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% glucose (SLAD) with and without sub-inhibitory concentrations of glycolysis inhibitors (2-Deoxy-D-Glucose (2DG) and sodium citrate (NaCi)), incubated for 10 days at 30 °C. Whole colony imaging was done using Olympus MVX10 stereo microscope and single cell imaging was done using Zeiss Apotome microscope. Scale bar represents 1mm for whole colony images and 5µm for single cell images. (C) Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of pseudohyphal cells were calculated using ImageJ. 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. Whole colony imaging was done using Olympus MVX10 stereo microscope and single cell imaging was done using Zeiss Apotome microscope. Scale bar represents 1mm for whole colony images and 5µm for single cell images. (F) Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of pseudohyphal cells were calculated using ImageJ. More than 500 cells were counted for each strains. 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%) or NaCi (0.5%) and allowed to grow at 30 °C for 24 hours. OD600 was recorded at 3-h intervals (H) Growth curve was performed to monitor overall growth of wild-type and knockout strains on SLAD. Overnight grown culture of deletion strains (ΔΔpfk1 or ΔΔadh1) 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 hours. OD600 was recorded at 3-h intervals. (I) Schematic overview of the role of glucose and glycolytic intermediates in the activation of 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 (1mM), incubated for 10 days at 30 °C. After 10 days, whole colony imaging was done using Olympus MVX10 stereo microscope. Scale bar represents 1mm 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 (1mM), incubated for 10 days at 30 °C. ΔΔgpa2 strain was used as a control. After 10 days, whole colony imaging was done using Olympus MVX10 stereo microscope. Scale bar represents 1mm for whole colony images. This figure was created using BioRender.com.

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 RNA-sequencing. (B) Volcano plots represent differentially expressed genes in 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 (n=3). β-actin was used as loading control. This figure was created using BioRender.com.

Glycolysis-mediated Regulation of Sulfur Metabolism is Critical for Pseudohyp-hal Differentiation in S. cerevisiae.

(A) Wild-type ∑1278b was spotted on SLAD containing sub-inhibitory concentration of 2DG with and without sulfur-containing compounds including cysteine (500µM) or methionine (20µM), incubated for 10 days at 30 °C. After 10 days, whole colony imaging was done using Olympus MVX10 stereo microscope. Scale bar represents 1mm for whole colony images. (B) Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of pseudohyphal cells were calculated using ImageJ. 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 which lack genes encoding for enzymes involved in glycolysis such as pfk1 and adh1 along with wild-type were spotted on SLAD with and without sulfur-containing compounds including cysteine (200µM) or methionine (20µM), incubated for 10 days at 30 °C. After 10 days, whole colony imaging was done using Olympus MVX10 stereo microscope. Scale bar represents 1mm for whole colony images. (D) Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of pseudohyphal cells were calculated using ImageJ. 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) and *(P<0.05). Error bars represent SEM. (E) Pertinent knockout strains which lack genes encoding for transcription factor involved in de novo biosynthesis of sulfur-containing amino acids including met32 along with wild-type ∑1278b were spotted on SLAD and imaged after 10 days using Olympus MVX10 stereo microscope. Scale bar represents 1mm for whole colony images. Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of pseudohyphal cells were calculated using ImageJ. 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 ΔΔmet32 was spotted on SLAD with and without sulfur-containing compounds including cysteine (200µM) or methionine (20µM), incubated for 10 days at 30 °C. Wild-type spotted on SLAD was used as control. After 10 days, imaging was done using Olympus MVX10 stereo microscope. Scale bar represents 1mm for whole colony images. Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of pseudohyphal cells were calculated using ImageJ. 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 Δmet30 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 (n=3). β-actin was used as loading control. (I) Raw images of Western blots were analysed 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 (n=3). β-actin was used as loading control. Raw images of Western blots were analysed 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.

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 single cells were imaged using Zeiss Apotome microscope and percentage of hyphal cells were calculated using ImageJ. More than 500 cells were counted for each condition. Scale bar represents 10µm 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 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 (100µM) and, incubated for 7 days at 37 °C. Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of hyphal cells were calculated using ImageJ. Scale bar represents 10µm 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 (20µM) and, incubated for 7 days at 37 °C. Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of hyphal cells were calculated using ImageJ. More than 500 cells were counted for each condition. Scale bar represents 10µm 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 (ΔΔpfk1) were spotted on SLAD, incubated for 7 days at 37 °C. Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of hyphal cells were calculated using ImageJ. Scale bar represents 10µm 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 ΔΔpfk1 on SLAD. Overnight grown culture of wild-type strain (SC5314) were diluted to OD600=0.01 in fresh SLAD medium with and without 2DG (0.2%) and allowed to grow at 30 °C for 24 hours. OD600 was recorded at 3-h intervals. For ΔΔpfk1, overnight grown culture of ΔΔpfk1 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 hours. OD600 was recorded at 3-h intervals. (I) ΔΔpfk1 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 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) ΔΔpfk1 was spotted on SLAD in the presence and absence of cysteine (100µM), incubated for 7 days at 37 °C. Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of hyphal cells were calculated using ImageJ. 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) ΔΔpfk1 was spotted on SLAD in the presence and absence of methionine (10µM), incubated for 7 days at 37 °C. Cells from colonies were isolated and single cells were imaged using Zeiss Apotome microscope and percentage of hyphal cells were calculated using ImageJ. 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.

Perturbation of Glycolysis Leads to Attenuated Fungal Virulence in C. albicans.

(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 2DG or wild-type SC5314 and ΔΔpfk1 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 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 ΔΔpfk1 strain with MOI=10. After 1 hour of incubation, macrophages were lysed and plating was done to enumerate CFU. 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 ΔΔpfk1. To determine 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 ΔΔpfk1. 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 ΔΔpfk1. Fungal cells appear black in colour against the colored background of the kidney tissue. Bright field imaging was done using Axioplan 2 microscope at 40X magnification. Scale bar represents 50 µm. (H) Schematic overview of N-acetyl cysteine (NAC) administration to mice. (I) 6mg/ml of NAC was dissolved in distilled water and administered orally to 6-8-week-old C57BL/6 mice before 72 hours of C. albicans infection. Mice administered with normal distilled water were used as controls. After 72 hours of treatment, mice were infected intravenously via lateral tail vein injection with 1 × 107 CFU of SC5314 or 1 × 107 CFU of ΔΔpfk1. To determine 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 ΔΔpfk1. Fungal cells appear black in colour against the colored background of the kidney tissue. Bright field imaging was done using Axioplan 2 microscope at 40X magnification. Scale bar represents 50 µm. This figure was created using BioRender.com.

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.

Confirmation of pfk1 and adh1 Deletion in S. cerevisiae.

(A) Confirmation of ΔΔpfk1 strain using whole genome sequencing. IGV software (version 2.16.2) was used for data visualisation. Red box represents gene locus of pfk1 in wild-type (∑1278b) and ΔΔpfk1 strains. (B) Confirmation of ΔΔadh1 strain using whole genome sequencing. IGV software (version 2.16.2) was used for data visualisation. Red box represents gene locus of adh1 in wild-type (∑1278b) and ΔΔadh1 strains. This figure was created using BioRender.com.

Comparative Transcriptomics Identifies the Role of Glycolysis During Pseudohypal 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), using Olympus MVX10 stereo microscope. Scale bar represents 1mm for whole colony images. (B) Heatmaps represent the differentially expressed genes involved in amino acid biosynthesis and transporters, in 2DG treated cells compared to untreated cells isolated from D-5 and D-10 colonies, respectively (n=3). Scale bar represents Z-Score. This figure was created using BioRender.com.

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 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 analysed 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 hours after which protein expression was checked using Western blotting (n=3). Pgk1 was used as loading control. (C) Raw images of Western blots were analysed 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.

Monitoring of Overall Growth Rate of ΔΔmet32 Strain.

(A) Growth curve analysis was performed to monitor overall growth of wild-type and ΔΔmet32 knockout strain on SLAD. Overnight grown cultures of deletion strain (ΔΔmet32) along with wild-type strain (∑1278b) were diluted to OD600=0.01 in fresh SLAD and allowed to grow at 30 °C for 24 hours. OD600 was recorded at 3-h intervals. This figure was created using BioRender.com.

Confirmation of pfk1 Deletion in C. albicans.

(A) Confirmation of ΔΔpfk1 strain using whole genome sequencing. IGV software (version 2.16.2) was used for data visualisation. Red box represents gene locus of pfk1 in wild-type (SC5314) and ΔΔpfk1 strains. This figure was created using BioRender.com.

Yeast Strains Used in this Study

Oligonucleotides Used in this Study

List of Reagents and Tools Used in this Study