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      Transcriptional changes of proteins of the thioredoxin and glutathione systems in Acanthamoeba spp. under oxidative stress – an RNA approach Translated title: Modifications transcriptionnelles des protéines des système thiorédoxine et glutathion chez Acanthamoeba spp. sous stress oxydatif – une approche par l’ARN

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          Abstract

          The thioredoxin (Trx) and the glutathione (GSH) systems represent important antioxidant systems in cells and in particular thioredoxin reductase (TrxR) has been shown to constitute a promising drug target in parasites. For the facultative protozoal pathogen Acanthamoeba, it was demonstrated that a bacterial TrxR as well as a TrxR, characteristic of higher eukaryotes, mammals and humans is expressed on the protein level. However, only bacterial TrxR is strongly induced by oxidative stress in Acanthamoeba castellanii. In this study, the impact of oxidative stress on key enzymes involved in the thioredoxin and the glutathione system of A. castellanii under different culture conditions and of clinical Acanthamoeba isolates was evaluated on the RNA level employing RT-qPCR. Additionally, the effect of auranofin, a thioredoxin reductase inhibitor, already established as a potential drug in other parasites, on target enzymes in A. castellanii was investigated. Oxidative stress induced by hydrogen peroxide led to significant stimulation of bacterial TrxR and thioredoxin, while diamide had a strong impact on all investigated enzymes. Different strains displayed distinct transcriptional responses, rather correlating to sensitivity against the respective stressor than to respective pathogenic potential. Culture conditions appear to have a major effect on transcriptional changes in A. castellanii. Treatment with auranofin led to transcriptional activation of the GSH system, indicating its role as a potential backup for the Trx system. Altogether, our data provide more profound insights into the complex redox system of Acanthamoeba, preparing the ground for further investigations on this topic.

          Translated abstract

          Les systèmes de la thiorédoxine (Trx) et du glutathion (GSH) représentent des systèmes antioxydants importants dans les cellules et, en particulier, la thiorédoxine réductase (TrxR) s’est avérée constituer une cible médicamenteuse prometteuse chez les parasites. Pour le pathogène protozoaire facultatif Acanthamoeba, il a été démontré qu’une TrxR bactérienne ainsi qu’une TrxR, caractéristique des eucaryotes supérieurs, des mammifères et des humains, s’expriment au niveau protéique. Cependant, seule la TrxR bactérienne est fortement induite par le stress oxydatif chez Acanthamoeba castellanii. Dans cette étude, l’impact du stress oxydatif sur les enzymes clés impliquées dans la thiorédoxine et le système glutathion d’ A. castellanii dans différentes conditions de culture et d’isolats cliniques d’ Acanthamoeba a été évalué au niveau de l’ARN en utilisant la RT-qPCR. De plus, l’effet de l’auranofine, un inhibiteur de la thiorédoxine réductase déjà établi comme médicament potentiel chez d’autres parasites, a été étudié sur les enzymes cibles chez A. castellanii. Le stress oxydatif induit par le peroxyde d’hydrogène a conduit à une stimulation significative du TrxR bactérien et de la thiorédoxine tandis que le diamide a eu un fort impact sur toutes les enzymes étudiées. Différentes souches ont affiché des réponses transcriptionnelles distinctes, plutôt corrélées à la sensibilité contre le facteur de stress respectif qu’à leur potentiel pathogène respectif. Les conditions de culture semblent avoir un effet majeur sur les changements transcriptionnels chez A. castellanii. Le traitement à l’auranofine a conduit à une activation transcriptionnelle du système GSH, indiquant son rôle de sauvegarde potentielle pour le système Trx. Dans l’ensemble, nos données fournissent des informations plus approfondies sur le système redox complexe d’ Acanthamoeba, préparant le terrain pour de nouvelles investigations sur ce sujet.

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          Most cited references45

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          A new mathematical model for relative quantification in real-time RT-PCR.

          M. Pfaffl (2001)
          Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
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            Genomic expression programs in the response of yeast cells to environmental changes.

            We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.
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              Glutathione peroxidases.

              With increasing evidence that hydroperoxides are not only toxic but rather exert essential physiological functions, also hydroperoxide removing enzymes have to be re-viewed. In mammals, the peroxidases inter alia comprise the 8 glutathione peroxidases (GPx1-GPx8) so far identified. Since GPxs have recently been reviewed under various aspects, we here focus on novel findings considering their diverse physiological roles exceeding an antioxidant activity. GPxs are involved in balancing the H2O2 homeostasis in signalling cascades, e.g. in the insulin signalling pathway by GPx1; GPx2 plays a dual role in carcinogenesis depending on the mode of initiation and cancer stage; GPx3 is membrane associated possibly explaining a peroxidatic function despite low plasma concentrations of GSH; GPx4 has novel roles in the regulation of apoptosis and, together with GPx5, in male fertility. Functions of GPx6 are still unknown, and the proposed involvement of GPx7 and GPx8 in protein folding awaits elucidation. Collectively, selenium-containing GPxs (GPx1-4 and 6) as well as their non-selenium congeners (GPx5, 7 and 8) became key players in important biological contexts far beyond the detoxification of hydroperoxides. This article is part of a Special Issue entitled Cellular functions of glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.
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                Author and article information

                Journal
                Parasite
                Parasite
                parasite
                Parasite
                EDP Sciences
                1252-607X
                1776-1042
                2022
                09 May 2022
                : 29
                : ( publisher-idID: parasite/2022/01 )
                : 24
                Affiliations
                [1 ] Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna 1090 Vienna Austria
                Author notes
                Author information
                http://orcid.org/0000-0003-2273-4163
                Article
                parasite220014 10.1051/parasite/2022025
                10.1051/parasite/2022025
                9083255
                35532265
                629498b0-e5a9-4fc3-b28d-4725e9bfd981
                © M. Köhsler et al., published by EDP Sciences, 2022

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 31 January 2022
                : 19 April 2022
                Page count
                Figures: 4, Tables: 1, Equations: 0, References: 42, Pages: 11
                Funding
                Funded by: Austrian Science Fund, doi 10.13039/501100002428;
                Award ID: P 30239
                Categories
                Research Article

                acanthamoeba castellanii,oxidative stress,thioredoxin system,glutathione system,auranofin

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