Increasing NADPH impairs fungal H₂O₂ resistance by perturbing transcriptional regulation of peroxiredoxin

Abstract

NADPH provides the reducing power for decomposition of reactive oxygen species (ROS), making it an indispensable part during ROS defense. It remains uncertain, however, if living cells respond to the ROS challenge with an elevated intracellular NADPH level or a more complex NADPH-mediated manner. Herein, we employed a model fungus Aspergillus nidulans to probe this issue. A conditional expression of glucose-6-phosphate dehydrogenase (G6PD)-strain was constructed to manipulate intracellular NADPH levels. As expected, turning down the cellular NADPH concentration drastically lowered the ROS response of the strain; it was interesting to note that increasing NADPH levels also impaired fungal H₂O₂ resistance. Further analysis showed that excess NADPH promoted the assembly of the CCAAT-binding factor AnCF, which in turn suppressed NapA, a transcriptional activator of PrxA (the key NADPH-dependent ROS scavenger), leading to low antioxidant ability. In natural cell response to oxidative stress, we noticed that the intracellular NADPH level fluctuated “down then up” in the presence of H₂O₂. This might be the result of a co-action of the PrxA-dependent NADPH consumption and NADPH-dependent feedback of G6PD. The fluctuation of NADPH is well correlated to the formation of AnCF assembly and expression of NapA, thus modulating the ROS defense. Our research elucidated how A. nidulans precisely controls NADPH levels for ROS defense.

Keywords

Peroxiredoxin / NADPH / Oxidative stress / Aspergillus / Glucose-6-phosphate dehydrogenase / AnCF

References

[1] Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev, 2007, 87: 245-313.

[2] Boronat S, Domenech A, Paulo E, Calvo IA, Garcia-Santamarina S, Garcia P, . Thiol-based H₂O₂ signalling in microbial systems. Redox Biol, 2014, 2: 395-399.

[3] Brakhage AA, Andrianopoulos A, Kato M, Steidl S, Davis MA, Tsukagoshi N, . HAP-Like CCAAT-binding complexes in filamentous fungi: implications for biotechnology. Fungal Genet Biol, 1999, 27: 243-252.

[4] Brandes RP, Weissmann N, Schroder K. Nox family NADPH oxidases: molecular mechanisms of activation. Free Radic Biol Med, 2014, 76: 208-226.

[5] Brown JD, Day AM, Taylor SR, Tomalin LE, Morgan BA, Veal EA. A peroxiredoxin promotes H₂O₂ signaling and oxidative stress resistance by oxidizing a thioredoxin family protein. Cell Rep, 2013, 5: 1425-1435.

[6] Day AM, Brown JD, Taylor SR, Rand JD, Morgan BA, Veal EA. Inactivation of a peroxiredoxin by hydrogen peroxide is critical for thioredoxin-mediated repair of oxidized proteins and cell survival. Mol Cell, 2012, 45: 398-408.

[7] Gibbs PA, Seviour RJ, Schmid F. Growth of filamentous fungi in submerged culture: problems and possible solutions. Crit Rev Biotechnol, 2000, 20: 17-48.

[8] Gu Y, Jiao X, Ye L, Yu H. Metabolic engineering strategies for de novo biosynthesis of sterols and steroids in yeast. Bioresour Bioprocess, 2021

[9] Gupte RS, Vijay V, Marks B, Levine RJ, Sabbah HN, Wolin MS, . Upregulation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase activity increases oxidative stress in failing human heart. J Card Fail, 2007, 13: 497-506.

[10] Handy DE, Loscalzo J. Redox regulation of mitochondrial function. Antioxid Redox Signal, 2012, 16: 1323-1367.

[11] Hillmann F, Bagramyan K, Strassburger M, Heinekamp T, Hong TB, Bzymek KP, . The crystal structure of peroxiredoxin Asp f3 provides mechanistic insight into oxidative stress resistance and virulence of Aspergillus fumigatus. Sci Rep, 2016, 6: 33396.

[12] Hortschansky P, Haas H, Huber EM, Groll M, Brakhage AA. The CCAAT-binding complex (CBC) in Aspergillus species. Biochim Biophys Acta Gene Regul Mech, 2017, 1860: 560-570.

[13] Imrichova D, Sarinova M, Cernicka J, Gbelska Y, Subik J. YAP1-mediated KNQ1 expression in Kluyveromyces lactis. FEMS Yeast Res, 2005, 5: 323-329.

[14] Kadooka C, Onitsuka S, Uzawa M, Tashiro S, Kajiwara Y, Takashita H, . Marker recycling system using the sC gene in the white koji mold, Aspergillus luchuensis mut. kawachii. J Gen Appl Microbiol, 2016, 62: 160-163.

[15] Kalyanaraman B, Cheng G, Hardy M, Ouari O, Bennett B, Zielonka J. Teaching the basics of reactive oxygen species and their relevance to cancer biology: mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies. Redox Biol, 2018, 15: 347-362.

[16] Kawasaki L, Aguirre J. Multiple catalase genes are differentially regulated in Aspergillus nidulans. J Bacteriol, 2001, 183: 1434-1440.

[17] Kawasaki L, Wysong D, Diamond R, Aguirre J. Two divergent catalase genes are differentially regulated during Aspergillus nidulans development and oxidative stress. J Bacteriol, 1997, 179: 3284-3292.

[18] Kitamoto K. Molecular biology of the Koji molds. Adv Appl Microbiol, 2002, 51: 129-153.

[19] Kuehne A, Emmert H, Soehle J, Winnefeld M, Fischer F, Wenck H, . Acute activation of oxidative pentose phosphate pathway as first-line response to oxidative stress in human skin cells. Mol Cell, 2015, 59: 359-371.

[20] Lee JW, Choi AH, Ham M, Kim JW, Choe SS, Park J, . G6PD up-regulation promotes pancreatic beta-cell dysfunction. Endocrinology, 2011, 152: 793-803.

[21] Legan SK, Rebrin I, Mockett RJ, Radyuk SN, Klichko VI, Sohal RS, . Overexpression of glucose-6-phosphate dehydrogenase extends the life span of Drosophila melanogaster. J Biol Chem, 2008, 283: 32492-32499.

[22] Leopold JA, Zhang YY, Scribner AW, Stanton RC, Loscalzo J. Glucose-6-phosphate dehydrogenase overexpression decreases endothelial cell oxidant stress and increases bioavailable nitric oxide. Arterioscler Thromb Vasc Biol, 2003, 23: 411-417.

[23] Leopold JA, Dam A, Maron BA, Scribner AW, Liao R, Handy DE, . Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity. Nat Med, 2007, 13: 189-197.

[24] Li Q, Bai Z, O'Donnell A, Harvey LM, Hoskisson PA, McNeil B. Oxidative stress in fungal fermentation processes: the roles of alternative respiration. Biotechnol Lett, 2011, 33: 457-467.

[25] Loussert C, Schmitt C, Prevost MC, Balloy V, Fadel E, Philippe B, . In vivo biofilm composition of Aspergillus fumigatus. Cell Microbiol, 2010, 12: 405-410.

[26] Lu J, Holmgren A. The thioredoxin antioxidant system. Free Radic Biol Med, 2014, 66: 75-87.

[27] Marchegiani E, Sidhu Y, Haynes K, Lebrun MH. Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters. Fungal Genet Biol, 2015, 79: 174-179.

[28] McNabb DS, Xing Y, Guarente L. Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding. Genes Dev, 1995, 9: 47-58.

[29] McNabb DS, Tseng KA, Guarente L. The Saccharomyces cerevisiae Hap5p homolog from fission yeast reveals two conserved domains that are essential for assembly of heterotetrameric CCAAT-binding factor. Mol Cell Biol, 1997, 17: 7008-7018.

[30] Mendoza-Martinez AE, Lara-Rojas F, Sanchez O, Aguirre J. NapA mediates a redox regulation of the antioxidant response, carbon utilization and development in Aspergillus nidulans. Front Microbiol, 2017, 8: 516.

[31] Miller CG, Holmgren A, Arner ESJ, Schmidt EE. NADPH-dependent and -independent disulfide reductase systems. Free Radic Biol Med, 2018, 127: 248-261.

[32] Mulder W, Scholten IH, de Boer RW, Grivell LA. Sequence of the HAP3 transcription factor of Kluyveromyces lactis predicts the presence of a novel 4-cysteine zinc-finger motif. Mol Gen Genet, 1994, 245: 96-106.

[33] Nathan C, Cunningham-Bussel A. Beyond oxidative stress: an immunologist's guide to reactive oxygen species. Nat Rev Immunol, 2013, 13: 349-361.

[34] Oldfield AJ, Yang P, Conway AE, Cinghu S, Freudenberg JM, Yellaboina S, . Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors. Mol Cell, 2014, 55: 708-722.

[35] Pais P, Costa C, Cavalheiro M, Romao D, Teixeira MC. Transcriptional control of drug resistance, virulence and immune system evasion in pathogenic fungi: a cross-species comparison. Front Cell Infect Microbiol, 2016, 6: 131.

[36] Papagiannopoulos P, Andrianopoulos A, Sharp JA, Davis MA, Hynes MJ. The hapC gene of Aspergillus nidulans is involved in the expression of CCAAT-containing promoters. Mol Gen Genet, 1996, 251: 412-421.

[37] Pohl C, Kiel JA, Driessen AJ, Bovenberg RA, Nygard Y. CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synth Biol, 2016, 5: 754-764.

[38] Poole LB, Hall A, Nelson KJ. Overview of peroxiredoxins in oxidant defense and redox regulation. Curr Protoc Toxicol, 2011, 49: 7.9.1-7.9.15.

[39] Ramos-Martinez JI. The regulation of the pentose phosphate pathway: remember Krebs. Arch Biochem Biophys, 2017, 614: 50-52.

[40] Rhee SG. Overview on peroxiredoxin. Mol Cells, 2016

[41] Rocha MC, de Godoy KF, Bannitz-Fernandes R, Fabri JHTM, Barbosa MMF, de Castro PA, . Analyses of the three 1-Cys peroxiredoxins from Aspergillus fumigatus reveal that cytosolic Prx1 is central to H₂O₂ metabolism and virulence. Sci Rep, 2018

[42] Rodrigues-Pousada C, Devaux F, Caetano SM, Pimentel C, da Silva S, Cordeiro AC, . Yeast AP-1 like transcription factors (Yap) and stress response: a current overview. Microb Cell, 2019, 6: 267-285.

[43] Rodriguez-Segade S, Ramos Martinez JI, Freire M. Reversal effect of oxidized glutathione on the inhibition of glucose-6-phosphate dehydrogenase by NADPH. Biochem J, 1985, 231: 805-806.

[44] Salvemini F, Franze A, Iervolino A, Filosa S, Salzano S, Ursini MV. Enhanced glutathione levels and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression. J Biol Chem, 1999, 274: 2750-2757.

[45] Sarsour EH, Kumar MG, Chaudhuri L, Kalen AL, Goswami PC. Redox control of the cell cycle in health and disease. Antioxid Redox Signal, 2009, 11: 2985-3011.

[46] Sato I, Shimatani K, Fujita K, Abe T, Shimizu M, Fujii T, . Glutathione reductase/glutathione is responsible for cytotoxic elemental sulfur tolerance via polysulfide shuttle in fungi. J Biol Chem, 2011, 286: 20283-20291.

[47] Stincone A, Prigione A, Cramer T, Wamelink MM, Campbell K, Cheung E, . The return of metabolism: biochemistry and physiology of the pentose phosphate pathway. Biol Rev Camb Philos Soc, 2015, 90: 927-963.

[48] Thomas D, Cherest H, Surdin-Kerjan Y. Identification of the structural gene for glucose-6-phosphate dehydrogenase in yeast. Inactivation leads to a nutritional requirement for organic sulfur. EMBO J, 1991, 10: 547-553.

[49] Thon M, Al Abdallah Q, Hortschansky P, Scharf DH, Eisendle M, Haas H, . The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes. Nucleic Acids Res, 2010, 38: 1098-1113.

[50] Tome ME, Johnson DB, Samulitis BK, Dorr RT, Briehl MM. Glucose 6-phosphate dehydrogenase overexpression models glucose deprivation and sensitizes lymphoma cells to apoptosis. Antioxid Redox Signal, 2006, 8: 1315-1327.

[51] Umemura M, Kuriiwa K, Dao LV, Okuda T, Terai G. Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production. Fungal Biol Biotechnol, 2020, 7: 3.

[52] Van Acker H, Coenye T. The role of reactive oxygen species in antibiotic-mediated killing of bacteria. Trends Microbiol, 2017, 25: 456-466.

[53] Wang YP, Zhou LS, Zhao YZ, Wang SW, Chen LL, Liu LX, . Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress. EMBO J, 2014, 33: 1304-1320.

[54] Wennekes LM, Goosen T, van den Broek PJ, van den Broek HW. Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus niger and Aspergillus nidulans. J Gen Microbiol, 1993, 139: 2793-2800.

[55] Xia Y, Yu H, Zhou Z, Takaya N, Zhou S, Wang P. Peroxiredoxin system of Aspergillus nidulans resists inactivation by high concentration of hydrogen peroxide-mediated oxidative stress. J Microbiol Biotechnol, 2018, 28: 145-156.

[56] Xiao W, Loscalzo J. Metabolic responses to reductive stress. Antioxid Redox Signal, 2020, 32: 1330-1347.

[57] Xiao W, Wang RS, Handy DE, Loscalzo J. NAD(H) and NADP(H) redox couples and cellular energy metabolism. Antioxid Redox Signal, 2018, 28: 251-272.

[58] Xue J, Balamurugan S, Li DW, Liu YH, Zeng H, Wang L, . Glucose-6-phosphate dehydrogenase as a target for highly efficient fatty acid biosynthesis in microalgae by enhancing NADPH supply. Metab Eng, 2017, 41: 212-221.

[59] Yang Y, Bazhin AV, Werner J, Karakhanova S. Reactive oxygen species in the immune system. Int Rev Immunol, 2013, 32: 249-270.

[60] Yu Q, Lee CF, Wang W, Karamanlidis G, Kuroda J, Matsushima S, . Elimination of NADPH oxidase activity promotes reductive stress and sensitizes the heart to ischemic injury. J Am Heart Assoc, 2014, 3.

[61] Yu T, Zhou YJ, Huang M, Liu Q, Pereira R, David F, . Reprogramming yeast metabolism from alcoholic fermentation to lipogenesis. Cell, 2018, 174(1549–1558

[62] Zhang Z, Yang Z, Zhu B, Hu J, Liew CW, Zhang Y, . Increasing glucose 6-phosphate dehydrogenase activity restores redox balance in vascular endothelial cells exposed to high glucose. PLoS ONE, 2012, 7.

[63] Zhang L, King E, Luo R, Li H. Development of a high-throughput, in vivo selection platform for NADPH-dependent reactions based on redox balance principles. ACS Synth Biol, 2018, 7: 1715-1721.

[64] Zheng H, Kim J, Liew M, Yan John K, Herrera O, Bok Jin W, . Redox metabolites signal polymicrobial biofilm development via the NapA oxidative stress cascade in Aspergillus. Curr Biol, 2015, 25: 29-37.

[65] Zhou S, Fushinobu S, Kim SW, Nakanishi Y, Maruyama J, Kitamoto K, . Functional analysis and subcellular location of two flavohemoglobins from Aspergillus oryzae. Fungal Genet Biol, 2011, 48: 200-207.

[66] Zhou L, Wang F, Sun R, Chen X, Zhang M, Xu Q, . SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense. EMBO Rep, 2016, 17: 811-822.

Funding

International Science and Technology Cooperation Programme(2017YFE0129600)

National Natural Science Foundation of China(21672065)

National Major Science and Technology Projects of China(2019ZX09739001)

Fundamental Research Funds for the Central Universities(22221818014)

Overseas Expertise Introduction Project for Discipline Innovation(B18022)