Candida glabrata Med3 Plays a Role in Altering Cell Size and Budding Index To Coordinate Cell Growth

Abstract

Candida glabrata is a promising microorganism for the production of organic acids. Here, we report deletion and quantitative-expression approaches to elucidate the role of C. glabrata Med3AB (CgMed3AB), a subunit of the mediator transcriptional coactivator, in regulating cell growth. Deletion of CgMed3AB caused an 8.6% decrease in final biomass based on growth curve plots and 10.5% lower cell viability. Based on transcriptomics data, the reason for this growth defect was attributable to changes in expression of genes involved in pyruvate and acetyl-coenzyme A (CoA)-related metabolism in a Cgmed3abΔ strain. Furthermore, the mRNA level of acetyl-CoA synthetase was downregulated after deleting Cgmed3ab, resulting in 22.8% and 21% lower activity of acetyl-CoA synthetase and cellular acetyl-CoA, respectively. Additionally, the mRNA level of CgCln3, whose expression depends on acetyl-CoA, was 34% lower in this strain. As a consequence, the cell size and budding index in the Cgmed3abΔ strain were both reduced. Conversely, overexpression of Cgmed3ab led to 16.8% more acetyl-CoA and 120% higher CgCln3 mRNA levels, as well as 19.1% larger cell size and a 13.3% higher budding index than in wild-type cells. Taken together, these results suggest that CgMed3AB regulates cell growth in C. glabrata by coordinating homeostasis between cellular acetyl-CoA and CgCln3.IMPORTANCE This study demonstrates that CgMed3AB can regulate cell growth in C. glabrata by coordinating the homeostasis of cellular acetyl-CoA metabolism and the cell cycle cyclin CgCln3. Specifically, we report that CgMed3AB regulates the cellular acetyl-CoA level, which induces the transcription of Cgcln3, finally resulting in alterations to the cell size and budding index. In conclusion, we report that CgMed3AB functions as a wheel responsible for driving cellular acetyl-CoA metabolism, indirectly inducing the transcription of Cgcln3 and coordinating cell growth. We propose that Mediator subunits may represent a vital regulatory target modulating cell growth in C. glabrata.

Keywords: Candida glabrata; Cgcln3; Mediator; acetyl-CoA; cell growth regulation.

FIG 1

Schematic diagram of the study. CgMed3AB functions as a wheel that drives cellular acetyl-CoA metabolism and then regulates transcription of Cgcln3.

FIG 2

CgMed3AB changes C. glabrata growth performance. (A) Wild-type, Cgmed3abΔ, and Cgmed3abΔ/(Cgmed3ab)_OE strains were spotted on YNB plates. (B) Growth curves for the wild-type, Cgmed3abΔ, and Cgmed3abΔ/(Cgmed3ab)_OE strains. (C) Cell viability in the wild-type, Cgmed3abΔ, and Cgmed3abΔ/(Cgmed3ab)_OE strains. The data refer to biological repeats; the error bars represent standard deviations (SD). *, P < 0.05; **, P < 0.01.

FIG 3

Transcriptome analysis of the wild type and the Cgmed3abΔ strain at pH 6.0. (A) Comparison of differentially expressed genes between the wild type and the Cgmed3abΔ strain as annotated using the KEGG database. (B) Heat map of the carbohydrate metabolism module at pH 6.0 following comparison between the wild type and the Cgmed3abΔ strain.

FIG 4

Deletion of Cgmed3ab affects gene expression of cellular acetyl-CoA and pyruvate-related metabolism. Pyruvate dehydrogenase (PDH), ACS, and acetyl-CoA acyltransferase (pot1) are the main pathways in cellular acetyl-CoA biosynthesis, whereas acetyl-CoA carboxylase (ACC), citrate synthase (CS), and malate synthase (MS) are the main pathways in cellular acetyl-CoA biodegradation. Pyruvate kinase (PYK) and pyruvate decarboxylase (PDC) are the main pathways in pyruvate-related metabolism. TCA, tricarboxylic acid cycle. Each experiment was followed by three biological repeats; the error bars represent SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

CgMed3AB affects acetyl-CoA synthetase activity by activating the transcription of Cgacs2. (A) Acetyl-CoA synthetase activity in wild-type, Cgmed3abΔ, and Cgmed3abΔ/(med3ab)_OE strains. (B) Cgino4 mRNA levels in the wild type and the Cgmed3abΔ strain. (C) Cgacs2 mRNA levels in Cgmed3abΔ, Cgino4Δ, and Cgmed3abΔ Cgino4Δ strains. (D) Yeast two-hybrid assays confirmed the interaction between CgMed3AB and CgIno4. P53-simian virus 40 (SV40) large T antigen (Clontech) protein interaction was used as a positive control. (E) Coimmunoprecipitation assays were used to detect the interaction between CgMed3AB and CgIno4 in vivo. (F) EMSAs of CgIno4 protein with upstream promoter regions of Cgacs2. The DNA probe (10 nM) was incubated with a protein concentration gradient (0, 0.5, 1, and 1.5 μM). EMSAs with a 200-fold excess of unlabeled specific probe and nonspecific competitor DNA (salmon sperm DNA) were conducted as controls. (G) Association of CgIno4 with the core promoter of Cgacs2 as determined by ChIP analysis and RT-PCR to measure occupancy. Signals were normalized to the input DNA, ChrV was used as a negative control, and the promoter was the core region of the Cgacs2 promoter. All experiments were repeated three times; the error bars represent SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

CgMed3AB affects transcription by regulating the cellular acetyl-CoA level. (A) Cellular acetyl-CoA levels in wild-type, Cgmed3abΔ, and Cgmed3abΔ/(Cgmed3ab)_OE strains. (B) Cells were cultivated in YNB medium for 48 h, washed three times with PBS, and added to prewarmed medium containing only 10 mM sodium acetate. The graph shows Cgcln3 mRNA levels measured every 5 min by RT-PCR and normalized to act1. (C) Cgcln3 mRNA levels in the Cgacs1Δ Cgacs2-ts strain at 25°C and 37°C following repletion with 1 mM acetate (cells were collected and added to 1 mM acetate and then grown at 30°C). (D) Cgcln3 mRNA levels in the Cgicl1Δ strain. (E) Median cell sizes (femtoliters [fl]) and percentages of budding were measured in the wild type and the CAGL0M11990gΔ strain. (F) Cgcln3 mRNA levels following in vitro regulation of the cellular acetyl-CoA level by adding 5 mM, 10 mM, and 15 mM acetate. (G) Cgmed3ab mRNA levels following in vivo regulation of the cellular acetyl-CoA level. The histograms represent cellular acetyl-CoA, and the circles represent the mRNA levels of Cgcln3. All experiments were repeated three times; the error bars represent SD. *, P < 0.05; **, P < 0.01.

FIG 7

CgCln3 changes cell size and budding. (A) Cgcln3 mRNA levels and cell sizes in the wild type and the Cgmed3abΔ strain. (B) Cell sizes were measured in the wild-type, Cgmed3abΔ, and Cgmed3abΔ/(Cgmed3ab)_OE strains. (C) Cells were treated with centrifugal elutriation and hydroxyurea, and the cell cycle was synchronized in G₁ phase. After release into fresh YNB medium, the budding index was determined by microscopic observation; the cell number was at least 200 cells. (D) Specific growth rates in the wild-type, Cgmed3abΔ, and Cgmed3abΔ/(Cgmed3ab)_OE strains. Each experiment was repeated three times; the error bars represent SD. *, P < 0.05; ***, P < 0.001.