Glycolysis-dependent sulfur metabolism orchestrates morphological plasticity and virulence in fungi
- CSIR-Centre for Cellular and Molecular Biology, India
- Academy of Scientific and Innovative Research (AcSIR), India
Figures
Figure 1 with 1 supplement
Glycolysis is critical for pseudohyphal differentiation in a cAMP-PKA-independent manner in S. cerevisiae.
(A) Schematic overview of glycolysis and its inhibition by glycolysis inhibitors (2-Deoxy-D-Glucose (2DG) and sodium citrate (NaCi)). (B) Wild-type ∑1278b or CEN.PK were spotted on nitrogen-limiting medium containing 2% (w/v) glucose (SLAD) with and without sub-inhibitory concentrations of glycolysis inhibitors (2DG or NaCi), incubated for 10 days at 30 °C. Scale bar represents 1 mm for whole colony images and 5 µm for single cell images. (C) Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of pseudohyphal cells from the total population was determined. More than 500 cells were counted for each condition. Statistical analysis was done using one-way ANOVA test, ***(p<0.001). Error bars represent SEM. (D) Schematic overview of glycolysis showing targeted gene deletions. (E) Pertinent knockout strains which lack genes encoding for enzymes involved in glycolysis, such as pfk1 and adh1 were spotted on SLAD along with wild-type ∑1278b or CEN.PK. Scale bar represents 1 mm for whole colony images and 5 µm for single cell images. (F) Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of pseudohyphal cells from the total population was determined. More than 500 cells were counted for each strain. Statistical analysis was done using one-way ANOVA test, **(p<0.01) and *(p<0.05). Error bars represent SEM. (G) Growth curve was performed to monitor overall growth of wild-type strains on SLAD, SLAD+2DG and SLAD+NaCi. Overnight grown culture of wild-type strains (∑1278b or CEN.PK) were diluted to OD600=0.01 in fresh SLAD medium with and without 2DG (0.05% (w/v)) or NaCi (0.5% (w/v)) and allowed to grow at 30 °C for 24 hr. OD600 was recorded at 3 hr intervals. (H) Growth curve was performed to monitor overall growth of wild-type and knockout strains on SLAD. Overnight grown culture of deletion strains (∆∆pfk1 or ∆∆adh1) along with wild-type strains (∑1278b or CEN.PK) were diluted to OD600=0.01 in fresh SLAD and allowed to grow at 30 °C for 24 hr. OD600 was recorded at 3 hr intervals. (I) Schematic overview of the role of glucose and glycolytic intermediates in the activation of the cAMP-PKA pathway during pseudohyphal differentiation. (J) Wild-type ∑1278b was spotted on SLAD containing sub-inhibitory concentration of 2DG or NaCi with and without cAMP (1 mM), incubated for 10 days at 30 °C. Scale bar represents 1 mm for whole colony images. (K) Pertinent knockout strains which lack genes that encode for enzymes involved in glycolysis, such as pfk1 and adh1 were spotted on SLAD with and without cAMP (1 mM), incubated for 10 days at 30 °C. ∆∆gpa2 strain was used as a control. Scale bar represents 1 mm for whole colony images. (L) Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of pseudohyphal cells from the total population was determined. More than 500 cells were counted for each condition. Statistical analysis was done using one-way ANOVA test, ****(p<0.0001), **(p<0.01) and ns (non-significant). Error bars represent SEM. This figure was created using Biorender.com.
Figure 1—figure supplement 1
Confirmation of phosphofructokinase-1 (pfk1) and alcohol dehydrogenase-1 (adh1) deletion in S. cerevisiae.
(A) Confirmation of ∆∆pfk1 strain using whole genome sequencing. IGV software was used for data visualization. Red box represents gene locus of pfk1 in wild-type (∑1278b) and ∆∆pfk1 strains. (B) Confirmation of ∆∆adh1 strain using whole genome sequencing. IGV software was used for data visualization. Red box represents gene locus of adh1 in wild-type (∑1278b) and ∆∆adh1 strains. This figure was created using Biorender.com.
Figure 2 with 2 supplements
Comparative transcriptomics identifies the role of glycolysis in regulating sulfur metabolism during pseudohyphal differentiation in S. cerevisiae.
(A) Schematic overview of steps involved in RNA isolation and sequencing. (B) Volcano plots represent differentially expressed genes in 2-Deoxy-D-Glucose (2DG)-treated cells compared to untreated cells isolated from D-5 and D-10 colonies, respectively. Genes which are upregulated (Log2 fold change ≥ 1) and downregulated (Log2 fold change ≤ –1) are highlighted in red. Some of the significantly upregulated and downregulated genes involved in the biosynthesis of various amino acids are highlighted in green. (C) Heatmaps represent the differentially expressed genes involved in de novo biosynthesis and transport of sulfur-containing amino acids, in 2DG-treated cells compared to untreated cells isolated from D-5 and D-10 colonies, respectively (n=3). Scale bar represents Z-Score. (D) Schematic overview of de novo biosynthesis of sulfur-containing amino acids. (E) Schematic overview of steps involved in the Western blotting experiments. Epitope-tagged strains of various proteins involved in the de novo biosynthesis of sulfur-containing amino acids, including Met4, Met32, Met16, Met10, Cys4, and Cys3 were spotted on SLAD in the presence and absence of sub-inhibitory concentration of 2DG, following which, cells from colonies were isolated on D-5 and levels of these proteins were assessed using Western blotting. β-actin was used as loading control. This figure was created using Biorender.com.
-
Figure 2—source data 1
Uncropped and labeled blots for Figure 2.
- https://cdn.elifesciences.org/articles/109075/elife-109075-fig2-data1-v1.zip
-
Figure 2—source data 2
Raw unedited blots for Figure 2.
- https://cdn.elifesciences.org/articles/109075/elife-109075-fig2-data2-v1.zip
Figure 2—figure supplement 1
Comparative transcriptomics identifies the role of glycolysis during pseudohyphal differentiation in S. cerevisiae.
(A) Wild-type ∑1278b was spotted on SLAD, incubated for 10 days at 30 °C. Colonies were imaged every day, for 10 consecutive days (D-1 to D-10). Scale bar represents 1 mm for whole colony images. (B) Heatmaps represent the differentially expressed genes involved in amino acid biosynthesis and transporters, in 2-Deoxy-D-Glucose (2DG)-treated cells compared to untreated cells isolated from D-5 and D-10 colonies, respectively (n=3). Scale bar represents Z-Score. (C) ∆∆pfk1 and ∆∆adh1 along with wild-type ∑1278b were spotted on SLAD and cells from colonies were isolated after ~4 days, for RNA isolation. We then performed comparative RT-qPCR to check for the relative expression of genes (each gene was normalized to its respective control group) involved in the de novo biosynthesis of sulfur-containing amino acids pathway, including met32, met3, met5, and met17. Statistical analysis was done using one-way ANOVA test, **(p<0.01) and *(p<0.05). Error bars represent SEM. This figure was created using Biorender.com.
Figure 2—figure supplement 2
Expression of proteins involved in the de novo biosynthesis of sulfur-containing amino acids in response to glycolysis perturbation.
(A) Epitope-tagged strains of various proteins involved in the de novo biosynthesis of sulfur-containing amino acids, including Met4, Met32, Met16, Met10, Cys4, and Cys3 were spotted on SLAD in the presence and absence of sub-inhibitory concentration of 2-Deoxy-D-Glucose (2DG) following which, cells from colonies were isolated at day 5 and levels of these proteins were assessed using Western blotting. Raw images of Western blots were analyzed using ImageJ to normalize targeted protein expression with the expression of housekeeping protein- β-actin, in order to generate densitometric graphs. Statistical analysis was done using unpaired t-test, **(p<0.01) and *(p<0.05). Error bars represent SEM. (B) Epitope-tagged strains of various proteins involved in the de novo biosynthesis of sulfur-containing amino acids, including Met32, Met16, Met10, and Cys3 were grown in liquid SLAD and then treated with sub-inhibitory concentration of 2DG for 24 hr after which protein expression was checked using Western blotting. Pgk1 was used as loading control. (C) Raw images of Western blots were analyzed using ImageJ to normalize targeted protein expression with the expression of housekeeping protein-Pgk1, in order to generate densitometric graphs. Statistical analysis was done using unpaired t-test, ***(p<0.001), **(p<0.01) and *(p<0.05). Error bars represent SEM. This figure was created using Biorender.com.
-
Figure 2—figure supplement 2—source data 1
Uncropped and labeled blots for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/109075/elife-109075-fig2-figsupp2-data1-v1.zip
-
Figure 2—figure supplement 2—source data 2
Raw unedited blots for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/109075/elife-109075-fig2-figsupp2-data2-v1.zip
Figure 3 with 1 supplement
Glycolysis-mediated regulation of sulfur metabolism is critical for pseudohyphal differentiation in S. cerevisiae.
(A) Wild-type ∑1278b was spotted on SLAD containing sub-inhibitory concentration of 2-Deoxy-D-Glucose (2DG) with and without sulfur-containing compounds, including cysteine (500 µM) or methionine (20 µM), incubated for 10 days at 30 °C. Scale bar represents 1 mm for whole colony images. (B) Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ, and the percentage of pseudohyphal cells from the total population was determined. More than 500 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001) and ns (non-significant). Error bars represent SEM. (C) Pertinent knockout strains (∆∆pfk1 or ∆∆adh1) along with wild-type ∑1278b were spotted on SLAD with and without sulfur-containing compounds, including cysteine (200 µM) or methionine (20 µM), incubated for 10 days at 30 °C. Scale bar represents 1 mm for whole colony images. (D) Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ, and the percentage of pseudohyphal cells from the total population was determined. More than 500 cells were counted for each condition. Statistical analysis was done using one-way ANOVA test, ****(p<0.0001), ***(p<0.001), **(p<0.01), *(p<0.05) and ns (non-significant). Error bars represent SEM. (E) Pertinent knockout strain which lack genes encoding for a transcription factor involved in de novo biosynthesis of sulfur-containing amino acids, including met32 along with wild-type ∑1278b were spotted on SLAD. Scale bar represents 1 mm for whole colony images. Cells from colonies were isolated, and the length/width ratio of individual cells was measured using ImageJ, and the percentage of pseudohyphal cells from the total population was determined. More than 500 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM. (F) Pertinent knockout strain (∆∆met32) was spotted on SLAD with and without sulfur-containing compounds, including cysteine (200 µM) or methionine (20 µM), incubated for 10 days at 30 °C. Wild-type spotted on SLAD was used as control. Scale bar represents 1 mm for whole colony images. Cells from colonies were isolated, and the length/width ratio of individual cells was measured using ImageJ and the percentage of pseudohyphal cells from the total population was determined. More than 500 cells were counted for each condition. Statistical analysis was done using one-way ANOVA test, ***(p<0.001) and **(p<0.01). Error bars represent SEM. (G) Schematic overview of the proposed role of SCFMet30 in glycolysis-dependent regulation of sulfur metabolism during pseudohyphal differentiation under nitrogen-limiting conditions in S. cerevisiae. (H) Epitope-tagged strains of various proteins involved in the de novo biosynthesis of sulfur-containing amino acids, including Met4, Met32, Met16, and Cys3 in the ∆met30 background were spotted on SLAD in the presence and absence of sub-inhibitory concentration of 2DG, following which, cells from colonies were isolated on D-5 and levels of these proteins were assessed using Western blotting. β-actin was used as loading control. (I) Raw images of Western blots were analyzed using ImageJ to normalize targeted protein expression with the expression of housekeeping protein- β-actin, in order to generate densitometric graphs. Statistical analysis was done using unpaired t-test, ns (non-significant). Error bars represent SEM. (J) Epitope-tagged strain of Met30 was spotted on SLAD in the presence and absence of sub-inhibitory concentration of 2DG following which cells from colonies were isolated on D-5 and levels of these proteins were assessed using Western blotting. β-actin was used as loading control. Raw images of Western blots were analyzed using ImageJ to normalize targeted protein expression with the expression of housekeeping protein-β-actin, in order to generate densitometric graphs. Statistical analysis was done using unpaired t-test, ns (non-significant). Error bars represent SEM. This figure was created using Biorender.com.
-
Figure 3—source data 1
Uncropped and labeled blots for Figure 3.
- https://cdn.elifesciences.org/articles/109075/elife-109075-fig3-data1-v1.zip
-
Figure 3—source data 2
Raw unedited blots for Figure 3.
- https://cdn.elifesciences.org/articles/109075/elife-109075-fig3-data2-v1.zip
Figure 3—figure supplement 1
Monitoring of overall growth rate of ∆∆met32 and ∆met30 strains.
(A) Growth curve analysis was performed to monitor overall growth of wild-type and ∆∆met32 strain as well as wild-type and ∆met30 strain on SLAD. Overnight grown cultures of deletion strains (∆∆met32 or ∆met30) along with wild-type strain (∑1278b) were diluted to OD600=0.01 in fresh SLAD and allowed to grow at 30 °C for 24 hr. OD600 was recorded at 3 hr intervals. This figure was created using Biorender.com.
Figure 4 with 1 supplement
Glycolysis-dependent sulfur metabolism is critical for hyphal differentiation in C. albicans.
(A) Schematic overview of glycolysis and its inhibition by glycolysis inhibitor 2-Deoxy-D-Glucose (2DG). (B) Wild-type SC5314 was spotted on SLAD with and without sub-inhibitory concentration of glycolysis inhibitor 2DG, incubated for 7 days at 37 °C. Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of hyphal cells from the total population was determined. More than 500 cells were counted for each condition. Scale bar represents 10 µm for single cell images. Statistical analysis was done using unpaired t-test, **(p<0.01). Error bars represent SEM. (C) Schematic overview of de novo biosynthesis of sulfur-containing amino acids. (D) Wild-type SC5314 was spotted on SLAD, in the presence and absence of sub-inhibitory concentration of 2DG and cells from colonies were isolated after ~4 days, for RNA isolation. We then performed comparative RT-qPCR to check for the relative expression of genes (each gene was normalized to its respective control group) involved in the de novo biosynthesis of sulfur-containing amino acids pathway, including met32, met3, met5 (ecm17), met10, and met17 (met15). Statistical analysis was done using one-way ANOVA test, ****(p<0.0001) and ***(p<0.001). Error bars represent SEM. (E) Wild-type SC5314 was spotted on SLAD containing sub-inhibitory concentration of 2DG with and without cysteine (100 µM) and incubated for 7 days at 37 °C. Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of hyphal cells from the total population was determined. Scale bar represents 10 µm for single cell images. More than 500 cells were counted for each condition. Statistical analysis was done using unpaired t-test, **(p<0.01). Error bars represent SEM. (F) Wild-type SC5314 was spotted on SLAD containing sub-inhibitory concentration of 2DG with and without methionine (20 µM) and incubated for 7 days at 37 °C. Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of hyphal cells from the total population was determined. More than 500 cells were counted for each condition. Scale bar represents 10 µm for single cell images. Statistical analysis was done using unpaired t-test, **(p<0.01). Error bars represent SEM. (G) Wild-type SC5314 and pertinent knockout strain which lacks the genes encoding for a glycolytic enzyme, pfk1 (∆∆pfk1) were spotted on SLAD, incubated for 7 days at 37 °C. Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of hyphal cells from the total population was determined. Scale bar represents 10 µm for single cell images. More than 500 cells were counted for each strain. Statistical analysis was done using unpaired t-test, **(p<0.01). Error bars represent SEM. (H) Growth curve was performed to monitor overall growth of wild-type strain on SLAD and SLAD+2DG and ∆∆pfk1 on SLAD. Overnight grown culture of wild-type strain (SC5314) was diluted to OD600=0.01 in fresh SLAD medium with and without 2DG (0.2% (w/v)) and allowed to grow at 30 °C for 24 hr. OD600 was recorded at 3 h intervals. For ∆∆pfk1, overnight grown culture of ∆∆pfk1 strains along with wild-type strain (SC5314) were diluted to OD600=0.01 in fresh SLAD and allowed to grow at 30 °C for 24 hr. OD600 was recorded at 3 hr intervals. (I) ∆∆pfk1 along with Wild-type SC5314 was spotted on SLAD and cells from colonies were isolated after ~4 days, for RNA isolation. We then performed comparative RT-qPCR to check for the relative expression of genes (each gene was normalized to its respective control group) involved in the de novo biosynthesis of sulfur-containing amino acids pathway, including met32, met3, met5 (ecm17), met10, and met17 (met15). Statistical analysis was done using one-way ANOVA test, ****(p<0.0001), ***(p<0.001), **(p<0.01), and *(p<0.05). Error bars represent SEM. (J) ∆∆pfk1 was spotted on SLAD in the presence and absence of cysteine (100 µM), incubated for 7 days at 37 °C. Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of hyphal cells from the total population was determined. Scale bar represents 10 µm for single cell images. More than 500 cells were counted for each condition. Statistical analysis was done using unpaired t-test, **(p<0.01). Error bars represent SEM. (K) ∆∆pfk1 was spotted on SLAD in the presence and absence of methionine (10 µM), incubated for 7 days at 37 °C. Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of hyphal cells from the total population was determined. Scale bar represents 10 µm for single cell images. More than 500 cells were counted for each condition. Statistical analysis was done using unpaired t-test, **(p<0.01). Error bars represent SEM. This figure was created using Biorender.com.
Figure 4—figure supplement 1
Confirmation of pfk1 deletion in C. albicans and glycolysis is critical for hyphal differentiation in a cAMP-PKA-independent manner in C. albicans.
(A) Confirmation of ∆∆pfk1 strain using whole genome sequencing. IGV software was used for data visualization. Red box represents gene locus of pfk1 in wild-type (SC5314) and ∆∆pfk1 strains. (B) Wild-type SC5314 was spotted on SLAD containing sub-inhibitory concentration of 2-Deoxy-D-Glucose (2DG) with and without cAMP (5 mM), incubated for 7 days at 37 °C. Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of hyphal cells from the total population was determined. More than 500 cells were counted for each condition. Scale bar represents 10 µm for single cell images. Statistical analysis was done using one-way ANOVA test, **(p<0.01). Error bars represent SEM. (C) Pertinent knockout strain which lacks the gene that encodes for the glycolytic enzyme phosphofructokinase-1 (pfk1) was spotted on SLAD with and without cAMP (5 mM), along with wild-type and incubated for 7 days at 37 °C. Cells from colonies were isolated and the length/width ratio of individual cells was measured using ImageJ and the percentage of hyphal cells from the total population was determined. More than 500 cells were counted for each condition. Scale bar represents 10 µm for single cell images. Statistical analysis was done using one-way ANOVA test, ***(p<0.001) and **(p<0.01). Error bars represent SEM. This figure was created using Biorender.com.
Figure 5
Perturbation of glycolysis leads to attenuated fungal virulence in C. albicans which is rescued by sulfur supplementation.
(A) Schematic overview of various proteins involved in hyphal differentiation and virulence of C. albicans. (B) Wild-type SC5314 was spotted on SLAD, in the presence and absence of sub-inhibitory concentration of 2-Deoxy-D-Glucose (2DG) or wild-type SC5314 and ∆∆pfk1 were spotted on SLAD and cells from these colonies were isolated after ~4 days, for RNA isolation. We then performed comparative RT-qPCR to check for the relative expression of genes (each gene was normalized to its respective control group) involved in hyphal differentiation and virulence of C. albicans, including als3, ece1, hwp1, hyr1, ihd1, rbt1, and sap6. Statistical analysis was done using one-way ANOVA test, ****(p<0.0001) and ***(p<0.001). Error bars represent SEM. (C) Schematic overview of in vitro microbial survival assay. RAW 264.7 macrophages were incubated with the wild-type and ∆∆pfk1 strain with MOI = 10. After 1 hr of incubation, macrophages were lysed and plating was done to enumerate colony-forming units (CFU). The percentage of survival was expressed as the number of CFU in the presence of macrophages divided by the number of CFU in the absence of macrophages. Statistical analysis was done using unpaired t-test, ***(p<0.001) and **(p<0.01). Error bars represent SEM. (D) 6–8 week-old C57BL/6 mice were infected intravenously via lateral tail vein injection with 1×107 CFU of wild-type SC5314 or 1×107 CFU of ∆∆pfk1. To determine the survival rate, mice were monitored for 21 days post-infection for clinical signs of illness or mortality. The results are representative of seven mice per injected strain. Survival percentage were statistically evaluated by the Log-rank Mantel-Cox test, ***(p<0.001) (n=7). (E) Schematic overview of kidney isolation to check fungal burden using CFU counting and histology. (F) Fungal burden was measured by CFU counting after kidney isolation from mice injected with either wild-type or ∆∆pfk1. Statistical analysis was done using unpaired t-test, **(p<0.01). Error bars represent SEM. (G) Histopathological analysis of kidney sections using Grocott Methanamine Silver (GMS) staining was done for kidneys isolated from mice injected with either wild-type SC5314 or ∆∆pfk1. Fungal cells appear black in colour against the colored background of the kidney tissue. Bright field imaging was done using a Zeiss Axioplan 2 microscope at 40X magnification. Scale bar represents 50 µm. (H) Schematic overview of N-acetyl cysteine (NAC) administration to mice. (I) 6 mg/ml of NAC was dissolved in distilled water and administered orally to 6–8 week-old C57BL/6 mice before 72 hr of C. albicans infection. Mice administered with normal distilled water were used as controls. After 72 hr of treatment, mice were infected intravenously via lateral tail vein injection with 1×107 CFU of SC5314 or 1×107 CFU of ∆∆pfk1. To determine the survival rate, mice were monitored for 21 days post-infection for clinical signs of illness or mortality. The results are representative of six mice per condition. Survival percentage were statistically evaluated by the Log-rank Mantel-Cox test, ***(p<0.001) and **(p<0.01) (n=6). (J) Histopathological analysis of kidney sections using GMS staining was done for kidneys isolated from mice administrated with normal distilled water or NAC-containing distilled water and injected with ∆∆pfk1. Fungal cells appear black in colour against the colored background of the kidney tissue. Bright field imaging was done using a Zeiss Axioplan 2 microscope at 40X magnification. Scale bar represents 50 µm. This figure was created using Biorender.com.
Figure 6
Glycolysis-dependent sulfur metabolism orchestrates morphological transitions and virulence in fungi.
Active glycolysis plays a key regulatory role in the de novo biosynthesis of sulfur-containing amino acids by modulating the activity of SCFMet30 complex (SCFMet30 complex includes Met30, Skp1, Cdc53, Hrt1, and Cdc34) which in turn affects the expression of genes involved in this process (In S. cerevisiae, MET transcription complex includes Met4, Met28, Cbf1, Met31, and Met32, whereas in C. albicans, the characterized components of this complex include Met4, Cbf1, and Met32. Hence, Met28 and Met31, unique to the MET transcription complex of S. cerevisiae are represented with dotted borders). Glycolysis-dependent sulfur metabolism, in turn, is critical for fungal morphogenesis in both species and the ability of C. albicans to survive within macrophages and cause systemic infection in a murine model of candidiasis. Perturbation of active glycolysis increases Met30 activity, which leads to the increased degradation of Met4, resulting in the reduced expression of genes involved in the de novo biosynthesis of sulfur-containing amino acids. This, in turn, attenuates fungal morphogenesis in both species and the ability of C. albicans to survive within macrophages and cause systemic infection in a murine model of candidiasis. This figure was created using Biorender.com.
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | pFA6a-kanMX | Gift from Dr. Sunil Laxman | ||
| Recombinant DNA reagent | pFA6a-Hyg | Gift from Dr. Sunil Laxman | ||
| Recombinant DNA reagent | pFA6a-Nat | Gift from Dr. Sunil Laxman | ||
| Recombinant DNA reagent | pFA6a-Ctag-HA-G418 | Gift from Dr. Sunil Laxman | ||
| Recombinant DNA reagent | pFA6a-Ctag-HA-NAT | Gift from Dr. Sunil Laxman | ||
| Recombinant DNA reagent | pSFS2a-NAT | Gift from Dr. Kaustuv Sanyal | ||
| Antibody | Rabbit anti-HA | Cell Signaling Technology | Cat. #3724S | (1:2000 dilution) |
| Antibody | Mouse anti-β-actin | Cell Signaling Technology | Cat. #3700S | (1:2000 dilution) |
| Antibody | Mouse anti-PGK1 | Santa Cruz Biotechnology | Cat. #sc-130335 | (1:2000 dilution) |
| Antibody | Goat anti-Rabbit IgG-HRP conjugate | Cell Signaling Technology | Cat. #7074S | (1:4000 dilution) |
| Antibody | Horse anti-Mouse IgG-HRP conjugate | Cell Signaling Technology | Cat. #7076S | (1:4000 dilution) |
| Chemical compound, drug | BD Bacto agar | Becton Dickinson | Cat. #214010 | |
| Chemical compound, drug | Agarose | MP Biomedicals | Cat. #100267 | |
| Chemical compound, drug | Ammonium sulfate | Sisco Research Laboratories | Cat. #0149175 | |
| Chemical compound, drug | Blotto, non-fat dry milk | Santa Cruz Biotechnology | Cat. #sc-2324 | |
| Chemical compound, drug | β-mercaptoethanol | Sigma-Aldrich | Cat. #444203 | |
| Chemical compound, drug | Ethanol | HiMedia | Cat. #MB228 | |
| Chemical compound, drug | D-(+)-Glucose anhydrous | HiMedia | Cat. #GRM016 | |
| Chemical compound, drug | Lithium acetate | Sigma-Aldrich | Cat. #517992 | |
| Chemical compound, drug | Peptone | Thermo Fisher Scientific | Cat. #211677 | |
| Chemical compound, drug | RNaseZAP | Sigma-Aldrich | Cat. #R2020 | |
| Chemical compound, drug | SDS | G Biosciences | Cat. #RC1184 | |
| Chemical compound, drug | Trichloroacetic acid | Sigma-Aldrich | Cat. #100807 | |
| Chemical compound, drug | Tris, free base | HiMedia | Cat. #MB029 | |
| Chemical compound, drug | Tween 20 | Sigma-Aldrich | Cat. #P7949 | |
| Chemical compound, drug | Yeast extract | Thermo Fisher Scientific | Cat. #212750 | |
| Chemical compound, drug | Yeast nitrogen base w/o amino acids and ammonium sulfate | Becton Dickinson | Cat. #233520 | |
| Chemical compound, drug | L-Cysteine | Sigma-Aldrich | Cat. #30089 | |
| Chemical compound, drug | L-Methionine | Sigma-Aldrich | Cat. #64319 | |
| Chemical compound, drug | Sodium citrate | Sigma-Aldrich | Cat. #C8532 | |
| Chemical compound, drug | Glycerol | HiMedia | Cat. #MB060 | |
| Chemical compound, drug | Potassium acetate | MP Biomedicals | Cat. #191425 | |
| Chemical compound, drug | 2-Deoxy-D-Glucose | Sigma-Aldrich | Cat. #D8375 | |
| Chemical compound, drug | Nourseothricin | Jena Bioscience | Cat. #AB-102XL | |
| Chemical compound, drug | G418 | MP Biomedicals | Cat. #158782 | |
| Chemical compound, drug | Ethidium bromide | HiMedia | Cat. #MB074 | |
| Chemical compound, drug | Pico-ECL substrate | Thermo Fisher Scientific | Cat. #34579 | |
| Chemical compound, drug | Femto-ECL substrate | Thermo Fisher Scientific | Cat. #34094 | |
| Chemical compound, drug | TEMED | Sisco Research Laboratories | Cat. #52145 | |
| Chemical compound, drug | Hygromycin | Sigma-Aldrich | Cat. #H0654 | |
| Commercial assay or kit | Ribopure RNA purification Kit-Yeast | Thermo Fisher Scientific | Cat. #AM1926 | |
| Commercial assay or kit | BCA Protein Assay Kit | Thermo Fisher Scientific | Cat. #23227 | |
| Commercial assay or kit | PrimeScript cDNA Synthesis Kit | Takara | Cat. #6110A | |
| Commercial assay or kit | GMS Staining Kit | ABcam | Cat. #AB287884 | |
| Software, algorithm | RStudio v4.3.2 | Posit PBC | ||
| Software, algorithm | GraphPad Prism v9 | GraphPad Software | ||
| Software, algorithm | ImageJ | Open-source Software | ||
| Software, algorithm | IGV v2.16.2 | Open-source Software |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/109075/elife-109075-mdarchecklist1-v1.docx
-
Supplementary file 1
Yeast strains used in this study.
- https://cdn.elifesciences.org/articles/109075/elife-109075-supp1-v1.docx
-
Supplementary file 2
Oligonucleotides used in this sUsed in this Study.
- https://cdn.elifesciences.org/articles/109075/elife-109075-supp2-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Glycolysis-dependent sulfur metabolism orchestrates morphological plasticity and virulence in fungi
eLife 14:RP109075.
https://doi.org/10.7554/eLife.109075.3
