Purpurin, an anthraquinone in madder root, interferes with pH homeostasis in Candida albicans via specific inhibition of plasma membrane ATPase Pma1p

Abstract

Objectives To investigate the in vitro effects of purpurin on Candida albicans membrane-bound ATPases Methods Plasma membrane ATPase (Pma1p) was isolated from a Saccharomyces cerevisiae strain that overexpresses C. albicans Pma1p by step-wise sucrose centrifugation (from 3,500 g to 105,000 g). Vacuolar membrane ATPase (Vma7p) was isolated from C. albicans spheroplasts in a discontinuous Ficoll gradient. ATPase activity was determined by quantifying the amount of liberated inorganic phosphate (Pi) using an ammonium molybdate method. Protein was quantified by BCA Protein Assay Kit. C. albicans PMA1 null mutants were created by a PCR-based gene targeting method using gene-specific primers. C. albicans PMA1 reintegrants were created by transforming the full length PMA1 gene into the null mutants. Cytosolic pH was determined in C. albicans that carries a pH-sensitive GFP (pHluorin). Fluorescence was measured at excitation: 485 nm and 410 nm, and at emission: 520 nm. Results C. albicans ATPases Pma1p and Vma7p were isolated. A reduction of ~8%, ~32%, ~57%, and ~61% of ATPase activity was detected in Pma1p in the presence of 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL, and 3.5 μg/mL purpurin respectively in a concentration-dependent manner; whereas no inhibitory effects of purpurin was detected in Vma7p (Fig. 1). C. albicans PMA1 null mutants were unable to regulate cytosolic pH and lost the ability to acidify growth medium; the ability to acidify growth medium was restored in the reintegrants. Conclusions Our findings revealed that purpurin specfically inhibits C. albicans plasma membrane ATPase Pma1p. The results of the present study dictate a new research initiatives that focus on improved antifungal regimens. One aspect could be a combinational use of purpurin with other antifungal agents with different modes of action to minimize the occurrence of resistance in clinical settings.