Growth inhibition and ultrastructural alterations induced by Δ24(25)-sterol methyltransferase inhibitors in Candida spp. isolates, including non-albicans organisms

Kelly Ishida,Wanderley De Souza,Marcos Dornelas Ribeiro,Juliany Cola Fernandes Rodrigues,Celso Vataru Nakamura,Sonia Rozental,Julio A Urbina,Taíssa Vieira Machado Vila

doi:10.1186/1471-2180-9-74

Abstract

BackgroundAlthough Candida species are commensal microorganisms, they can cause many invasive fungal infections. In addition, antifungal resistance can contribute to failure of treatment.The purpose of this study was to evaluate the antifungal activity of inhibitors of Δ24(25)-sterol methyltransferase (24-SMTI), 20-piperidin-2-yl-5α-pregnan-3β-20(R)-diol (AZA), and 24(R,S),25-epiminolanosterol (EIL), against clinical isolates of Candida spp., analysing the ultrastructural changes.ResultsAZA and EIL were found to be potent growth inhibitors of Candida spp. isolates. The median MIC50 was 0.5 μg.ml-1 for AZA and 2 μg.ml-1 for EIL, and the MIC90 was 2 μg.ml-1 for both compounds. All strains used in this study were susceptible to amphotericin B; however, some isolates were fluconazole- and itraconazole-resistant. Most of the azole-resistant isolates were Candida non-albicans (CNA) species, but several of them, such as C. guilliermondii, C. zeylanoides, and C. lipolytica, were susceptible to 24-SMTI, indicating a lack of cross-resistance. Reference strain C. krusei (ATCC 6258, FLC-resistant) was consistently susceptible to AZA, although not to EIL. The fungicidal activity of 24-SMTI was particularly high against CNA isolates. Treatment with sub-inhibitory concentrations of AZA and EIL induced several ultrastructural alterations, including changes in the cell-wall shape and thickness, a pronounced disconnection between the cell wall and cytoplasm with an electron-lucent zone between them, mitochondrial swelling, and the presence of electron-dense vacuoles. Fluorescence microscopy analyses indicated an accumulation of lipid bodies and alterations in the cell cycle of the yeasts. The selectivity of 24-SMTI for fungal cells versus mammalian cells was assessed by the sulforhodamine B viability assay.ConclusionTaken together, these results suggest that inhibition of 24-SMT may be a novel approach to control Candida spp. infections, including those caused by azole-resistant strains.

Highlights

Candida species are commensal microorganisms, they can cause many invasive fungal infections
Resistance to ITC was detected in 6 isolates (3 C. tropicalis, 1 C. albicans, 1 C. glabrata, and 1 C. krusei)
Most of the isolates had MIC50 and MIC90 for ITC lower than 0.03 μg.ml-1 (62%, and During the last two decades, some studies have described a new class of antifungals called azasterols, which are inhibitors of the Δ24(25)-sterol methyltransferase (24SMT), another key enzyme of the ergosterol biosynthesis pathway, which is absent in the mammalian host cells [68]

Summary

Introduction

Candida species are commensal microorganisms, they can cause many invasive fungal infections. Antifungal resistance can contribute to failure of treatment. The purpose of this study was to evaluate the antifungal activity of inhibitors of Δ24(25)-sterol methyltransferase (24-SMTI), 20-piperidin-2-yl-5α-pregnan-3β-20(R)-diol (AZA), and 24(R,S),25-epiminolanosterol (EIL), against clinical isolates of Candida spp., analysing the ultrastructural changes. Candida species are commensal microorganisms of vertebrate hosts that can cause infections ranging from nonlife-threatening to invasive illnesses. Candidiasis is the fourth most common cause of nosocomial bloodstream infections in Brazil and the U.S.A., with a mortality rate of approximately 40% [1,2]. Nowadays Candida non-albicans (CNA) species such as Candida glabrata, Candida krusei, and Candida parapsilosis have increased in importance and number among fungal infections [1]

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