ABSTRACT
BACKGROUND: Although previous studies have shown that vitamin A deficiency is associated with incident tuberculosis (TB) disease, the direction of the association has not been established. We investigated the impact of vitamin A deficiency on TB disease progression. METHODS: We conducted a longitudinal cohort study nested within a randomized clinical trial among HIV-infected patients in Haiti. We compared serial vitamin A levels in individuals who developed TB disease to controls matched on age, gender, follow-up time, and time to antiretroviral therapy initiation. We also evaluated histopathology, bacterial load, and immune outcomes in TB infection in a guinea pig model of dietary vitamin A deficiency. RESULTS: Among 773 participants, 96 developed incident TB during follow-up, 62.5% (60) of whom had stored serum samples obtained 90-365 days before TB diagnosis. In age- and sex- adjusted and multivariate analyses, respectively, incident TB cases were 3.99 times (95% confidence interval [CI], 2.41 to 6.60) and 3.59 times (95% CI, 2.05 to 6.29) more likely to have been vitamin A deficient than matched controls. Vitamin A-deficient guinea pigs manifested more extensive pulmonary pathology, atypical granuloma morphology, and increased bacterial growth after experimental TB infection. Reintroduction of dietary vitamin A to deficient guinea pigs after established TB disease successfully abrogated severe disease manifestations and altered cellular immune profiles. CONCLUSIONS: Human and animal studies support the role of baseline vitamin A deficiency as a determinant of future TB disease progression.
Subject(s)
Latent Tuberculosis , Tuberculosis , Vitamin A Deficiency , Vitamin D Deficiency , Humans , Animals , Guinea Pigs , Vitamin A , Risk Factors , Longitudinal Studies , Vitamin D Deficiency/complications , Tuberculosis/complications , Latent Tuberculosis/complications , Disease ProgressionABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Benzimidazoles/pharmacology , Lactams/pharmacology , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , beta-Lactamase Inhibitors/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Benzimidazoles/chemistry , Drug Discovery , Lactams/chemical synthesis , Lactams/chemistry , Molecular Structure , Mycobacterium smegmatis/enzymology , Mycobacterium tuberculosis/enzymology , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/metabolismABSTRACT
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.
Subject(s)
Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/immunology , Tuberculosis/complications , Tuberculosis/immunology , Animals , Comorbidity , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Flow Cytometry , Guinea Pigs , Reverse Transcriptase Polymerase Chain Reaction , Tuberculosis/pathologyABSTRACT
The formation of advanced glycation end-products (AGE) as a result of the action of reducing sugars on host macromolecules plays a role in increased morbidity of diabetic patients. There are currently no clinically available therapeutics for the prevention or eradication of AGEs. Following our previous identification of 2-aminoimidazole (2-AI) based AGE inhibitors and breakers, we now report the use of a rapid, scalable, two-step procedure to access a second generation of 2-AI based anti-AGE compounds from commercially available amino acids. Several second generation compounds exhibit increased AGE inhibition and breaking activty compared to the first generation compounds and to the known AGE inhibitor aminoguanidine.
Subject(s)
Glycation End Products, Advanced/antagonists & inhibitors , Imidazoles/pharmacology , Glycation End Products, Advanced/metabolism , Imidazoles/chemical synthesis , Imidazoles/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
Advanced glycation end-products (AGEs), unregulated modifications to host macromolecules that occur as a result of metabolic dysregulation, play a role in many diabetes related complications, inflammation and aging, and may lead to increased cardiovascular risk. Small molecules that have the ability to inhibit AGE formation, and even break preformed AGEs have enormous therapeutic potential in the treatment of these disease states. We report the screening of a series of 2-aminoimidazloles for anti-AGE activity, and the identification of a bis-2-aminoimidazole lead compound that possesses superior AGE inhibition and breaking activity compared to the known AGE inhibitor aminoguanidine.
ABSTRACT
Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy.
Subject(s)
Copper/pharmacology , Drug Resistance, Bacterial , Mycobacterium tuberculosis/drug effects , Tuberculosis/microbiology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Copper/metabolism , Copper Sulfate/metabolism , Copper Sulfate/pharmacology , Guinea Pigs , Lung/microbiology , Lung/pathology , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Mutation , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/pathogenicity , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Spleen/microbiology , Spleen/pathology , Virulence/geneticsABSTRACT
Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. We formulated a vaccine based on the H1 antigen (fusion protein of Ag85b and ESAT-6) adjuvanted with NanoSTING. Intranasal immunization of NanoSTING-H1 elicited a strong T-cell response in the lung of vaccinated animals characterized by (a) CXCR3+ KLRG1- lung resident T cells that are known to be essential for controlling bacterial infection, (b) IFNγ-secreting CD4+ T cells which is necessary for intracellular bactericidal activity, and (c) IL17-secreting CD4+ T cells that can confer protective immunity against multiple clinically relevant strains of Mtb. Upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain, intranasal NanoSTING-H1 provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille-Calmette-Guérin (BCG). Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that protects from infection by M. tuberculosis.
Subject(s)
Administration, Intranasal , Antigens, Bacterial , Mice, Inbred C57BL , Mycobacterium tuberculosis , Nanoparticles , Tuberculosis Vaccines , Animals , Tuberculosis Vaccines/immunology , Tuberculosis Vaccines/administration & dosage , Mycobacterium tuberculosis/immunology , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Antigens, Bacterial/immunology , Antigens, Bacterial/administration & dosage , Mice , Adjuvants, Immunologic/administration & dosage , Female , Tuberculosis/prevention & control , Tuberculosis/immunology , Lung/immunology , Lung/microbiology , Bacterial Proteins/immunology , Acyltransferases/immunology , Acyltransferases/genetics , CD4-Positive T-Lymphocytes/immunology , Adjuvants, Vaccine/administration & dosage , Immunity, Mucosal/immunology , Nanovaccines , Recombinant Fusion ProteinsABSTRACT
Tuberculosis, caused by Mycobacterium tuberculosis infection, is an ongoing epidemic with an estimated ten million active cases of the disease worldwide. Pulmonary tuberculosis is associated with cognitive and memory deficits, and patients with this disease are at an increased risk for Parkinson's disease and dementia. Although epidemiological data correlates neurological effects with peripheral disease, the pathology in the central nervous system is unknown. In an established guinea pig model of low-dose, aerosolized Mycobacterium tuberculosis infection, we see behavior changes and memory loss in infected animals. We correlate these findings with pathological changes within brain regions related to motor, cognition, and sensation across disease progression. This includes microglial and astrocytic proliferation and reactivity. These cellular changes are followed by the aggregation of neurotoxic amyloid ß and phosphorylated tau and, ultimately, neuronal degeneration in the hippocampus. Through these data, we have obtained a greater understanding of the neuropathological effects of a peripheral disease that affects millions of persons worldwide.
ABSTRACT
CD1 is an antigen presenting glycoprotein homologous to MHC I; however, CD1 proteins present lipid rather than peptide antigen. CD1 proteins are well established to present lipid antigens of Mycobacterium tuberculosis (Mtb) to T cells, but understanding the role of CD1-restricted immunity in vivo in response to Mtb infection has been limited by availability of animal models naturally expressing the CD1 proteins implicated in human response: CD1a, CD1b and CD1c. Guinea pigs, in contrast to other rodent models, express four CD1b orthologs, and here we utilize the guinea pig to establish the kinetics of gene and protein expression of CD1b orthologs, as well as the Mtb lipid-antigen and CD1b-restricted immune response at the tissue level over the course of Mtb infection. Our results indicate transient upregulation of CD1b expression during the effector phase of adaptive immunity that wanes with disease chronicity. Gene expression indicates that upregulation of CD1b is the result of transcriptional induction across all CD1b orthologs. We show high CD1b3 expression on B cells, and identify CD1b3 as the predominant CD1b ortholog in pulmonary granuloma lesions. We identify ex vivo cytotoxic activity directed against CD1b that closely paralleled the kinetic changes in CD1b expression in Mtb infected lung and spleen. This study confirms that CD1b expression is modulated by Mtb infection in lung and spleen, leading to pulmonary and extrapulmonary CD1b-restricted immunity as a component of the antigen-specific response to Mtb infection.
ABSTRACT
BACKGROUND: Cigarette smoke (CS) exposure is an epidemiological risk factor for tuberculosis, although the biological basis has not been elucidated. METHODS: We exposed C57BL/6 mice to CS for 14 weeks and examined their ability to control an aerosol infection of Mycobacterium tuberculosis Erdman. RESULTS: CS-exposed mice had more M. tuberculosis isolated from the lungs and spleens after 14 and 30 d, compared with control mice. The CS-exposed mice had worse lung lesions and less lung and splenic macrophages and dendritic cells (DCs) producing interleukin12 and tumor necrosis factor α (TNF-α). There were significantly more interleukin 10-producing macrophages and DCs in the spleens of infected CS-exposed mice than in non-CS-exposed controls. CS-exposed mice also showed a diminished influx of interferon γ-producing and TNF-α-producing CD4(+) and CD8(+) effector and memory T cells into the lungs and spleens. There was a trend toward an increased number of viable intracellular M. tuberculosis in macrophages isolated from humans who smoke compared with nonsmokers. THP-1 human macrophages and primary human alveolar macrophages exposed to CS extract, nicotine, or acrolein showed an increased burden of intracellular M. tuberculosis. CONCLUSION: CS suppresses the protective immune response to M. tuberculosis in mice, human THP-1 cells, and primary human alveolar macrophages.
Subject(s)
Disease Susceptibility , Mycobacterium tuberculosis/immunology , Smoking/adverse effects , Tuberculosis/immunology , Animals , Disease Models, Animal , Female , Mice , Mice, Inbred C57BLABSTRACT
The experimental compound TMC207 is showing promise against infections caused by Mycobacterium tuberculosis both in a variety of animal studies and in the field. In this study, we used the guinea pig model, a species that shows several similarities to human tuberculosis, including the hallmark of primary granuloma necrosis, to determine the efficacy of a combination regimen combining TMC207 with rifampin and pyrazinamide. This drug regimen rapidly reduced the bacterial load in the lungs to undetectable levels by 8 weeks of treatment. This reduction was associated with a substantial improvement in lung pathology, but despite this effect areas of residual necrosis still remained. In the draining lymph nodes, however, tissue damage was rapid and not significantly reversed by the drug treatment. Approximately 10 to 11 months after the treatment had ended, the animals began to trigger a Karnovsky scale indicating bacterial regrowth and potential relapse, an event confirmed by the new development of both pulmonary and extrapulmonary granulomatous lesions. Interestingly, a similar rate of relapse was also seen in animals receiving 24 weeks of rifampin, pyrazinamide, and isoniazid standard chemotherapy. These data indicate that TMC207 could be a useful addition to current treatment regimens for tuberculosis.
Subject(s)
Antitubercular Agents/therapeutic use , Pyrazinamide/therapeutic use , Quinolines/therapeutic use , Rifampin/therapeutic use , Tuberculosis/drug therapy , Animals , Diarylquinolines , Female , Flow Cytometry , Guinea Pigs , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/pathogenicity , Tuberculosis/microbiologyABSTRACT
The purpose of this study was 2-fold. First, we evaluated standard chemotherapy in the guinea pig model of tuberculosis to determine if this animal species could productively be used for this purpose. Second, given the similarities of the pathology of disease in guinea pigs and humans, we wished to evaluate additional parameters, including magnetic resonance imaging, microscopy, and cytokine expression and lymphocyte phenotypes, in response to an infection treated with drug therapy. This study shows that conventional rifampin-isoniazid-pyrazinamide chemotherapy significantly decreased the numbers of the highly virulent Erdman K01 strain of Mycobacterium tuberculosis, with most of the bacilli being eliminated in a month. Despite this result, bacteria could still be detected in the lungs and other tissues for at least another 3 to 4 months. Resolution of the nonnecrotic granulomas in the lungs and lymph nodes could be clearly visualized by magnetic resonance imaging at the macroscopic level. Microscopically, the majority of the pulmonary and extrapulmonary inflammation resolved spontaneously, leaving residual lesions composed of dystrophic calcification and fibrosis marking the site of necrosis of the primary lesion. Residual calcified lesions, which were also associated with pulmonary lymphangitis, contained acid-fast bacilli even following aggressive chemotherapy. The presence of intact extracellular bacilli within these lesions suggests that these could serve as the primary sites of disease reactivation. The chemotherapy reduced the level of T-cell influx into infected tissues and was accompanied by a large and sustained increase in TH1 cytokine expression. Chemotherapy also prevented the emergence in lung tissues of high levels of interleukin-10 and Foxp3-positive cells, known markers of regulatory T cells.
Subject(s)
Antitubercular Agents/pharmacology , Disease Models, Animal , Guinea Pigs , Rifampin/pharmacology , Tuberculosis, Pulmonary/drug therapy , Animals , Animals, Outbred Strains , Biomarkers/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Drug Therapy, Combination , Female , Flow Cytometry , Forkhead Transcription Factors/metabolism , Interleukin-10/metabolism , Isoniazid/pharmacology , Leukocyte Common Antigens/metabolism , Lung/pathology , Lymph Nodes/pathology , Magnetic Resonance Imaging , Pyrazinamide/pharmacology , Th1 Cells/immunology , Th1 Cells/metabolism , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/pathologyABSTRACT
OBJECTIVE: The purpose of this paper is to demonstrate the use of 2-D impedance spectroscopy to identify areas of biofilm growth on a CMOS biosensor microelectrode-array. METHODS: This paper presents the design and use of a novel multichannel impedance spectroscopy instrument to allow 2-D spatial and temporal evaluation of biofilm growth. The custom-designed circuits can provide a wide range of frequencies (1 Hz-100 kHz) to allow customization of impedance measurements, as the frequency of interest varies based on the type and state of biofilm under measurement. The device is capable of taking measurements as fast as once per second on the entire set of impedance sensors, allowing real-time observation. It also supports adjustable stimulus voltages. The distance between neighboring sensors is 220 micrometers which provides reasonable spatial resolution for biofilm study. RESULTS: Biofilm was grown on the surface of the chip, occupancy was measured using the new tool, and the results were validated optically using fluorescent staining. The results show that the developed tool can be used to determine the bacterial biofilm presence at a given location. CONCLUSION: This paper confirms that 2-D impedance spectroscopy can be used to measure biofilm occupancy. The new tool developed to perform the measurements was able to display real-time results, and determine biofilm coverage of the array electrodes. SIGNIFICANCE: The system presented in this report is the first fully integrated 2-D EIS measurement system with full software support for capturing biofilm growth dynamics in real-time. Due to its ability to nondestructively monitor biofilms over time, 2-D impedance spectroscopy using a microelectrode-array is a useful tool for studying biofilms.
Subject(s)
Biofilms , Biosensing Techniques , Dielectric Spectroscopy , Electric Impedance , MicroelectrodesABSTRACT
Dengue virus infection is associated with the upregulation of metabolic pathways within infected cells. This effect is common to infection by a broad array of viruses. These metabolic changes, including increased glucose metabolism, oxidative phosphorylation and autophagy, support the demands of viral genome replication and infectious particle formation. The mechanisms by which these changes occur are known to be, in part, directed by viral nonstructural proteins that contact and control cellular structures and metabolic enzymes. We investigated the roles of host proteins with overarching control of metabolic processes, the transcriptional regulators, cyclin-dependent kinase 8 (CDK8) and its paralog, CDK19, as mediators of virally induced metabolic changes. Here, we show that expression of CDK8, but not CDK19, is increased during dengue virus infection in Huh7 human hepatocellular carcinoma cells, although both are required for efficient viral replication. Chemical inhibition of CDK8 and CDK19 with Senexin A during infection blocks virus-induced expression of select metabolic and autophagic genes, hexokinase 2 (HK2) and microtubule-associated protein 1 light chain 3 (LC3), and reduces viral genome replication and infectious particle production. The results further define the dependence of virus replication on increased metabolic capacity in target cells and identify CDK8 and CDK19 as master regulators of key metabolic genes. The common inhibition of CDK8 and CDK19 offers a host-directed therapeutic intervention that is unlikely to be overcome by viral evolution.
Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Dengue Virus/growth & development , Energy Metabolism/physiology , Virus Replication/genetics , Autophagy/physiology , Cell Line, Tumor , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Dengue/pathology , Dengue Virus/metabolism , Gene Knockdown Techniques , Genome, Viral/genetics , Glucose/metabolism , Hexokinase/biosynthesis , Humans , Male , Microtubule-Associated Proteins/biosynthesis , Middle Aged , Oxidative PhosphorylationABSTRACT
The formation of bacterial biofilms significantly decreases the efficacy of antibiotic treatments. Herein, we've investigated the antibiofilm properties of the natural product meridianin D and a library of analogues against Mycobacterium smegmatis. As a result, we discovered several analogues that both inhibit and disperse M. smegmatis biofilms.
ABSTRACT
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.
Subject(s)
Hypoglycemic Agents/therapeutic use , Metformin/therapeutic use , Tuberculosis, Pulmonary/drug therapy , Animals , Cytokines/metabolism , Energy Metabolism/drug effects , Glucose/metabolism , Glucose Intolerance/drug therapy , Guinea Pigs , Insulin Resistance , Lung/pathology , Male , Membrane Potential, Mitochondrial/drug effects , Superoxides/metabolism , T-Lymphocytes/metabolism , Tuberculosis, Pulmonary/metabolism , Tuberculosis, Pulmonary/pathologyABSTRACT
Patients with type 2 diabetes (T2D) have a lower risk of Mycobacterium tuberculosis infection, progression from infection to tuberculosis (TB) disease, TB morality and TB recurrence, when being treated with metformin. However, a detailed mechanistic understanding of these protective effects is lacking. Here, we use mass cytometry to show that metformin treatment expands a population of memory-like antigen-inexperienced CD8+CXCR3+ T cells in naive mice, and in healthy individuals and patients with T2D. Metformin-educated CD8+ T cells have increased (i) mitochondrial mass, oxidative phosphorylation, and fatty acid oxidation; (ii) survival capacity; and (iii) anti-mycobacterial properties. CD8+ T cells from Cxcr3-/- mice do not exhibit this metformin-mediated metabolic programming. In BCG-vaccinated mice and guinea pigs, metformin enhances immunogenicity and protective efficacy against M. tuberculosis challenge. Collectively, these results demonstrate an important function of CD8+ T cells in metformin-derived host metabolic-fitness towards M. tuberculosis infection.
Subject(s)
CD8-Positive T-Lymphocytes/drug effects , Diabetes Mellitus, Type 2/drug therapy , Hypoglycemic Agents/administration & dosage , Metformin/administration & dosage , Animals , BCG Vaccine/administration & dosage , BCG Vaccine/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/immunology , Diabetes Mellitus, Type 2/metabolism , Female , Guinea Pigs , Humans , Male , Mice , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/physiology , Tuberculosis/etiology , Tuberculosis/immunology , Tuberculosis/microbiology , Tuberculosis/prevention & controlABSTRACT
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.
Subject(s)
Biofilms/drug effects , Electron Transport/drug effects , Imidazoles/pharmacology , Mycobacterium tuberculosis/drug effects , Proton-Motive Force/drug effects , Tuberculosis/drug therapy , Adenosine Triphosphate/metabolism , Antitubercular Agents/pharmacology , Biofilms/growth & development , Humans , Microbial Sensitivity Tests , Mycobacterium tuberculosis/growth & development , Oxygen Consumption/drug effects , Tuberculosis/microbiologyABSTRACT
Type 2 diabetes is a leading cause of morbidity and mortality among noncommunicable diseases, and additional animal models that more closely replicate the pathogenesis of human type 2 diabetes are needed. The goal of this study was to develop a model of type 2 diabetes in guinea pigs, in which diet-induced glucose intolerance precedes ß-cell cytotoxicity, two processes that are crucial to the development of human type 2 diabetes. Guinea pigs developed impaired glucose tolerance after 8 weeks of feeding on a high-fat, high-carbohydrate diet, as determined by oral glucose challenge. Diet-induced glucose intolerance was accompanied by ß-cell hyperplasia, compensatory hyperinsulinemia, and dyslipidemia with hepatocellular steatosis. Streptozotocin (STZ) treatment alone was ineffective at inducing diabetic hyperglycemia in guinea pigs, which failed to develop sustained glucose intolerance or fasting hyperglycemia and returned to euglycemia within 21 days after treatment. However, when high-fat, high-carbohydrate diet-fed guinea pigs were treated with STZ, glucose intolerance and fasting hyperglycemia persisted beyond 21 days post-STZ treatment. Guinea pigs with diet-induced glucose intolerance subsequently treated with STZ demonstrated an insulin-secretory capacity consistent with insulin-independent diabetes. This insulin-independent state was confirmed by response to oral antihyperglycemic drugs, metformin and glipizide, which resolved glucose intolerance and extended survival compared with guinea pigs with uncontrolled diabetes. In this study, we have developed a model of sequential glucose intolerance and ß-cell loss, through high-fat, high-carbohydrate diet and extensive optimization of STZ treatment in the guinea pig, which closely resembles human type 2 diabetes. This model will prove useful in the study of insulin-independent diabetes pathogenesis with or without comorbidities, where the guinea pig serves as a relevant model species.
Subject(s)
Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/complications , Glucose Intolerance/complications , Administration, Oral , Adrenergic alpha-2 Receptor Antagonists/pharmacology , Animals , Cell Death/drug effects , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/pathology , Diet , Dietary Carbohydrates , Disease Models, Animal , Dyslipidemias/complications , Dyslipidemias/drug therapy , Dyslipidemias/pathology , Feeding Behavior , Female , Glucose Intolerance/drug therapy , Glucose Intolerance/pathology , Guinea Pigs , Hyperglycemia/complications , Hyperglycemia/drug therapy , Hyperglycemia/pathology , Hyperinsulinism/complications , Hyperinsulinism/drug therapy , Hyperplasia , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/therapeutic use , Insulin/pharmacology , Insulin/therapeutic use , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Streptozocin , Survival Analysis , Weight Gain/drug effectsABSTRACT
There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment.