Med15B Regulates Acid Stress Response and Tolerance in Candida glabrata by Altering Membrane Lipid Composition

Abstract

Candida glabrata is a promising producer of organic acids. To elucidate the physiological function of the Mediator tail subunit Med15B in the response to low-pH stress, we constructed a deletion strain, C. glabrata med15BΔ, and an overexpression strain, C. glabrata HTUΔ/CgMED15B. Deletion of MED15B caused biomass production, glucose consumption rate, and cell viability to decrease by 28.3%, 31.7%, and 26.5%, respectively, compared with those of the parent (HTUΔ) strain at pH 2.0. Expression of lipid metabolism-related genes was significantly downregulated in the med15BΔ strain, whereas key genes of ergosterol biosynthesis showed abnormal upregulation. This caused the proportion of C 18:1 fatty acids, the ratio of unsaturated to saturated fatty acids (UFA/SFA), and the total phospholipid content to decrease by 11.6%, 27.4%, and 37.6%, respectively. Cells failed to synthesize fecosterol and ergosterol, leading to the accumulation and a 60.3-fold increase in the concentration of zymosterol. Additionally, cells showed reductions of 69.2%, 11.6%, and 21.8% in membrane integrity, fluidity, and H ϩ -ATPase activity, respectively. In contrast, overexpression of Med15B increased the C 18:1 levels, total phospholipids, ergosterol content, and UFA/SFA by 18.6%, 143.5%, 94.5%, and 18.7%, respectively. Membrane integrity, fluidity, and H ϩ -ATPase activity also increased by 30.2%, 6.9%, and 51.8%, respectively. Furthermore, in the absence of pH buffering, dry weight of cells and pyruvate concentrations were 29.3% and 61.2% higher, respectively, than those of the parent strain. These results indicated that in C. glabrata, Med15B regulates tolerance toward low pH via transcriptional regulation of acid stress response genes and alteration in lipid composition. IMPORTANCE This study explored the role of the Mediator tail subunit Med15B in the metabolism of Candida glabrata under acidic conditions. Overexpression of MED15B enhanced yeast tolerance to low pH and improved biomass production, cell viability, and pyruvate yield. Membrane lipid composition data indicated that Med15B might play a critical role in membrane integrity, fluidity, and H ϩ -ATPase activity homeostasis at low pH. Thus, controlling membrane composition may serve to increase C. glabrata productivity at low pH.KEYWORDS Candida glabrata, Mediator subunit Med15B, low-pH stress, transcriptomics, membrane lipid T he Mediator coactivator complex is required for transcription initiation in eukaryotes (1, 2). It is recruited by transcription activators and conveys regulatory information from gene-specific regulators to promoters (3, 4). Mediator can influence almost all stages of transcription and coordinated processes such as chromatin remodeling, transcription elongation, and posttranslational modifications (5, 6). Mediator is a multisubunit assembly comprising four modules: head, middle, tail, and cyclin-dependent kinase 8 (CDK8) (7). The head and middle modules are highly

Cg Hog1-Mediated Cg Rds2 Phosphorylation Alters Glycerophospholipid Composition To Coordinate Osmotic Stress in Candida glabrata

“…Third, sterols, abundant apolar membrane lipids, can increase membrane thickness and impermeability by reducing the flexibility of the surrounding acyl chains (59). In our previous study, we also showed that increasing the ergosterol content by overexpression of Cg-MED15 may enhance membrane fluidity, thereby promoting the resistance to low pH (60). Importantly, to manipulate the ratio of these membrane lipids, compensatory mechanisms exist in yeast.…”

“…The marker genes were amplified from the C. glabrata strain ATCC 2001 genome and fused between the upstream and downstream regions of the CgRDS2 or CgHOG1 gene open reading frame by fusion PCR. PCR products were transformed in the Candida glabrata strain CgHTUΔ as described previously (60), and the deletion strains were confirmed by genomic PCR and DNA sequencing. Overexpression strains were constructed using the pY26 plasmid carrying the target genes.…”

Copper blocks V‐ATPase activity and SNARE complex formation to inhibit yeast vacuole fusion

“…This as other H + /V + -ATPases have been shown to be inhibited when membrane fluidity is reduced. 49,[51][52][53][54] Separately, we and others have found that SNARE function can be altered by membrane fluidity. 32,33,55 Together, these notions and the data presented add another facet to pleiotropic effects of this metal to include membrane fusion.…”

Engineering of membrane phospholipid component enhances salt stress tolerance in Saccharomyces cerevisiae

“…PssA overexpression increases PE zwitterionic phospholipid content (Tan et al, ). Other methods of regulating membrane lipids include transcription factors such as Rds2 and Crz1 (Wu et al, ; Yan et al, ) and mediators such as Med15B (Qi, Liu, Yu, Chen, & Liu, ), which are applied to manipulate gene transcription level. These approaches do not directly engineer cell membranes and are not strongly dependent on phospholipid pathways.…”

“…Other methods of regulating membrane lipids include transcription factors such as Rds2 and Crz1 (Wu et al, 2019;Yan et al, 2016) and mediators such as Med15B (Qi, Liu, Yu, Chen, & Liu, 2017), which are applied to manipulate gene transcription level.…”