Downloaded from www.microbiologyresearch.org by IP: 107.23.175.76 On: Thu, 21 Jun 2018 09:23:23 The fungicide fludioxonil antagonizes fluconazole activity in the human fungal pathogen Candida albicans Anna Buschart, Anna Burakowska3 and Ursula Bilitewski Correspondence Ursula Bilitewski ursula.bilitewski@helmholtz-hzi.de Received 13 August 2012 Accepted 21 August 2012 Biological Systems Analysis, Helmholtz Centre for Infection Research, Braunschweig, Germany The fungicide fludioxonil is widely used in agriculture. Residua of this fungicide are occasionally detected in fruits and can therefore be ingested by humans. The human fungal pathogen Candida albicans expresses the target of fludioxonil, Nik1p, a type III histidine kinase involved in stress response. Inhibition of yeast and hyphae growth was hardly observable after treatment of C. albicans SC5314 with fludioxonil. As a side effect, however, we observed a concentration- dependent induction of the expression of the genes CDR1 and CDR2, encoding ATP-binding cassette (ABC) transporters. This was independent of the presence of the target of fludioxonil as induction was also observed in a Dnik1 deletion mutant. Deletion of the CDR1 gene aggravated the inhibition of germ tube formation by fludioxonil, indicating that, in the wild-type, the fungicide was discharged from the cell by Cdr1p. Cdr1p is also known as a resistance factor of C. albicans against the commonly used antimycotic fluconazole. Thus, the effect of concurrent exposure to fludioxonil and known cargoes of ABC transporters on their extrusion and the growth of C. albicans was examined. Pre-incubation with fludioxonil decreased the export rate of rhodamine 6G. The resistance to fluconazole was increased by fludioxonil, independently of Nik1p. Therefore, exposure of C. albicans to fludioxonil may lead to increased resistance to fluconazole treatment. INTRODUCTION The oligomorphic yeast C. albicans can cause localized superficial infections as well as life-threatening systemic candidiasis in immunocompromised individuals. C. albi- cans infections accompany severe diseases, are hospital- acquired, in the majority of cases, and are the fourth most frequent cause of nosocomial sepsis. Thus they have become a major complication particularly in intensive care units (Pfaller & Diekema, 2007). Fludioxonil is a phenylpyrrol fungicide that is widely used in agriculture, especially in the protection of grapes and berries from plant-pathogenic fungi, most prominently Botrytis cinerea. While fludioxonil is highly toxic to some aquatic organisms, the toxicity to mammals has been low or negligible in a wide range of toxicological studies. The fungicide interacts with the osmotic stress response of filamentous fungi and yeasts by inhibiting the activity of a type III histidine kinase (Pillonel & Meyer, 1997; Okada et al., 2005; Ochiai et al., 2002). A homologous histidine kinase, termed Nik1p, is expressed in the opportunistic human-pathogenic yeast Candida albicans (Alex et al., 1998; Yamada-Okabe et al., 1999) and is also targeted by fludioxonil (Buschart et al., 2012). Furthermore, Nik1p has been implicated in the yeast–hyphae transition (Alex et al., 1998; Yamada-Okabe et al., 1999), which is a major pathogenicity factor (Calderone & Fonzi, 2001). Therefore, we investigated the effect of fludioxonil on C. albicans. In a comprehensive gene expression analysis, we observed that fludioxonil induced the expression of the ATP-binding cassette (ABC) transporters Cdr1p and Cdr2p. The overexpression of such transporters can cause a higher resistance to antimycotic drugs, as the intracellular concen- tration of the antimycotic is decreased (Wirsching et al., 2000; Siikala et al., 2010). Active transport by ABC transporters, especially of the PDR-subfamily, is achieved by coupling ATP-hydrolysis to export (Prasad et al., 1995; Sipos & Kuchler, 2006). The genome of C. albicans contains nine genes for PDR-subfamily ABC transporters, according to Gaur et al. (2005) and the Candida Genome Database (Arnaud et al., 2012), and the relevance of the transporters Cdr1p and Cdr2p in resistance to fluconazole has been studied intensively (Prasad et al., 1995; Sanglard et al., 1997). Fluconazole is one of the azole antimycotics, which are used to treat systemic C. albicans infections, besides polyenes, echinocandins and flucytosin. Acquisition of 3Present address: Applied Biochemistry, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany Abbreviations: ABC, ATP-binding cassette; PDR, pleiotropic drug resistance; R6G, rhodamine 6G. Three supplementary tables are available with the online version of this paper. Journal of Medical Microbiology (2012), 61, 1696–1703 DOI 10.1099/jmm.0.050963-0 1696 050963 G 2012 Printed in Great Britain