RESUMEN
A fungal laccase-mediator system capable of high effectively oxidizing tetracyclines under a wide pH range will benefit environmental protection. This study reported a directed evolution of a laccase PIE5 to improve its performance on tetracyclines oxidization at alkaline conditions. Two mutants, namely MutA (D229N/A244V) and MutB (N123A/D229N/A244V) were obtained. Although they shared a similar optimum pH and temperature as PIE5, the two mutants displayed approximately 2- and 5-fold higher specific activity toward the mediators ABTS and guaiacol at pHs 4.0 to 6.5, respectively. Simultaneously, their catalytic efficiency increased by 8.0- and 6.4-fold compared to PIE5. At a pH range of 5-8 and 28 °C, MutA or MutB at a final concentration of 2.5 U·mL-1 degraded 77 % and 81 % of 100 mg·L-1 tetracycline within 10 min, higher than PIE5 (45 %). Furthermore, 0.1 U·mL-1 MutA or MutB completely degraded 100 mg·L-1 chlortetracycline within 6 min in the presence of 0.1 mM ABTS. At pH 8.0, MutA degraded tetracycline and chlortetracycline following the routine pathways were reported previously based on LC-MS analysis.
Asunto(s)
Lacasa , Tetraciclinas , Lacasa/genética , Lacasa/metabolismo , Lacasa/química , Tetraciclinas/química , Tetraciclinas/metabolismo , Concentración de Iones de Hidrógeno , Temperatura , Biodegradación Ambiental , Evolución Molecular Dirigida , Mutación , Cinética , Hongos/enzimología , Hongos/genética , Oxidación-ReducciónRESUMEN
The simultaneous development of antibiotic resistance in bacteria due to metal exposure poses a significant threat to the environment and human health. This study explored how exposure to both arsenic and antibiotics affects the ability of an arsenite oxidizer, Achromobacter xylosoxidans CAW4, to transform arsenite and its antibiotic resistance patterns. The bacterium was isolated from arsenic-contaminated groundwater in the Chandpur district of Bangladesh. We determined the minimum inhibitory concentration (MIC) of arsenite, cefotaxime, and tetracycline for A. xylosoxidans CAW4, demonstrating a multidrug resistance (MDR) trait. Following this determination, we aimed to mimic an environment where A. xylosoxidans CAW4 was exposed to both arsenite and antibiotics. We enabled the strain to grow in sub-MIC concentrations of 1 mM arsenite, 40 µg/mL cefotaxime, and 20 µg/mL tetracycline. The expression dynamics of the arsenite oxidase (aioA) gene in the presence or absence of antibiotics were analyzed. The findings indicated that simultaneous exposure to arsenite and antibiotics adversely affected the bacteria's capacity to metabolize arsenic. However, when arsenite was present in antibiotics-containing media, it promoted bacterial growth. The study observed a global downregulation of the aioA gene in arsenic-antibiotic conditions, indicating the possibility of increased susceptibility through co-resistance across the entire bacterial population of the environment. This study interprets that bacterial arsenic-metabolizing ability can rescue the bacteria from antibiotic stress, further disseminating environmental cross-resistance. Therefore, the co-selection of metal-driven antibiotic resistance in bacteria highlights the need for effective measures to address this emerging threat to human health and the environment.
Asunto(s)
Arsénico , Arsenitos , Humanos , Arsénico/farmacología , Arsénico/metabolismo , Arsenitos/farmacología , Arsenitos/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Bacterias , Metales/farmacología , Metales/metabolismo , Farmacorresistencia Microbiana , Cefotaxima/metabolismo , Cefotaxima/farmacología , Tetraciclinas/metabolismo , Tetraciclinas/farmacologíaRESUMEN
Regulation of gene expression at the level of RNA and/or by regulatory RNA is an integral part of the regulatory circuits in all living cells. In bacteria, transcription and translation can be coupled, enabling regulation by transcriptional attenuation, a mechanism based on mutually exclusive structures in nascent mRNA. Transcriptional attenuation gives rise to small RNAs that are well suited to act in trans by either base pairing or ligand binding. Examples of 5'-UTR-derived sRNAs in the alpha-proteobacterium Sinorhizobium meliloti are the sRNA rnTrpL of the tryptophan attenuator and SAM-II riboswitch sRNAs. Analyses addressing RNA-based gene regulation often include measurements of steady-state levels and of half-lives of specific sRNAs and mRNAs. Using such measurements, recently we have shown that the tryptophan attenuator responds to translation inhibition by tetracycline and that SAM-II riboswitches stabilize RNA. Here we discuss our experience in using alternative RNA purification methods for analysis of sRNA and mRNA of S. meliloti. Additionally, we show that other translational inhibitors (besides tetracycline) also cause attenuation giving rise to the rnTrpL sRNA. Furthermore, we discuss the importance of considering RNA stability changes under different conditions and describe in detail a robust and fast method for mRNA half-life determination. The latter includes rifampicin treatment, RNA isolation using commercially available columns, and mRNA analysis by reverse transcription followed by quantitative PCR (RT-qPCR). The latter can be performed as a one-step procedure or in a strand-specific manner using the same commercial kit and a spike-in transcript as a reference.
Asunto(s)
ARN Pequeño no Traducido , Sinorhizobium meliloti , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Triptófano/metabolismo , Semivida , ARN Pequeño no Traducido/metabolismo , Tetraciclinas/metabolismo , ARN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Numerous studies have demonstrated the vital roles of gut microbes in the health, immunity, nutrient metabolism, and behavior of adult worker honeybees. However, a few studies have been conducted on gut microbiota associated with the larval stage of honeybees. In the present study, we explored the role of a gut bacterium in larval development and larval-pupal transition in the Asian honeybee, Apis cerana. First, our examination of gut microbial profiling showed that Bombella apis, a larvae-associated bacterium, was the most dominant bacterium colonized in the fifth instar larvae. Second, we demonstrated that tetracycline, an antibiotic used to treat a honeybee bacterial brood disease, could cause the complete depletion of gut bacteria. This antibiotic-induced gut microbiome depletion in turn, significantly impacted the survivorship, pupation rate and emergence rate of the treated larvae. Furthermore, our analysis of gene expression pattens revealed noteworthy changes in key genes. The expression of genes responsible for encoding storage proteins vitellogenin (vg) and major royal jelly protein 1 (mrjp1) was significantly down-regulated in the tetracycline-treated larvae. Concurrently, the expression of krüppel homolog 1(kr-h1), a pivotal gene in endocrine signaling, increased, whilethe expression of broad-complex (br-c) gene that plays a key role in the ecdysone regulation decreased. These alterations indicated a disruption in the coordination of juvenile hormone and ecdysteroid synthesis. Finally, we cultivated B. apis isolated from the fifth instar worker larval of A. cerana and fed tetracycline-treated larvae with a diet replenished by B. apis. This intervention resulted in a significant improvement in the pupation rate, emergence rate, and overall survival rate of the treated larvae. Our findings demonstrate the positive impact of B. apis on honeybee larvae development, providing new evidence of the functional capacities of gut microbes in honeybee growth and development.
Asunto(s)
Acetobacteraceae , Antibacterianos , Proteínas de Insectos , Abejas , Animales , Larva/metabolismo , Pupa/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Tetraciclinas/metabolismoRESUMEN
Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
Asunto(s)
Virus de la Fiebre Aftosa , Fiebre Aftosa , Vacunas Virales , Animales , Virus de la Fiebre Aftosa/genética , Proteínas de la Cápside , Proteínas Virales/metabolismo , Fiebre Aftosa/prevención & control , Tetraciclinas/metabolismo , Anticuerpos Antivirales , Mamíferos/metabolismoRESUMEN
BACKGROUND: The protozoan parasite Toxoplasma gondii encodes a dozen Rab proteins, which are parts of the small GTPase superfamily and regulate intracellular membrane trafficking. Our previous study showed that depletion of Rab1B caused severe defects regarding parasite growth and morphological structure, yet early defects of endocytic trafficking and vesicle sorting to the rhoptry in T. gondii are not expected to have a strong effect. To understand this discrepancy, we performed an integrated analysis at the level of transcriptomics and metabolomics. METHODS: In the study, tetracycline-inducible TATi/Ty-Rab1B parasite line treated with ATc at three different time points (0, 18 and 24 h) was used. We first observed the morphological changes caused by Rab1B depletion via transmission electron technology. Then, high-throughput transcriptome along with non-targeted metabolomics were performed to analyze the RNA expression and metabolite changes in the Rab1B-depleted parasite. The essential nature of Rab1B in the parasite was revealed by the integrated omics approach. RESULTS: Transmission electron micrographs showed a strong disorganization of endo-membranes in the Rab1B-depleted parasites. Our deep analysis of transcriptome and metabolome identified 2181 and 2374 differentially expressed genes (DEGs) and 30 and 83 differentially expressed metabolites (DEMs) at 18 and 24 h of induction in the tetracycline-inducible parasite line, respectively. These DEGs included key genes associated with crucial organelles that contain the rhoptry, microneme, endoplasmic reticulum and Golgi apparatus. The analysis of qRT-PCR verified some of the key DEGs identified by RNA-Seq, supporting that the key vesicular regulator Rab1B was involved in biogenesis of multiple parasite organelles. Functional enrichment analyses revealed pathways related to central carbon metabolisms and lipid metabolisms, such as the TCA cycle, glycerophospholipid metabolism and fatty acid biosynthesis and elongation. Further correlation analysis of the major DEMs and DEGs supported the role of Rab1B in biogenesis of fatty acids (e.g. myrisoleic acid and oleic acid) (R > 0.95 and P < 0.05), which was consistent with the scavenging role in biotin via the endocytic process. CONCLUSIONS: Rab1B played an important role in parasite growth and morphology, which was supported by the replication assay and transmission electron microscopy observation. Our multi-omics analyses provided detailed insights into the overall impact on the parasite upon depletion of the protein. These analyses reinforced the role of Rab1B in the endocytic process, which has an impact on fatty acid biogenesis and the TCA cycle. Taken together, these findings contribute to our understanding of a key vesicular regulator, Rab1B, on parasite metabolism and morphological formation in T. gondii.
Asunto(s)
Parásitos , Toxoplasma , Animales , Toxoplasma/genética , Toxoplasma/metabolismo , Transcriptoma , Perfilación de la Expresión Génica , Parásitos/genética , Ácidos Grasos/metabolismo , Tetraciclinas/metabolismo , Proteínas Protozoarias/genéticaRESUMEN
Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations is redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. One method to rescue this cell death vulnerability is the inhibition of mitochondrial translation using tetracyclines. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that tetracyclines inhibit the mitochondrial ribosome and promote survival through suppression of endoplasmic reticulum (ER) stress. Tetracyclines increase mitochondrial levels of the mitoribosome quality control factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) and promote its recruitment to the mitoribosome large subunit, where MALSU1 is necessary for tetracycline-induced survival and suppression of ER stress. Glucose starvation induces ER stress to activate the unfolded protein response and IRE1α-mediated cell death that is inhibited by tetracyclines. These studies establish a new interorganelle communication whereby inhibition of the mitoribosome signals to the ER to promote survival, implicating basic mechanisms of cell survival and treatment of mitochondrial diseases.
Asunto(s)
Enfermedades Mitocondriales , Ribosomas Mitocondriales , Humanos , Ribosomas Mitocondriales/metabolismo , Ribosomas Mitocondriales/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Supervivencia Celular , Tetraciclinas/farmacología , Tetraciclinas/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Estrés del Retículo Endoplásmico/genética , Enfermedades Mitocondriales/genéticaRESUMEN
The reuse of water using effluents containing antibiotics from anthropogenic activities has been mainly linked to the development of antibiotic resistance. However, we report that the development of bacterial tolerance promotes plant growth. In the present study, we aimed to evaluate the efficiency of inoculation of a new antibiotic-degrading bacterium, Erwinia strain S9, in augmenting the tolerance of pea (Pisum sativum L.) plants to tetracycline (TET) (10 and 20 mg/L). Physiological parameters such as tissue elongation and biomass, as well as relative water content, were remarkably lower in plants exposed to TET than in the control. The inhibitory effects of TET were associated with reduced CO2 assimilation, stomatal conductance, transpiration, dark respiration, and light saturation point (LSP). High concentrations of TET-induced oxidative stress are attested by the overproduction of superoxide radicals (O2â¢-), hydrogen peroxide (H2O2), and hydroxyl radicals (HOâ¢), resulting in increased malondialdehyde content and cell death. The high activity of antioxidant enzymes such as catalase, ascorbate peroxidase, and guaiacol peroxidase validated the proposed mechanism. Under TET stress conditions, supplementation with Erwinia strain S9 was beneficial to pea plants through osmotic adjustment, increased nutrient uptake, gas exchange optimization, and increased antioxidant activities. Its presence not only ensures plant survival and growth during antibiotic stress but also degrades TET via significant antibiotrophy. This strategy is a cost-effective environmental chemical engineering tool that can be used to depollute wastewater or to improve crop resistance in rhizofiltration treatment when treated wastewater is reused for irrigation.
Asunto(s)
Antioxidantes , Peróxido de Hidrógeno , Antioxidantes/metabolismo , Peróxido de Hidrógeno/metabolismo , Aguas Residuales , Hojas de la Planta/metabolismo , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Agua/metabolismo , Tetraciclinas/metabolismo , Tetraciclinas/farmacologíaRESUMEN
Co-exposure of tetracycline (TC) and polyethylene microplastic (MP-PE) pollution might result in more intricate effects on rice growth and grain quality. In present study, two different rice cultivars of contrasting drought tolerance, Hanyou73 (H73, drought-resistant) and Quanyou280 (Q280, drought-sensitive) were grown on MP-PE and TC-contaminated soils under drought. It was found that drought stress had different influence on TC accumulation in the two rice cultivars. H73 accumulated more TC in leaves and grains without drought stress while Q280 accumulated more TC under drought stress. Furthermore, metabolomics results demonstrated that under drought stress, about 80 % of metabolites in H73 and 95 % in Q280 were down-regulated as compared to non-drought treatments. These findings provide insights into the effects of TC and MP-PE with and without drought stress on potential risks to rice growth and grain quality, which has implications on rice production and cultivar election under multiple-stress conditions.
Asunto(s)
Oryza , Oryza/metabolismo , Plásticos , Polietileno/toxicidad , Polietileno/metabolismo , Microplásticos , Grano Comestible , Tetraciclinas/metabolismo , Estrés FisiológicoRESUMEN
Antibiotic administration during early life has been shown to have lasting effects on the gut microbiota, which have been linked to sustained alterations in liver metabolism and adiposity. Recent investigations have discerned that the gut microbiota continues to develop toward an adult-like profile during adolescence. However, the impact of antibiotic exposure during adolescence on metabolism and adiposity is unclear. Herein, a retrospective analysis of Medicaid claims data was performed, which indicated that tetracycline class antibiotics are commonly prescribed for the systemic treatment of adolescent acne. The purpose of this was to discern the impact of a prolonged tetracycline antibiotic exposure during adolescence on the gut microbiota, liver metabolism, and adiposity. Male C57BL/6T specific pathogen-free mice were administered a tetracycline antibiotic during the pubertal/postpubertal adolescent growth phase. Groups were euthanized at different time points to assess immediate and sustained antibiotic treatment effects. Antibiotic exposure during adolescence caused lasting genera-level shifts in the intestinal bacteriome and persistent dysregulation of metabolic pathways in the liver. Dysregulated hepatic metabolism was linked to sustained disruption of the intestinal farnesoid X receptor-fibroblast growth factor 15 axis, a gut-liver endocrine axis that supports metabolic homeostasis. Antibiotic exposure during adolescence increased subcutaneous, visceral, and marrow adiposity, which intriguingly manifested following antibiotic therapy. This preclinical work highlights that prolonged antibiotic courses for the clinical treatment of adolescent acne may have unintended deleterious effects on liver metabolism and adiposity.
Asunto(s)
Adiposidad , Antibacterianos , Masculino , Ratones , Animales , Antibacterianos/efectos adversos , Estudios Retrospectivos , Ratones Endogámicos C57BL , Obesidad/metabolismo , Hígado/metabolismo , Tetraciclinas/metabolismoRESUMEN
Adipocytes and osteoblasts derive from a common mesenchymal progenitor present in a range of connective tissues. Differentiation of the progenitors toward the two cell lineages can be induced in vitro through well-established protocols, and leads to the appearance of lipid-laden adipocytes and osteoblasts embedded in a mineralized matrix. The formation of these two lineages in cell cultures can be monitored using lipophilic dyes such as Oil Red O and substances binding to mineral deposits such as Alizarin Red S, respectively. However, these common staining techniques require cell fixation and are thus incompatible with live analyses. Recently, alternative approaches using vital stains have allowed the dual visualization and fluorescence imaging of adipogenic and osteogenic lineages in live cultures. Here we present the concomitant analysis of cultures containing adipogenic and osteogenic cell types using live staining, combining LipidTox Red and tetracycline with NucRed nuclear counterstain for confocal imaging. This approach can be applied to visualize the kinetics and 3D structure of differentiating mesenchymal cultures over time and highlights the interaction of adipose and mineralized compartments associated with bone marrow stroma.
Asunto(s)
Células Madre Mesenquimatosas , Adipogénesis , Diferenciación Celular , Células Cultivadas , Colorantes/metabolismo , Lípidos , Minerales , Osteogénesis , Células Madre , Tetraciclinas/metabolismoRESUMEN
Nerve/glial antigen 2 (NG2) is a protein marker of NG2 glia and mural cells, and NG2 promoter activity is utilized to target these cells. However, the NG2 promoter cannot target NG2 glia and mural cells separately. This has been an obstacle for NG2 glia-specific manipulation. Here, we developed transgenic mice in which either cell type can be targeted using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type-specific transgene expression, and generated NG2-tetracycline transactivator (tTA) transgenic lines. We crossed tTA lines with the tetO-ChR2 (channelrhodopsin-2)-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA mouse lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in NG2 glia-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that tTA-mediated ChR2 expression was sufficient to impact cellular function. ChR2-mediated depolarization was observed in NG2 glia in acute hippocampal slices. In addition, ChR2-mediated depolarization of NG2 glia inhibited their proliferation but promoted their differentiation in juvenile mice. Since the tTA-tetO combination is expandable, the mural cell-specific NG2-tTA line and the NG2 glia-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.
Asunto(s)
Neuroglía , Optogenética , Animales , Ratones , Neuroglía/metabolismo , Ratones Transgénicos , Antígenos/genética , Antígenos/metabolismo , Tetraciclinas/metabolismoRESUMEN
Bacteria have a mechanism to rescue stalled ribosomes known as trans-translation consisting of SsrA, a transfer-messenger RNA (tmRNA), and the small protein SmpB. Other alternative rescue mechanisms mediated by ArfA and ArfB proteins are present only in some species. Ribosome rescue mechanisms also play a role in tolerance to antibiotics and various stresses such as heat. This study shows that the genome of the soil bacterium A. vinelandii harbours genes encoding for tmRNA, SmpB, two paralogs of ArfA (arfA1 and arfA2), and ArfB. A number of mutant strains carrying mutations in the ssrA, arfA1, arfA2, and arfB genes were constructed and tested for their growth and susceptibility to heat and the antibiotic tetracycline. We found that the inactivation of both ssrA and one or the two arfA genes was detrimental to growth and caused a higher susceptibility to heat and to the antibiotic tetracycline. Interestingly, the arfB mutant strain was unable to grow after 2 h of incubation at 45°C. Inactivation of arfB in the ssrA-arfA1-arfA2 strain caused a lethal phenotype since the quadruple mutant could not be isolated. Taken together, our data suggest that both arfA1 and arfA2, as well as arfB, are functional as back up mechanisms, and that the ArfB pathway has an essential role that confers A. vinelandii resistance to high temperatures.
Asunto(s)
Azotobacter vinelandii , Azotobacter vinelandii/genética , Antibacterianos/farmacología , Antibacterianos/metabolismo , Calor , Proteínas de Unión al ARN/genética , Ribosomas/genética , Ribosomas/metabolismo , ARN Bacteriano/genética , Biosíntesis de Proteínas , Tetraciclinas/metabolismoRESUMEN
Toxoplasma gondii (T. gondii) is a pathogen belonging to the apicomplexan phylum, and it threatens human and animal health. Calcium ions, a critical second messenger in cells, can regulate important biological processes, including parasite invasion and egress. Calmodulin (CaM) is a small, highly conserved, Ca2+-binding protein found in all eukaryotic cells. After binding to Ca2+, CaM can be activated to interact with various proteins. However, little is known about CaM's function and its interacting proteins in T. gondii. In this study, we successfully knocked down CaM in the T. gondii parent strain TATI using a tetracycline-off system with the Toxoplasma CaM promoter. The results indicated that CaM was required for tachyzoite proliferation, invasion, and egress and that CaM depletion resulted in apicoplast loss, thus threatening parasite survival in the next lytic cycle. In the tachyzoite stage, CaM loss caused significant anomalies in the parasite's basal constriction, motility, and parasite rosette-like arrangement in the parasitophorous vacuole (PV). These phenotypic defects caused by CaM depletion indicate the importance of CaM in T. gondii. Therefore, it is important to identify the CaM-interacting proteins in T. gondii. Applying BioID technology, more than 300 CaM's proximal interacting proteins were identified from T. gondii. These CaM partners were broadly distributed throughout the parasite. Furthermore, the protein interactome and transcriptome analyses indicated the potential role of CaM in ion binding, cation binding, metal ion binding, calcium ion binding, and oxidation-reduction. Our findings shed light on the CaM function and CaM-interactome in T. gondii and other eukaryotes. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that threatens human and animal health. This unicellular parasite is active in many biological processes, such as egress and invasion. The implementation efficiency of T. gondii biological processes is dependent on signal transmission. Ca2+, as a second messenger, is essential for the parasite's life cycle. Calmodulin, a ubiquitous Ca2+ receptor protein, is highly conserved and mediates numerous Ca2+-dependent events in eukaryotes. Few CaM functions or regulated partners have been characterized in T. gondii tachyzoites. Here, we reported the essential functions of calmodulin in T. gondii tachyzoite and the identification of its interacting partners using BioID technology, shedding light on the CaM function and CaM-interactome in Toxoplasma gondii and other eukaryotes.
Asunto(s)
Parásitos , Toxoplasma , Animales , Humanos , Toxoplasma/genética , Calmodulina/genética , Calmodulina/metabolismo , Calcio/metabolismo , Tecnología , Tetraciclinas/metabolismo , Cationes/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Although anaerobic bioengineering treatment of chicken manure has the advantages of low energy consumption, less pollution, and recyclable biomass energy, antibiotics are usually added in the process of modern intensive farming. And antibiotics still exist in feces and pose a threat to human health. Therefore, this study aimed to deeply understand the role of TC in the anaerobic digestion of chicken manure and to analyze the effect of tetracycline antibiotics on the anaerobic digestion of chicken manure based on data mining. In this study, chicken manure was used as raw material for anaerobic fermentation, and the effects of tetracycline (TC) on anaerobic and anaerobic fermentation of chicken manure were compared through batch and sequence experiments. Also, this study analyzes the fermentative transformation to elucidate the effect of TC on anaerobic manure conversion in chicken manure, which further studies the effect of TC on the anaerobic fermentation of chicken manure. The experimental results in this study show that when the TC concentration is 50-150 mg/L, the content of tryptophan and tyrosine proteins in the treatment group is also higher than that in the control group. It shows that at low concentrations (10, 25 mg/L), TC mainly promotes the degradation of LEPS by promoting the dissolution of intracellular substances in the sludge. With the increase of TC concentration (50-300 mg/L), TC mainly promotes the breakdown of the sludge cell membrane by promoting the rupture of the sludge cell membrane, thereby promoting the degradation of LEPS.
Asunto(s)
Pollos , Estiércol , Anaerobiosis , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Pollos/metabolismo , Minería de Datos , Aguas del Alcantarillado , Tetraciclinas/metabolismoRESUMEN
Cyromazine, a symmetrical triazine insecticide, is used to control dipteran larvae in chicken manure by feeding to the poultry, flies on animals, and leafminers in vegetables. Its extensive use has resulted in the widespread contamination in the environment. In the current study, a cyromazine degrading bacterium (designated strain ZX01) was isolated and characterized from a Chinese ginger cultivated soil by selective enrichment culture method. On the basis of morphological, biochemical characteristics, and 16S rRNA gene sequence, this bacterium showed strong similarity to the Pseudomonadales members and was closely related to the Acinetobacter baumannii group. Spectrophotometric and HPLC analyses revealed that strain ZX01 degraded cyromazine and utilized it as the sole carbon source for its growth. This process hydrolyzes cyromazine to melamine. Strain ZX01 degraded most of the cyromazine in 60 h. Besides, its substrate specificity against four symmetrical triazine herbicides, one triazinone herbicide, as well as 10 insecticides and its antibiotic sensitivity towards eight commercial antibiotics were also tested. At the concentration of 100 µg/mL for 60 h, it could effectively degrade a variety of different pesticides, including atrazine, prometon, simazine, prometryn, enitrothion, diazinon, cypermethrin, and acetamiprid, and the degradation was in the range of 71-87%. In particular, melamine, the main degradation product of cyromazine, was degraded by 47.3%. This microorganism was sensitive to chloramphenicol and tetracycline and intermediate to amoxicillin and trimethoprim. These results highlight that strain ZX01 can be used as a potential biological agent for the remediation of soil, water, or crop contaminated with cyromazine and other symmetrical triazine insecticides.
Asunto(s)
Acinetobacter , Atrazina , Herbicidas , Insecticidas , Plaguicidas , Zingiber officinale , Acinetobacter/metabolismo , Amoxicilina , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Atrazina/metabolismo , Biodegradación Ambiental , Factores Biológicos/metabolismo , Carbono/metabolismo , China , Cloranfenicol , Diazinón , Herbicidas/metabolismo , Insecticidas/metabolismo , Estiércol , Plaguicidas/metabolismo , Prometrina/metabolismo , ARN Ribosómico 16S/genética , Simazina , Suelo , Microbiología del Suelo , Tetraciclinas/metabolismo , Triazinas/metabolismo , Trimetoprim , Agua/metabolismoRESUMEN
BACKGROUND: Mitochondrial diseases represent one of the most common groups of genetic diseases. With a prevalence greater than 1 in 5000 adults, such diseases still lack effective treatment. Current therapies are purely palliative and, in most cases, insufficient. Novel approaches to compensate and, if possible, revert mitochondrial dysfunction must be developed. RESULTS: In this study, we tackled the issue using as a model fibroblasts from a patient bearing a mutation in the GFM1 gene, which is involved in mitochondrial protein synthesis. Mutant GFM1 fibroblasts could not survive in galactose restrictive medium for more than 3 days, making them the perfect screening platform to test several compounds. Tetracycline enabled mutant GFM1 fibroblasts survival under nutritional stress. Here we demonstrate that tetracycline upregulates the mitochondrial Unfolded Protein Response (UPRmt), a compensatory pathway regulating mitochondrial proteostasis. We additionally report that activation of UPRmt improves mutant GFM1 cellular bioenergetics and partially restores mitochondrial protein expression. CONCLUSIONS: Overall, we provide compelling evidence to propose the activation of intrinsic cellular compensatory mechanisms as promising therapeutic strategy for mitochondrial diseases.
Asunto(s)
Enfermedades Mitocondriales , Respuesta de Proteína Desplegada , Humanos , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Tetraciclinas/metabolismoRESUMEN
BACKGROUND: Tigecycline is one of the few last-resort antibiotics for the treatment of carbapenem-resistant Enterobacteriaceae infection, the incidence of which has been rapidly increasing. However, the emergence and spread of tigecycline resistance genes tet(X) (including tet(X3) and tet(X4)) has largely compromised the efficient usage of tetracyclines in the clinical settings. METHODS: The synergistic effect was determined by a checkerboard minimum inhibitory concentration (MIC) assay, a time-killing assay and scanning electron microscopy (SEM) analysis. In-depth mechanisms were defined using an enzyme inhibition assay, western blotting, RT-PCR analysis, molecular dynamics (MD) simulations, biolayer interferometry (BLI) assay and metabolomics analysis. FINDINGS: Herein, our work identified a natural compound, plumbagin, as an effective broad-spectrum inhibitor of Tet(X) (also known as monooxygenase) by simultaneously inhibiting the activity and the production of Tet(X3)/Tet(X4). Plumbagin in combination with tetracyclines showed a synergistic bactericidal effect against Tet(X3)/Tet(X4)-producing bacteria. Mechanistic studies revealed that direct engagement of plumbagin with the catalytic pocket of Tet(X3)/Tet(X4) induced an alternation in its secondary structure to inhibit the activity of these monooxygenases. As a consequence, monotherapy or combination therapy with plumbagin increases the oxidative stress and metabolism in bacteria. Moreover, in a mouse systemic infection model of tet(X4)-positive E. coli, the combination of plumbagin and methacycline exhibited remarkable treatment benefits, as shown by a reduced bacterial load and the alleviation of pathological injury. INTERPRETATION: Plumbagin, as an inhibitor of Tet(X3)/Tet(X4), represents a promising lead drug, as well as an adjunct with tetracyclines to treat bacterial infections, especially for extensively drug-resistant bacteria harbouring Tet(X3)/Tet(X4). FUNDING: The National Natural Science Foundation of China.
Asunto(s)
Escherichia coli , Tetraciclinas , Animales , Antibacterianos/farmacología , Bacterias/metabolismo , Escherichia coli/genética , Ratones , Pruebas de Sensibilidad Microbiana , Oxigenasas de Función Mixta/genética , Plásmidos , Tetraciclinas/metabolismo , Tetraciclinas/farmacología , Tigeciclina/metabolismo , Tigeciclina/farmacologíaRESUMEN
Phycoremediation of swine wastewater is an attractive treatment to remove contaminants and simultaneously produce valuable feedstock biomass. However, there is a lack of information about the application of phycoremediation on veterinary antibiotic removal. Thus, this research investigated the degradation of tetracycline, oxytetracycline, chlortetracycline and doxycycline in swine wastewater treated with phycoremediation. The tetracyclines degradation kinetics was adjusted to the pseudo-first-order kinetics model, with kinetic constant k1 in the following: 0.36 > 0.27>0.19 > 0.18 (d-1) for tetracycline, doxycycline, oxytetracycline and chlortetracycline, respectively. The maximum concentration of microalgae biomass (342.4 ± 20.3 mg L-1) was obtained after 11 days of cultivation, when tetracycline was completely removed. Chlortetracycline concentration decreased, generating iso-chlortetracycline and 4-epi-iso-chlortetracycline. Microalgae biomass harvested after antibiotics removal presented a carbohydrate-rich content of 52.7 ± 8.1, 50.1 ± 3.3, 51.4 ± 5.4 and 57.4 ± 10.4 (%) when cultured with tetracycline, oxytetracycline, chlortetracycline and doxycycline, respectively, while the control culture without antibiotics presented a carbohydrate content of 40 ± 6.5%. These results indicate that could be a valuable source for bioenergy conversion.
Asunto(s)
Microalgas , Animales , Antibacterianos , Microalgas/metabolismo , Porcinos , Tetraciclina , Tetraciclinas/metabolismo , Aguas ResidualesRESUMEN
Tigecycline is regarded as one of the few important last-resort antibiotics to treat complicated skin and intra-abdominal infections. Members of the genus Staphylococcus are zoonotic pathogens and pose a serious threat to public health. Tigecycline resistance in this species appears to be a rare phenomenon, and the mechanisms underlying tigecycline resistance have not been fully elucidated. Here, we report two novel variants of the tet(L) gene in Staphylococcus spp. from swine in China, designed as tet(L)F58L and tet(L)A117V. The tet(L)F58L was located within a 18,720 bp chromosomal multidrug resistance gene cluster flanked by two copies of IS257 in Staphylococcus cohnii 11-B-312, while the tet(L)A117V was located on a 6,292 bp plasmid in S. haemolyticus 11-B-93, which could be transferred to S. aureus by electrotransformation. Cloning of each of the two tet(L) variants into S. aureus RN4220 showed 16- or 8-fold increases in the minimal inhibition concentrations (MICs), which can fully confer the resistance to tigecycline (MICs from 0.125 to 2 mg/liter) and eravacycline (MICs from 0.125 to 1 or 2 mg/liter), but no increase in the MICs of omadacycline, compared with the MICs of the recipient strain S. aureus RN4220. In the in vivo murine sepsis and in the murine pneumonia models, an increase in CFU of S. aureus 29213_pT93 carrying the tet(L)A117V was seen despite tigecycline treatment. This observation suggests that the tet(L)A117V and its associated gene product compromise the efficacy of tigecycline treatment in vivo and may lead to clinical treatment failure. Our finding, that novel Tet(L) efflux pump variants which confer tigecycline and eravacycline resistance have been identified in Staphylococcus spp., requires urgent attention. IMPORTANCE Tigecycline and eravacycline are both important last-resort broad spectrum antimicrobial agents. The presence of novel Tet(L) efflux pump variants conferring the resistance to tigecycline and eravacycline in Staphylococcus spp. and its potential transmission to S. aureus will compromise the efficacy of tigecycline and eravacycline treatment for S. aureus associated infection in vivo and may lead to clinical treatment failure.