RESUMO
Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1(+)(-)) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1(+)(-) showed â¼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1(+)(-) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1(+)(-) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1(+)(-) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1(+)(-) were found to increase killing of Caenorhabditis elegans compared to WT.
Assuntos
Antifúngicos/farmacologia , Proteínas Fúngicas/genética , Mucor/efeitos dos fármacos , Mucor/genética , Genótipo , Mucor/patogenicidade , Mutação , Filogenia , Transporte Proteico , Esporos Fúngicos/patogenicidade , Proteínas de Transporte Vesicular/genética , VirulênciaRESUMO
The ethanol stress response in ethanologenic yeast during fermentation involves the swishing of several adaptation mechanisms. In Saccharomyces cerevisiae, the Jac1p and Isu1p proteins constitute the scaffold system for the Fe-S cluster assembly. This study was performed using the over-expression of the Jac1p and Isu1p in the industrially utilized S. cerevisiae UMArn3 strain, with the objective of improving the Fe-S assembly/recycling, and thus counteracting the toxic effects of ethanol stress during fermentation. The UMArn3 yeast was transformed with both the JAC1-His and ISU1-His genes-plasmid contained. The Jac1p and Isu1p His-tagged proteins over-expression in the engineered yeasts was confirmed by immunodetection, rendering increases in ethanol tolerance level from a DL50 = ~ 4.5% ethanol (v/v) to DL50 = ~ 8.2% ethanol (v/v), and survival up 90% at 15% ethanol (v/v) comparing to ~ 50% survival in the control strain. Fermentation by the engineered yeasts showed that the ethanol production was increased, producing 15-20% more ethanol than the control yeast. The decrease of ROS and free-iron accumulation was observed in the engineered yeasts under ethanol stress condition. The results indicate that Jac1p and Isu1p over-expression in the S. cerevisiae UMArn3.3 yeast increased its ethanol tolerance level and ethanol production by a mechanism that involves ROS and iron homeostasis related to the biogenesis/recycling of Fe-S clusters dependent proteins.
Assuntos
Etanol/metabolismo , Homeostase , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fermentação , Ferro/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Superóxidos/metabolismoRESUMO
The Cuatro Ciénegas Basin (CCB) within the Chihuahuan Desert in México is an extremely oligotrophic oasis with negligible phosphorous levels, described as a hot spot of biodiversity, not only in stromatolites and microbial mats, but also in living forms in general. The microorganisms possess the capability to produce a wide variety of virulence factors, antibiotics, and quorum-sensing (QS) crosstalk signals such as non-ribosomal cyclodipeptides (CDPs) which enables them to colonize diverse ecological niches. In the aquatic system of CCB known as Churince, a bacterial population was isolated from the Lagunita pond dominated by Gammaproteobacteria. In this work, we determined the relationships between the antagonism and CDPs production in this bacterial population. Results indicate that 68% of isolates showed antagonistic effects over other isolates, correlating with production of CDPs and the antibiotic 2,4-diacetylphloroglucinol (DAPG). Although a minority of the isolates were capable of inducing a QS biosensor strain, bacterial QS interference was not the main mechanism in the antagonism observed. Thus, our results indicate that CDPs primarily, and DAPG to a lesser degree, are involved with the growth-inhibition competition mechanisms of bacterial communities in the Lagunita pond and was associated with a Gammaproteobacteria dominancy phenomena.
Assuntos
Gammaproteobacteria/metabolismo , Microbiota , Peptídeos Cíclicos/metabolismo , Percepção de Quorum , Gammaproteobacteria/fisiologia , México , Microbiologia da ÁguaRESUMO
Diverse molecules mediate cross-kingdom communication between bacteria and their eukaryotic partners and determine pathogenic or symbiotic relationships. N-acyl-L-homoserine lactone-dependent quorum-sensing signaling represses the biosynthesis of bacterial cyclodipeptides (CDPs) that act as auxin signal mimics in the host plant Arabidopsis thaliana. In this work, we performed bioinformatics, biochemical, and plant growth analyses to identify non-ribosomal peptide synthase (NRPS) proteins of Pseudomonas aeruginosa, which are involved in CDP synthesis. A reverse genetics strategy allowed the identification of the genes encoding putative multi-modular-NRPS (MM-NRPS). Mutations in these genes affected the synthesis of the CDPs cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Leu), and cyclo(L-Pro-L-Tyr), while showing wild-type-like levels of virulence factors, such as violacein, elastase, and pyocyanin. When analyzing the bioactivity of purified, naturally produced CDPs, it was found that cyclo(L-Pro-L-Tyr) and cyclo(L-Pro-L-Val) were capable of antagonizing quorum-sensing-LasR (QS-LasR)-dependent signaling in a contrasting manner in the cell-free supernatants of the selected NRPS mutants, which showed QS induction. Using a bacteria-plant interaction system, we further show that the pvdJ, ambB, and pchE P. aeruginosa mutants failed to repress primary root growth, but improved root branching in A. thaliana seedlings. These results indicated that the CDP production in P. aeruginosa depended on the functional MM-NRPS, which influences quorum-sensing of bacteria and plays a role in root architecture remodeling.
Assuntos
Arabidopsis/microbiologia , Dipeptídeos/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Biossíntese de Peptídeos Independentes de Ácido Nucleico/genética , Peptídeos Cíclicos/metabolismo , Piperazinas/metabolismo , Raízes de Plantas/embriologia , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/fisiologia , Dipeptídeos/genética , Ácidos Indolacéticos/metabolismo , Indóis/metabolismo , Peptídeos Cíclicos/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Piocianina/metabolismo , Percepção de Quorum/genética , Transdução de Sinais , Fatores de Virulência/metabolismoRESUMO
Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0-G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD50 of 60-250 µM) and inducing apoptosis with EC50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~10³-105 fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K-Akt-mTOR-S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.
Assuntos
Dipeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudomonas aeruginosa/química , Proteínas Quinases S6 Ribossômicas/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Dipeptídeos/isolamento & purificação , Dipeptídeos/metabolismo , Células HeLa , Humanos , Dose Letal Mediana , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Ethanol accumulation during fermentation contributes to the toxic effects in Saccharomyces cerevisiae, impairing its viability and fermentative capabilities. The iron-sulfur (Fe-S) cluster biogenesis is encoded by the ISC genes. Reactive oxygen species (ROS) generation is associated with iron release from Fe-S-containing enzymes. We evaluated ethanol toxicity, ROS generation, antioxidant response and mitochondrial integrity in S. cerevisiae ISC mutants. These mutants showed an impaired tolerance to ethanol. ROS generation increased substantially when ethanol accumulated at toxic concentrations under the fermentation process. At the cellular and mitochondrial levels, ROS were increased in yeast treated with ethanol and increased to a higher level in the ssq1∆, isa1∆, iba57∆ and grx5∆ mutants - hydrogen peroxide and superoxide were the main molecules detected. Additionally, ethanol treatment decreased GSH/GSSG ratio and increased catalase activity in the ISC mutants. Examination of cytochrome c integrity indicated that mitochondrial apoptosis was triggered following ethanol treatment. The findings indicate that the mechanism of ethanol toxicity occurs via ROS generation dependent on ISC assembly system functionality. In addition, mutations in the ISC genes in S. cerevisiae contribute to the increase in ROS concentration at the mitochondrial and cellular level, leading to depletion of the antioxidant responses and finally to mitochondrial apoptosis.
Assuntos
Etanol/metabolismo , Ferro/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Enxofre/metabolismo , Apoptose/efeitos dos fármacos , Etanol/toxicidade , Fermentação , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The [Fe-S] late-acting subsystem comprised of Isa1p/Isa2p, Grx5p, and Iba57p proteins (Fe-S-IBG subsystem) is involved in [4Fe-4S]-cluster protein assembly. The effect of deleting IBA57 in Saccharomyces cerevisiae on mitochondrial respiratory complex integration and functionality associated with Rieske protein maturation was evaluated. The iba57Δ mutant showed decreased expression and maturation of the Rieske protein. The loss of Rieske protein caused by IBA57 deletion affected the structure of supercomplexes III2IV2 and III2IV1 and their integration into the mitochondria, causing dysfunction in the electron transport chain. These effects were correlated with decreased cytochrome functionality and content in the iba57Δ mutant. These findings suggest that Iba57p participates in maturation of the [2Fe-2S]-cluster into the Rieske protein and that Rieske protein plays important roles in the conformation and functionality of mitochondrial supercomplex III/IV in the electron transport chain.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citocromos/deficiência , Deleção de Genes , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mezcal, a traditional beverage that originated in Mexico, is produced from species of the Agavaceae family. The esters associated with the yeasts utilized during fermentation are important for improving the organoleptic properties of the beverage. We improved the ester contents in a mezcal beverage by using the yeast Kluyveromyces marxianus, which was engineered with the ATF1 gene. ATF1 expression in the recombinant yeast significantly increased compared with that in the parental yeast, but its fermentative parameters were unchanged. Volatile-organic-compound-content analysis showed that esters had significantly increased in the mezcal produced with the engineered yeast. In a sensory-panel test, 48% of the panelists preferred the mezcal produced from the engineered yeast, 30% preferred the mezcal produced from the wild type, and 15 and 7% preferred the two mezcal types produced following the routine procedure. Correlation analysis showed that the fruitiness/sweetness description of the mezcal produced using the ATF1-engineered K. marxianus yeast correlated with the content of the esters, whose presence improved the organoleptic properties of the craft mezcal beverage.
Assuntos
Bebidas Alcoólicas/análise , Asparagaceae/química , Ácidos Graxos/análise , Kluyveromyces/genética , Proteínas/genética , Sensação , Comportamento do Consumidor , Ésteres/análise , Fermentação , Expressão Gênica , Humanos , Kluyveromyces/metabolismo , México , Microrganismos Geneticamente Modificados/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The related study has confirmed that in Saccharomyces cerevisiae, iba57 protein participates in maturation of the [2Fe-2S] cluster into the Rieske protein, which plays important roles in the conformation and functionality of mitochondrial supercomplexes III/IV in the electron transport chain (Sánchez et al., 2018) [1]. We determined in S. cerevisiae the effects of mutation in the IBA57 gene on reactive oxygen species (ROS) and iron homeostasis. Flow cytometry and confocal microscopy analyses showed an increased generation of ROS, correlated with free Fe2+ release in the IBA57 mutant yeast. Data obtained support that a dysfunction in the Rieske protein has close relationship between ROS generation and free Fe2+ content, and which is possible that free Fe2+ release mainly proceeds from [Fe-S] cluster-containing proteins.
RESUMO
Pseudomonas aeruginosa is an opportunistic pathogen of plants and animals, which produces virulence factors in order to infect or colonize its eukaryotic hosts. Cyclodipeptides (CDPs) produced by P. aeruginosa exhibit cytotoxic properties toward human tumor cells. In this study, we evaluated the effect of a CDP mix, comprised of cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) that were isolated from P. aeruginosa, on two human cancer cell lines. Our results demonstrated that the CDP mix promoted cell death in cultures of the HeLa cervical adenocarcinoma and Caco-2 colorectal adenocarcinoma cell lines in a dose-dependent manner, with a 50% inhibitory concentration (IC50) of 0.53 and 0.66 mg/mL, for HeLa and Caco-2 cells, respectively. Flow cytometric analysis, using annexin V and propidium iodide as apoptosis and necrosis indicators, respectively, clearly showed that HeLa and Caco-2 cells exhibited apoptotic characteristics when treated with the CDP mix at a concentration <0.001 mg/mL. IC50 values for apoptotic cells in HeLa and Caco-2 cells were 6.5 × 10(-5) and 1.8 × 10(-4) mg/mL, respectively. Our results indicate that an apoptotic pathway is involved in the inhibition of cell proliferation caused by the P. aeruginosa CDP mix.
Assuntos
Apoptose/efeitos dos fármacos , Dipeptídeos/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/metabolismo , Antineoplásicos/administração & dosagem , Células CACO-2 , Linhagem Celular Tumoral , Dipeptídeos/metabolismo , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Especificidade da EspécieRESUMO
Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.