CaIPF7817 is involved in the regulation of redox homeostasis in Candida albicans

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Abstract

CaIPF7817, a functionally unknown gene in Candida albicans, was suggested to be involved in the redox system previously, but its exact role is unknown. In this study, ipf7817 null mutant was generated with the URA-blaster method. After the deletion of CaIPF7817, intracellular levels of reactive oxygen species were significantly increased; mitochondrial membrane potential, a direct indicator of mitochondrial function, was elevated; some important redox-related genes, including GLR1, SOD2, and TRR1, were up-regulated; and the GSH/GSSG ratio was raised. These changes indicated that CaIPF7817 played important roles in the regulation of redox homeostasis in C. albicans.

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Materials and methods

Strains, culture, and growth of C. albicans. Candida albicans strains used in this study were listed in Table 1. The strains were cultured at 30 °C in YPD medium (1% Difco yeast extract, 2% Difco peptone, 2% dextrose) or YPD medium supplemented with 0.005% uridine (Sigma, USA). Agar (1.5%) was added to prepare the solid media. The URA3 transformants were selected on MIN (2% glucose, 0.67% Difco yeast nitrogen base without amino acids) agar plates supplemented with 25 μg/mL histidine. The ura3

Generation and characterization of ipf7817 mutants

To investigate the roles of CaIPF7817 in C. albicans, we disrupted the two alleles of CaIPF7817 sequentially in RM1000 strain using the URA-blaster method and 5-FOA selection, yielding the following strains: the Ura+ipf7817/IPF7817 strain (JH1U), Uraipf7817/IPF7817 strain (JH1), Ura+ipf7817/ipf7817 strain (JH2U), and Uraipf7817/ipf7817 strain (JH2) (Table 1). The strategy to disrupt both copies of CaIPF7817 was depicted in Fig. 1A. Southern blot analysis of DNA from the constructed strains

Discussion

In this work, we interrupted both copies of CaIPF7817 in C. albicans and studied the role of CaIPF7817 in cellular redox regulation. Intracellular ROS level, mitochondrial membrane potential, expressions of some important redox-related genes and GSH/GSSG ratio were measured to reveal changes in cellular redox condition after CaIPF7817 deletion. Without CaIPF7817, the intracellular ROS level was increased; mitochondrial membrane potential was elevated; the expressions of some important

Acknowledgments

This work was funded by a grant from the National Natural Science Foundation of China (30500628) and the National 973 Program of China (2005CB523105). We thank J. Pla for kindly providing us the C. albicans strain RM1000 and W.A. Fonzi for plasmid p5921.

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    These authors contributed equally to this work.

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