RESUMEN
The observations that the innate immune system employs copper to eliminate bacterial infection and that resistance to copper enhances virulence of Mycobacterium tuberculosis (Mtb) prompted us to examine the effects the anti-cancer agent elesclomol on Mtb. As a bis-thionohydrazide, elesclomol chelates with copper to form a copper complex in situ that via redox cycling of the metal ion greatly enhances oxidative stress in tumour cells. Here, we demonstrate that elesclomol is relatively potent against Mtb H37Rv with minimum inhibitory concentration of 10 µM (4 mg/L) and against multidrug resistant clinical isolates of Mtb, displays additive interactions with known tuberculosis drugs such as isoniazid and ethambutol, and a synergistic interaction with rifampicin. Controlled supplementation of elesclomol with copper in culture medium increased Mtb sensitivity by >65 fold. Overall, the activities of elesclomol in principle indicate the possibility of repurposing elesclomol or designing new thionohydrazides as potential drugs for use against Mtb. © 2019 IUBMB Life, 71(5):532-538, 2019.
Asunto(s)
Antineoplásicos/farmacología , Antituberculosos/farmacología , Quelantes/farmacología , Cobre/química , Hidrazinas/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Cobre/metabolismo , Reposicionamiento de Medicamentos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Oxidación-Reducción , Tuberculosis/microbiologíaRESUMEN
Evidence to-date points to a detrimental role of the type I IFNs during tuberculosis. The mechanisms underpinning the IFNαß-mediated exacerbation of the disease is unclear. The 2'-5'-oligoadenylate synthetases (OAS), namely OAS1, OAS2 and OAS3 are part of the interferon-induced genes which until now have been synonymous with an anti-viral function. Blood transcriptome profiling has continuously observed their upregulation in a number of gene expression signatures which discriminate active TB from latent TB infection, however the role of the OASs and the effect that their expression has on the pathogenesis and persistence of TB is unknown. Evidence suggests that the OASs exhibit other cellular functions which include the induction of apoptosis, enhancement of IFNαß signalling, immune cell receptor modulation and autophagy. We propose that i) during the late stages of disease, sustained RNaseL expression through OAS activation enhances type I IFN signalling and, ii) that they may exhibit immune-modulatory capabilities.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Tuberculosis/metabolismo , Animales , Apoptosis/fisiología , Humanos , Interferón Tipo I/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a-c, 7 and 8; H37Rv-MIC99 ≤1.25⯵M, R5401-MIC99 ≤2.5⯵M, X_61-MIC99 ≤5⯵M) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a-c and 3b (MIC90 ≤13.42⯵M) and inhibition of EB efflux demonstrated by these compounds are encouraging.
Asunto(s)
Antituberculosos/síntesis química , Diseño de Fármacos , Isoniazida/química , Animales , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Células CHO , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Humanos , Bombas Iónicas/antagonistas & inhibidores , Bombas Iónicas/metabolismo , Isoniazida/síntesis química , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
A series of 2(5H)-furanone-based compounds were synthesized from commercially available mucohalic acids. From the first-generation compounds, three showed inhibitory activity (10 µg/mL) of at least 35% against Mycobacterium smegmatis mc(2) 155 growth (Bioscreen C system). In screening the active first-generation compounds for growth inhibition against Mycobacterium tuberculosis H37Rv, the most active compound was identified with a minimum inhibitory concentration (MIC99 ) of 8.07 µg/mL (15.8 µM) using BACTEC 460 system. No cross-resistance was observed with some current first-line anti-TB drugs, since it similarly inhibited the growth of multidrug resistant (MDR) clinical isolates. The compound showed a good selectivity for mycobacteria since it did not inhibit the growth of selected Gram-positive and Gram-negative bacteria. It also showed synergistic activity with rifampicin (RIF) and additive activity with isoniazid (INH) and ethambutol (EMB). Additional time-kill studies showed that the compound is bacteriostatic to mycobacteria, but cytotoxic to the Chinese Hamster Ovarian (CHO) cell line. From a second generation library, two compounds showed improved anti-TB activity against M. tuberculosis H37Rv and decreased CHO cell cytotoxicity. The compounds exhibited MIC values of 2.62 µg/mL (5.6 µM) and 3.07 µg/mL (5.6 µM) respectively. The improved cytotoxicity against CHO cell line of the two compounds ranged from IC50 = 38.24 µg/mL to IC50 = 45.58 µg/mL when compared to the most active first-generation compound (IC50 = 1.82 µg/mL). The two second generation leads with selectivity indices (SI) of 14.64 and 14.85 respectively, warrant further development as anti-TB drug candidates. © 2016 IUBMB Life, 68(8):612-620, 2016.
Asunto(s)
Antituberculosos/administración & dosificación , Furanos/administración & dosificación , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/síntesis química , Antituberculosos/química , Células CHO/efectos de los fármacos , Células CHO/microbiología , Cricetulus , Sinergismo Farmacológico , Etambutol/administración & dosificación , Furanos/síntesis química , Furanos/química , Humanos , Isoniazida/administración & dosificación , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/patogenicidad , Rifampin/administración & dosificación , Tuberculosis/microbiologíaRESUMEN
New drugs to treat drug-resistant tuberculosis are urgently needed. Extensively drug-resistant and probably the totally drug-resistant tuberculosis strains are resistant to fluoroquinolones like moxifloxacin, which target gyrase A, and most people infected with these strains die within a year. In this study, we found that a novel aminobenzimidazole, VXc-486, which targets gyrase B, potently inhibits multiple drug-sensitive isolates and drug-resistant isolates of Mycobacterium tuberculosis in vitro (MICs of 0.03 to 0.30 µg/ml and 0.08 to 5.48 µg/ml, respectively) and reduces mycobacterial burdens in lungs of infected mice in vivo. VXc-486 is active against drug-resistant isolates, has bactericidal activity, and kills intracellular and dormant M. tuberculosis bacteria in a low-oxygen environment. Furthermore, we found that VXc-486 inhibits the growth of multiple strains of Mycobacterium abscessus, Mycobacterium avium complex, and Mycobacterium kansasii (MICs of 0.1 to 2.0 µg/ml), as well as that of several strains of Nocardia spp. (MICs of 0.1 to 1.0 µg/ml). We made a direct comparison of the parent compound VXc-486 and a phosphate prodrug of VXc-486 and showed that the prodrug of VXc-486 had more potent killing of M. tuberculosis than did VXc-486 in vivo. In combination with other antimycobacterial drugs, the prodrug of VXc-486 sterilized M. tuberculosis infection when combined with rifapentine-pyrazinamide and bedaquiline-pyrazinamide in a relapse infection study in mice. Furthermore, the prodrug of VXc-486 appeared to perform at least as well as the gyrase A inhibitor moxifloxacin. These findings warrant further development of the prodrug of VXc-486 for the treatment of tuberculosis and nontuberculosis mycobacterial infections.
Asunto(s)
Antibacterianos/uso terapéutico , Bencimidazoles/uso terapéutico , Infecciones por Mycobacterium/tratamiento farmacológico , Inhibidores de Topoisomerasa II/uso terapéutico , Animales , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Sensibilidad MicrobianaRESUMEN
The synthesis and antimycobacterial activity of formononetin analogues is hereby reported. Formononetin and its analogue 11E showed 88% and 95% growth inhibition, respectively, against the H37Rv strain of Mycobacterium tuberculosis. Pharmacophore modeling studies indicated that the presence of a hydroxyl group in formononetin and its analogues, is crucial for maintaining activity.
Asunto(s)
Antituberculosos/síntesis química , Productos Biológicos/síntesis química , Isoflavonas/química , Isoflavonas/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Productos Biológicos/química , Productos Biológicos/farmacología , Diseño de Fármacos , Isoflavonas/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH.
Asunto(s)
Antioxidantes/metabolismo , Farmacorresistencia Bacteriana/genética , Ergotioneína/metabolismo , Mycobacterium smegmatis/metabolismo , Proteínas Bacterianas/metabolismo , Medios de Cultivo , Cisteína/metabolismo , Ergotioneína/genética , Glicopéptidos/metabolismo , Inositol/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Estrés OxidativoRESUMEN
OBJECTIVES: To investigate the effect of trimethoprim/sulfamethoxazole on the survival of Mycobacterium tuberculosis and trimethoprim and sulfamethoxazole individually and combined with the first-line tuberculosis drugs (isoniazid, rifampicin and ethambutol). METHODS: M. tuberculosis strains were exposed to either trimethoprim/sulfamethoxazole combination or sulfamethoxazole and trimethoprim alone at various concentrations. The strains were also exposed to sulfamethoxazole in combination with existing antibiotics to assess the combined effect on the growth of M. tuberculosis in the BACTEC 460TB system. The effect of the drugs was compared with vehicle-treated controls. Drug interactions were interpreted using quotient values obtained from the growth index of cultures treated with a single drug or the combination. RESULTS: Trimethoprim showed a negligible effect on the growth of M. tuberculosis while sulfamethoxazole inhibited 80% of the growth of M. tuberculosis at 4.75 mg/L. There was no synergistic activity between sulfamethoxazole and trimethoprim, although an additive effect was observed. A statistically significant synergistic effect was observed between sulfamethoxazole and rifampicin. Sulfamethoxazole also had an additive effect with ethambutol, but there was no interaction with isoniazid. CONCLUSIONS: Sulfamethoxazole is the main active compound against M. tuberculosis in the combination trimethoprim/sulfamethoxazole and has a synergistic effect with rifampicin. These findings suggest that sulfamethoxazole has potential in the multidrug regimen against M. tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Sinergismo Farmacológico , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Sulfametoxazol/farmacología , Etambutol/farmacología , Isoniazida/farmacología , Trimetoprim/farmacologíaRESUMEN
The synthesis of a novel series of thiolactone-isatin hybrids led to the discovery of tetracyclic by-products which displayed superior antiplasmodial activity. The tetracycles thus formed the basis of a more focused SAR study. Identified from this series is a compound with an IC(50) of 6.92 µM against the chloroquine-resistant (W2) strain of Plasmodium falciparum. Useful antimalarial SARs delineated include the need for substitution at C-5 of the isatin scaffold and the enhancement of activity by increasing the linker length. In contrast to their antimalarial activity, the tetracycles were devoid of antitubercular activity whereas the advanced intermediates displayed growth inhibitory activity against the H(37)Rv strain of Mycobacterium tuberculosis as revealed by BACTEC, MABA and LORA assays.
Asunto(s)
Antimaláricos/farmacología , Antituberculosos/farmacología , Isatina/farmacología , Lactonas/farmacología , Animales , Antimaláricos/química , Antituberculosos/química , Isatina/química , Lactonas/química , Mycobacterium tuberculosis/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
BACKGROUND: The assimilation of nitrogen is an essential process in all prokaryotes, yet a relatively limited amount of information is available on nitrogen metabolism in the mycobacteria. The physiological role and pathogenic properties of glutamine synthetase (GS) have been extensively investigated in Mycobacterium tuberculosis. However, little is known about this enzyme in other mycobacterial species, or the role of an additional nitrogen assimilatory pathway via glutamate dehydrogenase (GDH), in the mycobacteria as a whole. We investigated specific enzyme activity and transcription of GS and as well as both possible isoforms of GDH (NAD+- and NADP+-specific GDH) under varying conditions of nitrogen availability in Mycobacterium smegmatis as a model for the mycobacteria. RESULTS: It was found that the specific activity of the aminating NADP+-GDH reaction and the deaminating NAD+-GDH reaction did not change appreciably in response to nitrogen availability. However, GS activity as well as the deaminating NADP+-GDH and aminating NAD+-GDH reactions were indeed significantly altered in response to exogenous nitrogen concentrations. Transcription of genes encoding for GS and the GDH isoforms were also found to be regulated under our experimental conditions. CONCLUSIONS: The physiological role and regulation of GS in M. smegmatis was similar to that which has been described for other mycobacteria, however, in our study the regulation of both NADP+- and NAD+-GDH specific activity in M. smegmatis appeared to be different to that of other Actinomycetales. It was found that NAD+-GDH played an important role in nitrogen assimilation rather than glutamate catabolism as was previously thought, and is it's activity appeared to be regulated in response to nitrogen availability. Transcription of the genes encoding for NAD+-GDH enzymes seem to be regulated in M. smegmatis under the conditions tested and may contribute to the changes in enzyme activity observed, however, our results indicate that an additional regulatory mechanism may be involved. NADP+-GDH seemed to be involved in nitrogen assimilation due to a constitutive aminating activity. The deaminating reaction, however was observed to change in response to varying ammonium concentrations which suggests that NADP+-GDH is also regulated in response to nitrogen availability. The regulation of NADP+-GDH activity was not reflected at the level of gene transcription thereby implicating post-transcriptional modification as a regulatory mechanism in response to nitrogen availability.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glutamato Deshidrogenasa/biosíntesis , Glutamato-Amoníaco Ligasa/biosíntesis , Mycobacterium smegmatis/fisiología , Nitrógeno/metabolismo , Ácido Glutámico/metabolismo , Redes y Vías Metabólicas , Modelos Biológicos , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/metabolismo , NAD/metabolismo , NADP/metabolismoRESUMEN
BACKGROUND: With few exceptions, existing tuberculosis drugs were developed many years ago and resistance profiles have emerged. This has created a need for new drugs with discrete modes of action. There is evidence that tuberculosis (like other bacteria) is susceptible to oxidative pressure and this has yet to be properly utilised as a therapeutic approach in a manner similar to that which has proven highly successful in malaria therapy. OBJECTIVE: To develop an alternative approach to the incorporation of bacterial siderophores that results in the creation of antitubercular peroxidic leads for subsequent development as novel agents against tuberculosis. METHODS: Eight novel peroxides were prepared and the antitubercular activity (H37Rv) was compared to existing artemisinin derivatives in vitro. The potential for toxicity was evaluated against the L6 rat skeletal myoblast and HeLa cervical cancer lines in vitro. RESULTS: The addition of a pyrimidinyl residue to an artemisinin or, preferably, a tetraoxane peroxidic structure results in antitubercular activity in vitro. The same effect is not observed in the absence of the pyrimidine or with other heteroaromatic substituents. CONCLUSION: The incorporation of a pyrimidinyl residue adjacent to the peroxidic function in an organic peroxide results in anti-tubercular activity in an otherwise inactive peroxidic compound. This will be a useful approach for creating oxidative drugs to target tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Artemisininas/farmacología , Tetraoxanos/farmacología , Animales , Antituberculosos/síntesis química , Artemisininas/síntesis química , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Ratas , Tetraoxanos/síntesis químicaRESUMEN
BACKGROUND: Although the gene encoding for glutamine synthetase (glnA) is essential in several organisms, multiple glnA copies have been identified in bacterial genomes such as those of the phylum Actinobacteria, notably the mycobacterial species. Intriguingly, previous reports have shown that only one copy (glnA1) is essential for growth in M. tuberculosis, while the other copies (glnA2, glnA3 and glnA4) are not. RESULTS: In this report it is shown that the glnA1 and glnA2 encoded glutamine synthetase sequences were inherited from an Actinobacteria ancestor, while the glnA4 and glnA3 encoded GS sequences were sequentially acquired during Actinobacteria speciation. The glutamine synthetase sequences encoded by glnA4 and glnA3 are undergoing reductive evolution in the mycobacteria, whilst those encoded by glnA1 and glnA2 are more conserved. CONCLUSION: Different selective pressures by the ecological niche that the organisms occupy may influence the sequence evolution of glnA1 and glnA2 and thereby affecting phylogenies based on the protein sequences they encode. The findings in this report may impact the use of similar sequences as molecular markers, as well as shed some light on the evolution of glutamine synthetase in the mycobacteria.
Asunto(s)
Actinobacteria/genética , Proteínas Bacterianas/genética , Evolución Molecular , Especiación Genética , Glutamato-Amoníaco Ligasa/genética , Secuencia de Aminoácidos , Secuencia Conservada , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Marcadores Genéticos , Genoma Bacteriano , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Filogenia , ARN Ribosómico 16S/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The mechanisms governing the regulation of nitrogen metabolism in Corynebacterium glutamicum and Streptomyces coelicolor have been extensively studied. These Actinomycetales are closely related to the Mycobacterium genus and may therefore serve as a models to elucidate the cascade of nitrogen signalling in other mycobacteria. Some factors involved in nitrogen metabolism in Mycobacterium tuberculosis have been described, including glutamine synthetase and its adenylyltransferase, but not much data concerning the other components involved in the signalling cascade is available. In this review a comparative study of factors involved in nitrogen metabolism in C. glutamicum and S. coelicolor is made to identify similarities with M. tuberculosis on both a genomic and proteomic level. This may provide insight into a potential global mechanism of nitrogen control in Mycobacterium tuberculosis.
Asunto(s)
Corynebacterium glutamicum/metabolismo , Mycobacterium tuberculosis/metabolismo , Nitrógeno/metabolismo , Streptomyces coelicolor/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Compuestos de Amonio Cuaternario/metabolismo , Transducción de Señal/fisiología , Transcripción GenéticaRESUMEN
To evaluate the feasibility of developing drugs that may be active against both malaria and tuberculosis (TB) by using in part putative cholesterol transporters in the causative pathogens and through enhancement of passive diffusion in granulomatous TB, artemisinin-cholesterol conjugates were synthesized by connecting the component molecules through various linkers. The compounds were screened inâ vitro against Plasmodium falciparum (Pf) and Mycobacterium tuberculosis (Mtb). Antimalarial activities (IC50 ) against Pf drug-sensitive NF54, and drug-resistant K1 and W2 strains ranged from 0.03-2.6, 0.03-1.9, and 0.02-1.7â µm. Although the compounds are less active than the precursor artemisinin derivatives, the cholesterol moiety renders the compounds relatively insoluble in the culture medium, and variation in solubilities among the different compounds may reflect in the range of efficacies observed. Activities against Mtb H37Rv were assessed using a standardized colony-forming unit (CFU) assay after 24â h pretreatment of cultures with each of the compounds. Percentage inhibition ranged from 3-38 % and 18-52 % at 10 and 80â µm, respectively. Thus, in contrast to the comparator drug artemether, the conjugates display enhanced activities. The immediate aims include the preparation of conjugates with enhanced aqueous solubilities, assays against malaria and TB inâ vivo, and for TB, assays using an infected macrophage model and assessment of granuloma influx.
Asunto(s)
Antimaláricos/síntesis química , Antituberculosos/síntesis química , Artemisininas/química , Colesterol/química , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Malaria/tratamiento farmacológico , Malaria/patología , Mycobacterium tuberculosis/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Tuberculosis/patologíaRESUMEN
The type I IFN response quickly became associated with its role in the innate immune response to viral infection. The past few years have seen the significance of IFNs expand in breadth to include non-viral pathogens. Previous work has identified that following viral infection, type I IFN signaling induces the production of the 2'-5'-oligoadenylate synthetase (OAS) family, which include OAS1, OAS2, OAS3, and OAS-like (OASL) protein. OASL was identified to be strongly induced following viral infection through engaging the RNA sensor RIG-I and increasing signaling through this pathway to enhance the anti-viral type I IFN response. Surprisingly, infection with viral dsDNA revealed an IFN inhibitory role and therefore pro-viral function of OASL through the inhibition of the cGAS cytosolic DNA sensing mechanism. Intracellular bacteria are able to activate the cytosolic DNA sensing pathway, however the role of OASL during bacterial infection is largely unknown. Vacuolar pathogenic microbes such as mycobacteria induce OASL early post infection, where it functions in a prosurvival fashion by inhibiting autophagic mechanisms and antimicrobial peptide expression. This suggests an underestimated role of OASL in the innate immune response to infection with a variety of pathogens and points to OASL-associated modulation of the type I IFN response. OASL may therefore play a critical role in defining the outcome of infection. We provide a brief update on the recent developments of the OAS family of proteins in response to DNA and RNA virus infections, as well as discuss evidence of Oasl expression in response to a number of cytosolic and vacuolar replicating bacterial pathogens.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/inmunología , Bacterias/inmunología , Citoplasma/inmunología , Interacciones Huésped-Patógeno/inmunología , Interferón Tipo I/inmunología , 2',5'-Oligoadenilato Sintetasa/clasificación , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/fisiología , Autofagia/fisiología , Bacterias/crecimiento & desarrollo , Bacterias/patogenicidad , Citoplasma/genética , Citoplasma/microbiología , Citosol/inmunología , Citosol/microbiología , Regulación de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno/genética , Humanos , Inmunidad Innata , Interferón Tipo I/genética , Mycobacterium/inmunología , Mycobacterium/patogenicidad , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Transducción de Señal/inmunología , Vacuolas/microbiología , Virosis/inmunologíaRESUMEN
The distinguishing factors that characterize the host response to infection with virulent Mycobacterium tuberculosis (M.tb) are largely confounding. We present an infection study with 2 genetically closely related M.tb strains that have vastly different pathogenic characteristics. The early host response to infection with these detergent-free cultured strains was analyzed through RNAseq in an attempt to provide information on the subtleties which may ultimately contribute to the virulent phenotype. Murine bone marrow derived macrophages (BMDMs) were infected with either a hyper- (R5527) or hypovirulent (R1507) Beijing M. tuberculosis clinical isolate. RNAseq revealed 69 differentially expressed host genes in BMDMs during comparison of these 2 transcriptomes. Pathway analysis revealed activation of the stress-induced and growth inhibitory Gadd45 signaling pathway in hypervirulent infected BMDMs. Upstream regulators of interferon activation such as and IRF3 and IRF7 were predicted to be upregulated in hypovirulent-infected BMDMs. Additional analysis of the host immune response through ELISA and qPCR included the use of human THP-1 macrophages where a robust proinflammatory response was observed after infection with the hypervirulent strain. RNAseq revealed 2 early-response genes (ier3 and saa3) and 2 host-defense genes (oasl1 and slpi) that were significantly upregulated by the hypervirulent strain. The role of these genes under M.tb infection conditions are largely unknown but here we provide validation of their presence with use of qPCR and Western blot. Further analysis into their biological role during infection with virulent M.tb is required.
Asunto(s)
Interacciones Huésped-Patógeno , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/microbiología , Animales , Western Blotting , Proteínas de Ciclo Celular/genética , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Factor 3 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , Macrófagos/microbiología , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Proteínas Nucleares/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , VirulenciaRESUMEN
BACKGROUND: The emergence of drug-resistant strains of Mycobacterium tuberculosis has become a global public health problem. Polyherbal medicines offer great hope for developing alternative drugs for the treatment of tuberculosis. OBJECTIVE: To evaluate the anti-tubercular activity of polyherbal medicines used for the treatment of tuberculosis. METHODS: The remedies were screened against Mycobacterium tuberculosis H37Rv using Middlebrook 7H9 media and MGIT BACTEC 960 system. They were liquid preparations from King Williams Town site A (KWTa), King Williams Town site B (KWTb), King Williams Town site C (KWTc), Hogsback first site (HBfs), Hogsback second site (HBss), Hogsback third site (HBts), East London (EL), Alice (AL) and Fort Beaufort (FB). RESULTS: The susceptibility testing revealed that all the remedies contain anti-tubercular activity with KWTa, KWTb, KWTc, HBfs, HBts, AL and FB exhibiting more activity at a concentration below 25 µl/ml. Furthermore, MIC values exhibited inhibitory activity with the most active remedies from KWTa, HBfs and HBts at 1.562 µg/ml. However, isoniazid showed more inhibitory activity against M. tuberculosis at 0.05 µg/ml when compare to the polyherbal remedies. CONCLUSION: This study has indicated that these remedies could be potential sources of new anti-mycobacterial agents against M. tuberculosis. However, the activity of these preparations and their active principles still require in vivo study in order to assess their future as new anti-tuberculosis agents.
Asunto(s)
Antituberculosos/uso terapéutico , Contaminación de Medicamentos , Isoniazida/uso terapéutico , Fitoterapia , Extractos Vegetales/uso terapéutico , Plantas Medicinales/microbiología , Tuberculosis/tratamiento farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , SudáfricaRESUMEN
We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Glutamato Deshidrogenasa/genética , Mycobacterium bovis/genética , Transaminasas/genética , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Expresión Génica , Prueba de Complementación Genética , Glutamato Deshidrogenasa/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Macrófagos/microbiología , Ratones , Mutación , Mycobacterium bovis/efectos de los fármacos , Mycobacterium bovis/enzimología , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/química , Donantes de Óxido Nítrico/farmacología , Cultivo Primario de Células , Estrés Fisiológico , Transaminasas/metabolismoRESUMEN
During Mycobacterium tuberculosis (M.tb) infection, the initial interactions between the pathogen and the host cell determines internalization and innate immune response events. It is established that detergents such as Tween alter the mycobacterial cell wall and solubilize various lipids and proteins. The implication of this is significant since induced changes on the cell wall affect macrophage uptake and the immune response to M.tb. Importantly, during transmission between hosts, aerosolized M.tb enters the host in its native form, i.e. in a detergent-free environment, thus in vitro and in vivo studies should mimic this as closely as possible. To this end, we have optimized a procedure for growing and processing detergent-free M.tb and assessed the response of murine macrophages (BMDM) infected with multi drug-resistant M.tb (R179 Beijing 220 clinical isolate) using RNAseq. We compared the effects of the host response to M.tb cultured under standard laboratory conditions (Tween 80 containing medium -R179T), or in detergent-free medium (R179NT). RNAseq comparisons reveal 2651 differentially expressed genes in BMDMs infected with R179T M.tb vs. BMDMs infected with R179NT M.tb. A range of differentially expressed genes involved in BMDM receptor interaction with M.tb (Mrc1, Ifngr1, Tlr9, Fpr1 and Itgax) and pro-inflammatory cytokines/chemokines (Il6, Il1b, Tnf, Ccl5 and Cxcl14) were selected for analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory response. Interestingly, R179NT M.tb induce transcription of Fpr1, a receptor which detects bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show that the host components Cxcl14, with an unknown role in M.tb infection, and Tlr9, an emerging role player, are only stimulated by infection with R179NT M.tb. Taken together, our results suggest that the host response differs significantly in response to Tween 80 cultured M.tb and should therefore not be used in infection experiments.
Asunto(s)
Detergentes/farmacología , Perfilación de la Expresión Génica/métodos , Polisorbatos/farmacología , Tuberculosis/genética , Animales , Células Cultivadas , Medios de Cultivo/química , Medios de Cultivo/farmacología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Análisis de Secuencia de ARN/métodos , Tuberculosis/inmunología , Tuberculosis/microbiologíaRESUMEN
Guidelines for the diagnosis and management of Bacillus Calmette-Guérin (BCG) disease in children are lacking, and there are limited data on drug resistance of Mycobacterium bovis BCG. A 6-month-old HIV-infected infant presented with right axillary adenitis ipsilateral to the site of BCG immunization. M. tuberculosis complex was cultured from axillary lymph nodes and gastric aspirates, and M. bovis BCG was isolated. Susceptibility testing before initiation of therapy demonstrated inherent resistance to isoniazid. The organism acquired rifampin resistance during therapy. This was confirmed by the presence of a mutation in codon 531 (Ser531Tyr) of the rpoB gene. Treatment guidelines for BCG disease with consideration of inherent and possible acquired drug resistance should be established in settings with high rates of vertical HIV transmission and routine BCG vaccination.