Oxidative stress is a biological principle affecting all life on Earth and is also an important factor in the pathogen-host relationship. The pathogenic free-living amoeba Acanthamoeba castellanii has several pathways to cope with reactive oxygen species and the damage that they cause. In this study, we aimed to provide a comprehensive analysis of the amoeba's response to different sources of oxidative stress. Using whole-cell proteomic analysis, we obtained a complex picture of the changes in the proteome and identified potential key players in the defense against oxidative stress. Importantly, from the differential proteomics analysis, we identified a candidate efflux pump that may be involved in Acanthamoeba drug resistance.

Center for Pathophysiology Infectiology and Immunology Institute of Specific Prophylaxis and Tropical Medicine Medical University of Vienna Vienna 1090 Austria

Department of Parasitology Faculty of Science BIOCEV Charles University Vestec 25250 Czech Republic

Department of Zoology Faculty of Science BIOCEV Charles University Vestec 25250 Czech Republic

Université de Paris Cité CNRS Institut Jacques Monod Paris F 75013 France

Zobrazit více v PubMed

Kang D.; Hamasaki N. Mitochondrial Oxidative Stress and Mitochondrial DNA. Clin. Chem. Lab. Med. 2003, 41 (10), 1281–1288. 10.1515/CCLM.2003.195. PubMed DOI

Beckman K. B.; Ames B. N. The Free Radical Theory of Aging Matures. Physiol. Rev. 1998, 78 (2), 547–581. 10.1152/physrev.1998.78.2.547. PubMed DOI

Wang J.; Yi J. Cancer Cell Killing via ROS: To Increase or Decrease, That Is a Question. Cancer Biol. Ther. 2008, 7 (12), 1875–1884. 10.4161/cbt.7.12.7067. PubMed DOI

Chio I. I. C.; Tuveson D. A. ROS in Cancer: The Burning Question. Trends Mol. Med. 2017, 23 (5), 411–429. 10.1016/j.molmed.2017.03.004. PubMed DOI PMC

Yang H.; Villani R. M.; Wang H.; Simpson M. J.; Roberts M. S.; Tang M.; Liang X. The Role of Cellular Reactive Oxygen Species in Cancer Chemotherapy. J. Exp. Clin. Cancer Res. 2018, 37, 1.10.1186/s13046-018-0909-x. PubMed DOI PMC

Wigner P.; Czarny P.; Synowiec E.; Bijak M.; Białek K.; Talarowska M.; Galecki P.; Szemraj J.; Sliwinski T. Variation of Genes Involved in Oxidative and Nitrosative Stresses in Depression. Eur. Psychiatry 2018, 48, 38–48. 10.1016/j.eurpsy.2017.10.012. PubMed DOI

Christen Y. Oxidative Stress and Alzheimer Disease. Am. J. Clin. Nutr. 2000, 71 (2), 621S–629S. 10.1093/AJCN/71.2.621S. PubMed DOI

Halliwell B. Role of Free Radicals in the Neurodegenerative Diseases: Therapeutic Implications for Antioxidant Treatment. Drugs Aging 2001, 18 (9), 685–716. 10.2165/00002512-200118090-00004. PubMed DOI

Singh R. P.; Sharad S.; Kapur S.. Free Radicals and Oxidative Stress in Neurodegenerative Diseases: Relevance of Dietary Antioxidants. J. Indian Acad. Clin. Med. 2004, 5, (3), , 218, 225.

Landry W. D.; Cotter T. G. ROS Signalling, NADPH Oxidases and Cancer. Biochem. Soc. Trans. 2014, 42 (4), 934–938. 10.1042/BST20140060. PubMed DOI

Sies H. Hydrogen Peroxide as a Central Redox Signaling Molecule in Physiological Oxidative Stress: Oxidative Eustress. Redox Biol. 2017, 11, 613–619. 10.1016/j.redox.2016.12.035. PubMed DOI PMC

Massart C.; Hoste C.; Virion A.; Ruf J.; Dumont J. E.; Van Sande J. Cell Biology of H2O2 Generation in the Thyroid: Investigation of the Control of Dual Oxidases (DUOX) Activity in Intact Ex Vivo Thyroid Tissue and Cell Lines. Mol. Cell. Endocrinol. 2011, 343 (1–2), 32–44. 10.1016/j.mce.2011.05.047. PubMed DOI

Schieber M.; Chandel N. S. ROS Function in Redox Signaling and Oxidative Stress. Curr. Biol. 2014, 24 (10), R453–R462. 10.1016/J.CUB.2014.03.034. PubMed DOI PMC

Monaghan P.; Metcalfe N. B.; Torres R. Oxidative Stress as a Mediator of Life History Trade-Offs: Mechanisms, Measurements and Interpretation. Ecol Lett. 2009, 12 (1), 75–92. 10.1111/j.1461-0248.2008.01258.x. PubMed DOI

Hong Y.; Drlica K.; Zhao X.. Antimicrobial-Mediated Bacterial Suicide Antimicrobial Resistance In The 21st Century Springer; 2018619–64210.1007/978-3-319-78538-7_20 DOI

Kohanski M. A.; Dwyer D. J.; Hayete B.; Lawrence C. A.; Collins J. J. A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics. Cell 2007, 130, P797–810. 10.1016/j.cell.2007.06.049. PubMed DOI

Kruger P.; Saffarzadeh M.; Weber A. N. R.; Rieber N.; Radsak M.; von Bernuth H.; Benarafa C.; Roos D.; Skokowa J.; Hartl D. Neutrophils: Between Host Defence, Immune Modulation, and Tissue Injury. PloS Pathog. 2015, 11 (3), e100465110.1371/journal.ppat.1004651. PubMed DOI PMC

Mócsai A. Diverse Novel Functions of Neutrophils in Immunity, Inflammation, and Beyond. J. Exp. Med. 2013, 210 (7), 1283–1299. 10.1084/jem.20122220. PubMed DOI PMC

Dupré-Crochet S.; Erard M.; Nüβe O. ROS Production in Phagocytes: Why, When, and Where?. J. Leukoc. Biol. 2013, 94 (4), 657–670. 10.1189/jlb.1012544. PubMed DOI

Lass A.; Guerrero M.; Li X.; Karanis G.; Ma L.; Karanis P. Detection of Acanthamoeba spp. in Water Samples Collected from Natural Water Reservoirs, Sewages, and Pharmaceutical Factory Drains Using LAMP and PCR in China. Sci. Total Environ. 2017, 584–585, 489–494. 10.1016/j.scitotenv.2017.01.046. PubMed DOI

Ashton N.; Stamm W.. Amoebic Infection of the Eye. A Pathologic Report. Trans. Ophthalmol. Soc. U.K. 1975, 95, pp. 214–220. PubMed

Illingworth C. D.; Cook S. D. Acanthamoeba Keratitis. Surv. Ophthalmol. 1998, 42 (6), 493–508. 10.1016/S0039-6257(98)00004-6. PubMed DOI

Damhorst G. L.; Watts A.; Hernandez-Romieu A.; Mel N.; Palmore M.; Ali I. K. M.; Neill S. G.; Kalapila A.; Cope J. R. Acanthamoeba castellanii Encephalitis in a Patient with AIDS: A Case Report and Literature Review. Lancet Infect Dis. 2022, 22 (2), e59–e6510.1016/S1473-3099(20)30933-6. PubMed DOI PMC

Sarink M. J.; van der Meijs N. L.; Denzer K.; Koenderman L.; Tielens A. G. M.; van Hellemond J. J. Three Encephalitis-Causing Amoebae and Their Distinct Interactions with the Host. Trends Parasitol. 2022, 38 (3), 230–245. 10.1016/j.pt.2021.10.004. PubMed DOI

Czarna M.; Jarmuszkiewicz W. Activation of Alternative Oxidase and Uncoupling Protein Lowers Hydrogen Peroxide Formation in Amoeba Acanthamoeba castellanii Mitochondria. FEBS Lett. 2005, 579 (14), 3136–3140. 10.1016/j.febslet.2005.04.081. PubMed DOI

Hadas E.; Mazur T. S.. Studies on Biochemical Indicators of Virulence in the Amoeba of Acanthamoeba sp. Acta Protozool., 1991, 30, (3–4), , pp. 161–164.

Choi D. H.; Na B. K.; Seo M. S.; Song H. R.; Song C. Y. Purification and Characterization of Iron Superoxide Dismutase and Copper-Zinc Superoxide Dismutase from Acanthamoeba castellanii. J. Parasitol. 2000, 86 (5), 899–907. 10.1645/0022-3395(2000)086[0899:PACOIS]2.0.CO;2. PubMed DOI

Leitsch D.; Mbouaka A. L.; Köhsler M.; Müller N.; Walochnik J. An Unusual Thioredoxin System in the Facultative Parasite Acanthamoeba castellanii. Cell. Mol. Life Sci. 2021, 78 (7), 3673–3689. 10.1007/s00018-021-03786-x. PubMed DOI PMC

Köhsler M.; Leitsch D.; Mbouaka L. A.; Wekerle M.; Walochnik J. Transcriptional Changes of Proteins of the Thioredoxin and Glutathione Systems in Acanthamoeba spp. under Oxidative Stress -an RNA Approach. Parasite 2022, 29, 24.10.1051/parasite/2022025. PubMed DOI PMC

Grechnikova M.; Arbon D.; Ženíšková K.; Malych R.; Mach J.; Krejbichová L.; Šimáčková A.; Sutak R. Elucidation of Iron Homeostasis in Acanthamoeba castellanii. Int. J. Parasitol. 2022, 52 (8), 497–508. 10.1016/j.ijpara.2022.03.007. PubMed DOI

Perez-Riverol Y.; Bai J.; Bandla C.; García-Seisdedos D.; Hewapathirana S.; Kamatchinathan S.; Kundu D. J.; Prakash A.; Frericks-Zipper A.; Eisenacher M.; Walzer M.; Wang S.; Brazma A.; Vizcaíno J. A. The PRIDE Database Resources in 2022: A Hub for Mass Spectrometry-Based Proteomics Evidences. Nucleic Acids Res. 2022, 50 (D1), D543–D552. 10.1093/nar/gkab1038. PubMed DOI PMC

Vandesompele J.; De Preter K.; Pattyn F.; Poppe B.; Van Roy N.; De Paepe A.; Speleman F. Accurate Normalization of Real-Time Quantitative RT-PCR Data by Geometric Averaging of Multiple Internal Control Genes. Genome Biol. 2002, 3 (7), research0034.1.10.1186/gb-2002-3-7-research0034. PubMed DOI PMC

Köhsler M.; Leitsch D.; Müller N.; Walochnik J. Validation of Reference Genes for the Normalization of RT-QPCR Gene Expression in Acanthamoeba spp. Sci. Rep. 2020, 10 (1), 10362.10.1038/s41598-020-67035-0. PubMed DOI PMC

Schindelin J.; Arganda-Carreras I.; Frise E.; Kaynig V.; Longair M.; Pietzsch T.; Preibisch S.; Rueden C.; Saalfeld S.; Schmid B.; Tinevez J. Y.; White D. J.; Hartenstein V.; Eliceiri K.; Tomancak P.; Cardona A. Fiji: An Open-Source Platform for Biological-Image Analysis. Nat. Methods 2012, 9 (7), 676–682. 10.1038/nmeth.2019. PubMed DOI PMC

Yu R.; Mandlekar S.; Harvey K. J.; Ucker D. S.; Kong2 A.-N. T.. Chemopreventive Isothiocyanates Induce Apoptosis and Caspase-3-like Protease Activity. Cancer Res. 1998, 58, pp. 402–408. PubMed

Zhang Y.; Tang L.; Gonzalez V.. Selected Isothiocyanates Rapidly Induce Growth Inhibition of Cancer Cells. Mol. Cancer Ther. 2003, 2, (10), , pp. 1045–1052. PubMed

Wu S.-J.; Ng L. T.; Lin C.-C. Effects of Antioxidants and Caspase-3 Inhibitor on the Phenylethyl Isothiocyanate-Induced Apoptotic Signaling Pathways in Human PLC/PRF/5 Cells. Eur. J. Pharmacol. 2005, 518 (2–3), 96–106. 10.1016/j.ejphar.2005.06.021. PubMed DOI

Fato R.; Bergamini C.; Bortolus M.; Maniero A. L.; Leoni S.; Ohnishi T.; Lenaz G. Differential Effects of Mitochondrial Complex I Inhibitors on Production of Reactive Oxygen Species. Biochim. Biophys. Acta Bioenerg. 2009, 1787 (5), 384–392. 10.1016/j.bbabio.2008.11.003. PubMed DOI PMC

Li N.; Ragheb K.; Lawler G.; Sturgis J.; Rajwa B.; Melendez J. A.; Robinson J. P. Mitochondrial Complex I Inhibitor Rotenone Induces Apoptosis through Enhancing Mitochondrial Reactive Oxygen Species Production. J. Biol. Chem. 2003, 278 (10), 8516–8525. 10.1074/jbc.M210432200. PubMed DOI

Terwel D.; Nieland L. J. M.; Schutte B.; Reutelingsperger C. P. M.; Ramaekers F. C. S.; Steinbusch H. W. M. S-Nitroso-N-Acetylpenicillamine and Nitroprusside Induce Apoptosis in a Neuronal Cell Line by the Production of Different Reactive Molecules. Eur. J. Pharmacol. 2000, 400 (1), 19–33. 10.1016/S0014-2999(00)00379-4. PubMed DOI

Nazari Q. A.; Mizuno K.; Kume T.; Takada-Takatori Y.; Izumi Y.; Akaike A. In Vivo Brain Oxidative Stress Model Induced by Microinjection of Sodium Nitroprusside in Mice. J. Pharmacol. Sci. 2012, 120 (2), 105–111. 10.1254/jphs.12143FP. PubMed DOI

Heberle H.; Meirelles V. G.; da Silva F. R.; Telles G. P.; Minghim R. InteractiVenn: A Web-Based Tool for the Analysis of Sets through Venn Diagrams. BMC Bioinf. 2015, 16 (1), 169.10.1186/S12859-015-0611-3. PubMed DOI PMC

Liu X. ABC Family Transporters. Adv. Exp. Med. Biol. 2019, 1141, 13–100. 10.1007/978-981-13-7647-4_2. PubMed DOI

Willforss J.; Chawade A.; Levander F. NormalyzerDE: Online Tool for Improved Normalization of Omics Expression Data and High-Sensitivity Differential Expression Analysis. J. Proteome Res. 2019, 18 (2), 732–740. 10.1021/acs.jproteome.8b00523. PubMed DOI

Wang F.; Yuan Q.; Chen F.; Pang J.; Pan C.; Xu F.; Chen Y. Fundamental Mechanisms of the Cell Death Caused by Nitrosative Stress. Front. Cell Dev. Biol. 2021, 9, 9.10.3389/fcell.2021.742483. PubMed DOI PMC

Dowd P. F.; Johnson E. T. The Involvement of a PIG3 Homolog Quinone Oxidoreductase Gene in Maize Resistance to Insects and Fungi Demonstrated through Transgenic Expression in Maize Callus. Plant Gene 2023, 35, 100429.10.1016/j.plgene.2023.100429. DOI

Porté S.; Valencia E.; Yakovtseva E. A.; Borràs E.; Shafqat N.; Debreczeny J. E.; Pike A. C. W.; Opperman U.; Farrés J.; Fita I.; et al. Three-Dimensional Structure and Enzymatic Function of Proapoptotic Human P53-Inducible Quinone Oxidoreductase PIG3. J. Biol. Chem. 2009, 284 (25), 17194–17205. 10.1074/JBC.M109.001800. PubMed DOI PMC

Lee J. H.; Kang Y.; Khare V.; Jin Z. Y.; Kang M. Y.; Yoon Y.; Hyun J. W.; Chung M. H.; Cho S. I.; Jun J. Y.; Chang I. Y.; You H. J. The P53-Inducible Gene 3 (PIG3) Contributes to Early Cellular Response to DNA Damage. Oncogene 2010, 29 (10), 1431–1450. 10.1038/onc.2009.438. PubMed DOI

Motavallihaghi S.; Khodadadi I.; Goudarzi F.; Afshar S.; Shahbazi A. E.; Maghsood A. H. The Role of Acanthamoeba castellanii (T4 Genotype) Antioxidant Enzymes in Parasite Survival under H2O2-Induced Oxidative Stress. Parasitol. Int. 2022, 87, 102523.10.1016/J.PARINT.2021.102523. PubMed DOI

Woyda-Ploszczyca A.; Koziel A.; Antos-Krzeminska N.; Jarmuszkiewicz W. Impact of Oxidative Stress on Acanthamoeba castellanii Mitochondrial Bioenergetics Depends on Cell Growth Stage. J. Bioenerg. Biomembr. 2011, 43 (3), 217–225. 10.1007/s10863-011-9351-x. PubMed DOI

Telang U.; Ji Y.; Morris M. E. ABC Transporters and Isothiocyanates: Potential for Pharmacokinetic Diet-Drug Interactions. Biopharm. Drug Dispos. 2009, 30 (7), 335–344. 10.1002/bdd.668. PubMed DOI PMC

Pal C.; Bandyopadhyay U. Redox-Active Antiparasitic Drugs. Antioxid. Redox Signal. 2012, 17 (4), 555–582. 10.1089/ars.2011.4436. PubMed DOI

Jumper J.; Evans R.; Pritzel A.; Green T.; Figurnov M.; Ronneberger O.; Tunyasuvunakool K.; Bates R.; Žídek A.; Potapenko A.; Bridgland A.; Meyer C.; Kohl S. A. A.; Ballard A. J.; Cowie A.; Romera-Paredes B.; Nikolov S.; Jain R.; Adler J.; Back T.; Petersen S.; Reiman D.; Clancy E.; Zielinski M.; Steinegger M.; Pacholska M.; Berghammer T.; Bodenstein S.; Silver D.; Vinyals O.; Senior A. W.; Kavukcuoglu K.; Kohli P.; Hassabis D. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596 (7873), 583–589. 10.1038/s41586-021-03819-2. PubMed DOI PMC

van Kempen M.; Kim S. S.; Tumescheit C.; Mirdita M.; Lee J.; Gilchrist C. L. M.; Söding J.; Steinegger M. Fast and Accurate Protein Structure Search with Foldseek. Nat. Biotechnol. 2024, 42 (2), 243–246. 10.1038/s41587-023-01773-0. PubMed DOI PMC