Revealing Molecular Mechanisms of Fluconazole (Flc) Resistance in Candida tropicalis (Ct) Through Whole Genome Sequencing (Wgs) and Rna-Seq

J. M. Rybak1, C. M. Dickens2, P. D. Rogers1

Author address: 

1Univ. of Tennessee, Memphis, TN, 2Univ. of Texas A & M, College Station, TX


Background: Candida spp. are the most common cause of invasive fungal infections in North America and remain associated with mortality rates as high as 40%. Rising rates of resistance to FLC, the most commonly utilized antifungal, remains of clear concern. While much is known about the molecular mechanisms contributing to FLC resistance in Candida albicans (Ca), relatively little is known with regards to Candida tropicalis (Ct), where rates of FLC resistance are higher. We utilized WGS and RNA-seq to identify novel mutations and altered gene expression in genes related to fluconazole susceptibility.Methods: 13 clinical isolates of Ct with reduced susceptibility to FLC (MIC≥1mg/L) and 5 susceptible control clinical isolates were obtained from the University of Iowa repository. FLC MICs for each isolate were determined using Clinical Laboratory and Standards Institute methodology. RNA and genomic DNA were isolated from cultures of each isolate in mid-log growth phase. WGS and RNA-seq was performed using the Ion Proton System and aligned to the Ct MYA-3404 reference genome. RNA-seq analysis was performed using edgeR, and gene expression was compared to a composite of the 5 control isolates.Results: MIC ranged from 0.5 to ≥256mg/L. 12 isolates were observed to be FLC resistant (MIC 8 to ≥256mg/L). WGS revealed numerous novel mutations in several ergosterol biosynthetic genes including ERG3 and ERG11, as well as transcription factor genes UPC2, TAC1, and MRR1. RNA-seq results were consistent with increased transcription factor activity in Ct isolates with potential gain- of- function mutations in UPC2, TAC1, and MRR1. Overexpression of genes associated with reduced FLC susceptibility in Ca, ERG11 (>3 fold), CDR1 (≥10 fold), and MDR1 (20 fold) was observed in FLC resistant Ct isolates. Overexpression (>10 fold) of the efflux pump genes SNQ2 and CDR11, not previously reported in Ca, was also observed in FLC resistant isolates.Conclusions: These data reveal potential molecular mechanisms of FLC resistance in Ct which parallel mechanisms previously identified in Ca. Additional mechanisms of resistance such as greatly increased expression of the efflux pump genes SNQ2 and CDR11 were also identified. Further research is needed to definitively identify mechanisms of FLC resistance among Ct.

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Full conference title: 

ASM Microbe 2016
    • ASM microbe 1st (2016)