Candida albicans PPG1, a serine/threonine phosphatase, plays a vital role in central carbon metabolisms under filament-inducing conditions: A multi-omics approach

Candida albicansis the leading cause of life-threatening bloodstream candidiasis, especially among immunocompromised patients. The reversible morphological transition from yeast to hyphal filaments in response to host environmental cues facilitatesC.albicanstissue invasion, immune evasion, and dissemination. Hence, it is widely considered that filamentation represents one of the major virulence properties inC.albicans. We have previously characterized Ppg1, a PP2A-type protein phosphatase that controls filament extension and virulence inC.albicans. This study conducted RNA sequencing analysis of samples obtained fromC.albicanswild type andppg1?/? strains grown under filament-inducing conditions. Overall,ppg1?/? strain showed 1448 upregulated and 710 downregulated genes, representing approximately one-third of the entire annotatedC.albicansgenome. Transcriptomic analysis identified significant downregulation of well-characterized genes linked to filamentation and virulence, such asALS3,HWP1,ECE1,and RBT1.Expression analysis showed that essentialgenes involved inC.albicanscentral carbon metabolisms, includingGDH3,GPD1,GPD2,RHR2,INO1,AAH1, andMET14were among the top upregulated genes. Subsequent metabolomics analysis ofC.albicans ppg1?/? strain revealed a negative enrichment of metabolites with carboxylic acid substituents and a positive enrichment of metabolites with pyranose substituents. Altogether, Ppg1in vitroanalysis revealed a link between metabolites substituents and filament formation controlled by a phosphatase to regulate morphogenesis and virulence. Overall design: Yeast extract-peptone-dextrose (YEPD) medium at 30°C was used as the standard non-filament-inducing growth condition for all strains. Liquid serum and temperature induction experiments were performed by growing strains overnight in YEPD medium at 30°C to an optical density at 600 nm (OD600) of ~4.0 and diluting 1:10 into 50 ml of pre-warmed YEPD medium plus 10% serum at 37°C. Aliquots of cells harvested at specific post-induction time points 3 and 5 hours for RNA isolation. We selected 3 and 5 hours-time points as these times points show the most prominent phenotypic (filamentation) difference betweenC.albicanswild-type andppg1?/? strains. RNA extraction was performed using RNeasy Micro Kit (Qiagen GmbH, Germany), following the manufacturer's instructions. Three biological replicates were obtained for each condition (W.T. and mutant). RNA sequencing was performed at BGI Group, Shenzhen, China. RNA was extracted from 8 samples belonging to two different strains, DK318 and MAY34. RNA obtained from 8 samples (Table 1) was sequenced using the BGISEQ-500 platform (Shenzhen, China), generating on average about 23.93 million reads per sample. The average mapping ratio with reference genomes was 96.92%, and the average gene mapping ratio was 82.77%; a total of 6,113 genes were detected. Sequence reads containing low-quality, adaptor-polluted and/or unknown high base (N) content were excluded from any further analysis. Retained sequence reads were further filtered using internal software SOAPnuke to produce a set of "clean reads" that was stored in FASTQ format as per each sample. Composition filtering statistics of raw data and quality metrics of clean reads. Clean filtered sequence reads were mapped to the reference genome using HISAT (hierarchical indexing for spliced alignment of transcripts) to perform the genome mapping step. Reads were mapped to the C. albicans strain SC5314 reference genome (assembly 21) (http://www.candidagenome.org).