Acidic pH Reduces Fluconazole Susceptibility in Cryptococcus neoformans by Altering Iron Uptake and Enhancing Ergosterol Biosynthesis

The opportunistic fungal pathogen Cryptococcus neoformans faces a wide range of environmental pH within the host, and adaptation to different pH conditions plays a critical role in their survival and pathogenesis. In the current study, how environmental pH influences antifungal susceptibility and iron uptake in C. neoformans was investigated. We found that acidic pH conditions significantly reduced antifungal susceptibility of C. neoformans to fluconazole. Moreover, our study revealed that iron acquisition in C. neoformans at acidic pH is independent of the high-affinity iron uptake system, and leads to increase of intracellular iron accumulation, increased ergosterol and heme levels, which may contribute to reduced fluconazole susceptibility of the fungus. Transcriptome analysis further elucidated the molecular mechanisms underlying the pH-dependent shift of iron uptake and antifungal susceptibility of C. neoformans. Our findings highlight the importance of environmental pH in physiology and pathogenesis of C. neoformans and provide insights into developing novel treatment for cryptococcosis. RNA-seq was performed to analyze differentially expressed genes in the C. neoformans cells grown at pH 5.0 compared to that in the cells grown at pH 7.0. The cells were grown in YPD for overnight, harvested and reinoculated into YNB LIM pH 6.8 for overnight, and the cells were harvested and incubated to YNB LIM pH 6.8 or pH 5.0 at 1×107 cells/mL for 6 hr. The cells were harvested by centrifugation and the total RNA was extracted by the RNeasy Mini Kit following the manufacturer's instructions. Contaminating DNA was eliminated by RNase-free DNase (Invitrogen, Waltham, MA, USA). Libraries for RNA sequencing were prepared using high-quality RNA with the value of RNA integrity greater than 7.0, and TruSeq Stranded mRNA Sample Prep Kit (Illumina, US) following the manufacturer’s protocol. The libraries were submitted to an Illumina HiSeq2500 platform, and the 100-bp paired-end reads were generated and sequenced. Quality-filtered reads were mapped to the reference genome sequence (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) using CLC Genomics Workbench 4.0 (CLC bio). Mapped reads were counted by featureCounts in Subread package v1.4.3 and the relative transcript abundance was TPM-normalized.