A new regulator in the crossroads of oxidative stress resistance and virulence in Candida glabrata: The transcription factor CgTog1

Abstract

Candida glabrata is a prominent pathogenic yeast which exhibits a unique ability to survive the harsh environment of host immune cells. In this study, we describe the role of the transcription factor encoded by the gene CAGL0F09229g , here named CgTog1 after its Saccharomyces cerevisiae ortholog, as a new determinant of C. glabrata virulence. Interestingly, Tog1 is absent in the other clinically relevant Candida species ( C. albicans, C. parapsilosis, C. tropicalis, C. auris ), being exclusive to C. glabrata . CgTog1 was found to be required for oxidative stress resistance and for the modulation of reactive oxygen species inside C. glabrata cells. Also, CgTog1 was observed to be a nuclear protein, whose activity up-regulates the expression of 147 genes and represses 112 genes in C. glabrata cells exposed to H 2 O 2 , as revealed through RNA-seq-based transcriptomics analysis. Given the importance of oxidative stress response in the resistance to host immune cells, the effect of CgTOG1 expression in yeast survival upon phagocytosis by Galleria mellonella hemocytes was evaluated, leading to the identification of CgTog1 as a determinant of yeast survival upon phagocytosis. Interestingly, CgTog1 targets include many whose expression changes in C. glabrata cells after engulfment by macrophages, including those involved in reprogrammed carbon metabolism, glyoxylate cycle and fatty acid degradation. In summary, CgTog1 is a new and specific regulator of virulence in C. glabrata , contributing to oxidative stress resistance and survival upon phagocytosis by host immune cells.

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata

“…Candida glabrata CBS138, KUE100 [ 46 ], KUE100::URA- [ 47 ], ∆cgtpo4 [ 42 ] and L5U1 (cgura3∆0 cgleu2∆0) [ 16 ] strains, the latter kindly provided by John Bennett, NIAID, NIH, Bethesda, were used in this study. C. glabrata cells were cultivated in YPD medium, containing: 20 g/L D-(+)- glucose (Merck, Darmstadt, Germany), 20 g/L bacterial-peptone (LioChem, Conyers, GA, USA) and 10 g/L of yeast extract (Difco, BD, England, United Kingdom).…”

Characterization of the Candida glabrata Transcription Factor CgMar1: Role in Azole Susceptibility

“…The pGREG576 plasmid from the Drag&Drop collection was used as described before to clone and express the C. glabrata ORF CAGL0B03421g [26,[29][30][31]. pGREG576 was acquired from Euroscarf and contains a galactose inducible promoter (GAL1); the yeast selectable marker URA3; and the GFP gene, encoding a Green Fluorescent Protein (GFPS65T), which allows for monitoring of the expression and subcellular localization of the cloned fusion protein.…”

“…The deletion of the C. glabrata URA3 gene encoded by ORF CAGL0I03080g was carried out in the ∆cgmar1 mutant as described before [26] using the CRISPR-Cas9 system from Vyas et al [24]. Briefly, a CgURA3 gRNA sequence selected from the resources made available by Vyas et al [24] was cloned in the pV1382 plasmid, previously linearized with the restriction enzyme BsmBI (New England Biolabs, Ipswich, MA, USA).…”

“…Raw data for the wild type were obtained concurrently and has been previously submitted [29]. Bioinformatics analysis was performed as described previously [26,29]. Briefly, sample reads were trimmed using Skewer (v0.2.2) [34] and aligned to the C. glabrata CBS138 reference genome, obtained from the Candida Genome Database (CGD), using TopHat (v2.1.1) [35] with the parameters −p 12 (number of threads), −g 1 (maximum amount of times that a read can be mapped to the genome), −b2-very-sensitive (preset option), and −library-type fr-firststrand (to account for strand specificity).…”

“…Plasma membrane permeability was assessed by the passive uptake of propidium iodide (PI; 20 mM in DMSO, Invitrogen, Waltham, MA, USA) as described previously [26,29]. C. glabrata cell suspensions from strains KUE100, KUE100_∆cgmar1, and KUE100_∆cgrsb1 were prepared in BM until a standard culture OD600nm of 0.5 ± 0.05 was reached and transferred to the same medium with or without 150 mg/L of fluconazole.…”