Reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) is dependent on CaFRE10 ferric reductase for Candida albicans grown in unbuffered media

• Published in: Microbiology (Society for General Microbiology) 2006-08, Vol.152 (8), p.2301-2308
• Main Authors: Knight, Simon A. B, Dancis, Andrew
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Candida albicans; Candida albicans - growth & development; Candida albicans - metabolism; Culture Media; FMN Reductase - physiology; Fundamental and applied biological sciences. Psychology; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Hydrogen-Ion Concentration; Iron - metabolism; Microbiology; Mycology; Oxidation-Reduction; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Superoxide Dismutase - metabolism; Tetrazolium Salts - metabolism
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.28843-0

University of Pennsylvania School of Medicine, Department of Medicine, Division of Hematology/Oncology, 730 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160, USA Correspondence Simon A. B. Knight sknight{at}mail.med.upenn.edu The reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) and other tetrazolium salts is widely used as an assay for bacterial, fungal and mammalian cell viability, but the genes encoding the reductase activities have not been defined. Here, it was shown that XTT and plasma membrane ferric reductase activities were 10–40-fold greater in Candida albicans than in Saccharomyces cerevisiae . XTT reductase activity was induced fivefold in C. albicans grown in low-iron conditions compared with iron-replete conditions, and for cells grown in unbuffered (pH 4.0–4.4) medium, XTT reductase activity was largely dependent on CaFRE10 . XTT reductase activity of C. albicans grown in medium buffered to pH 6.8 was independent of CaFRE10 but, nonetheless, was upregulated in cells deprived of iron. Reduction of 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), a membrane-permeable tetrazolium salt, occurred at an intracellular location and was independent of CaFRE10 . However, MTT activity was induced by iron deprivation in C. albicans but not in S. cerevisiae . C. albicans possessed multiple iron- and pH-regulated reductase activities capable of reducing tetrazolium salts, but, when grown in unbuffered medium, CaFRE10 was required for XTT reductase activity. Abbreviations: 1-mPMS, 1-methoxy-5-methylphenazinium, methylsulfate; BPS, bathophenanthroline disulfonate; FRE, ferric reductase; mtDNA, mitochondrial DNA; MTT, 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide; NOX, NAD(P)H oxidase; SOD, superoxide dismutase; WST-1, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt; XTT, sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt