, guinea pigs were treated daily with the anti-diabetic drug metformin starting 4 weeks prior or concurrent with aerosol exposure to the H37Rv strain of Mtb. In the chronic stages of infection, Mtb infected metformin-treated animals had restored systemic insulin sensitivity but remained glucose intolerant as determined by oral glucose tolerance testing. Despite persistent glucose intolerance, metformin-treated guinea pigs had a 2.8-fold reduction in lung lesion burden and a 0.7 log decrease in CFUs. An alternative hypothesis that metformin treatment improved clinical disease by having a direct effect on immune cell energy metabolism was tested using extracellular flux analysis and flow cytometry. The proinflammatory immune response to Mtb infection in untreated guinea pigs was associated with a marked increase in energy metabolism (glycolysis and mitochondrial respiration) of peripheral blood mononuclear cells (PBMCs), which was normalized in metformin-treated guinea pigs. Moreover, both CD4+ and CD8+ T lymphocytes from Mtb infected, metformin treated animals maintained a more normal mitochondrial membrane potential while those isolated from untreated animals had persistent mitochondrial hyperpolarization. These data suggest that metformin promotes natural host resistance to Mtb infection by maintaining immune cell metabolic homeostasis and function during the chronic stages of active TB disease.
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Hipoglucemiantes/uso terapéutico , Metformina/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Citocinas/metabolismo , Metabolismo Energético/efectos de los fármacos , Glucosa/metabolismo , Intolerancia a la Glucosa/tratamiento farmacológico , Cobayas , Resistencia a la Insulina , Pulmón/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Superóxidos/metabolismo , Linfocitos T/metabolismo , Tuberculosis Pulmonar/metabolismo , Tuberculosis Pulmonar/patologíaRESUMEN
Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (TCM), follicular helper T cells (TFH) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted.
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Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculosis , Sistemas de Secreción Tipo VII , Animales , Antígenos Bacterianos , Vacuna BCG , Cobayas , Inmunización Secundaria , Ratones , Mycobacterium tuberculosis/genética , Tuberculosis/prevención & control , VacunaciónRESUMEN
Many vaccines against childhood diseases are administered early after birth, but vaccine development studies frequently test efficacy in adult rather than in neonatal animal models. In countries with endemic tuberculosis (TB), Bacillus Calmette-Guerin (BCG) is administered as part of the neonatal vaccine regimen because it prevents against the disseminated form of TB in children, although it has variable efficacy against pulmonary TB. Several promising new vaccines against TB are currently being tested in adult animal models. Here we evaluated neonatal piglets as an animal model to test vaccine efficacy. For this purpose, minipigs were vaccinated or not with BCG 48â¯h after birth and their immune response followed longitudinally until adolescence. We characterized the memory and activation phenotype of T cells, cytokine profile, and monocyte activation in response to BCG stimulation from 4 weeks of age into adolescence- age of 24 weeks. Immunological responses in vaccinated and non-vaccinated animals were further monitored upon infection with a low dose exposure to Mycobacterium tuberculosis strain HN878 via the aerosol route. Comparing the immunological response elicited by BCG vaccination in minipigs vs similar studies in infants, suggest that minipigs have the potential to serve as an effective neonatal animal model for vaccine development.
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Vacuna BCG/inmunología , Modelos Animales de Enfermedad , Mycobacterium tuberculosis/inmunología , Porcinos Enanos/inmunología , Tuberculosis Pulmonar/inmunología , Animales , Animales Recién Nacidos , Citocinas/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Inmunogenicidad Vacunal , Memoria Inmunológica , Inmunofenotipificación , Estudios Longitudinales , Activación de Linfocitos , Masculino , Monocitos/inmunología , Porcinos , Tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & controlRESUMEN
There is an urgent need to develop new drugs against tuberculosis. In particular, it is critical to target drug tolerant Mycobacterium tuberculosis (M. tuberculosis), responsible, in part, for the lengthy antibiotic regimen required for treatment. We previously postulated that the presence of in vivo biofilm-like communities of M. tuberculosis could contribute to this drug tolerance. Consistent with this hypothesis, certain 2-aminoimidazole (2-AIs) molecules with anti-biofilm activity were shown to revert mycobacterial drug tolerance in an in vitro M. tuberculosis biofilm model. While exploring their mechanism of action, it was serendipitously observed that these 2-AI molecules also potentiated ß-lactam antibiotics by affecting mycobacterial protein secretion and lipid export. As these two bacterial processes are energy-dependent, herein it was evaluated if 2-AI compounds affect mycobacterial bioenergetics. At low concentrations, 2B8, the lead 2-AI compound, collapsed both components of the proton motive force, similar to other cationic amphiphiles. Interestingly, however, the minimum inhibitory concentration of 2B8 against M. tuberculosis correlated with a higher drug concentration determined to interfere with the mycobacterial electron transport chain. Collectively, this study elucidates the mechanism of action of 2-AIs against M. tuberculosis, providing a tool to better understand mycobacterial bioenergetics and develop compounds with improved anti-mycobacterial activity.
Asunto(s)
Biopelículas/efectos de los fármacos , Transporte de Electrón/efectos de los fármacos , Imidazoles/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Fuerza Protón-Motriz/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Antituberculosos/farmacología , Biopelículas/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/crecimiento & desarrollo , Consumo de Oxígeno/efectos de los fármacos , Tuberculosis/microbiologíaRESUMEN
In endemic countries more than 20% of tuberculosis (TB) cases are in infants and children. Current animal models study TB during adulthood but animal models for infant TB are scarce. Here we propose that minipigs can be used as an animal model to study adult, adolescent and infant TB including natural transmission. In these studies, two-month old minipigs (representing infant age in humans) and six-month old minipigs (representing adolescence in humans) were infected via the aerosol route with hyper-virulent clinical strain W-Beijing Mycobacterium tuberculosis (Mtb) HN878 and were monitored for 11 or 36 weeks post-challenge, respectively. In the same studies, infected and unchallenged animals were housed together. Viable bacteria were recovered from pulmonary and thoracic lymph nodes from both -infected and their initially unchallenged natural contacts. Bacillary load, gross lesions and histopathology revealed similarities to the spectrum of disease observed in human TB. The study did not reach terminal end point, thus it was not possible to annotate definitive clinical symptoms of active TB. The results demonstrated that minipigs are experimental hosts of Mtb HN878, and the pathology developed in their lungs resembles pathological findings described in human TB. Importantly, within communities of Mtb infected minipigs natural transmission occurs.