Figure 1.
Effect of carbon starvation in transcripts and protein expression in Paracoccidioides yeast cells.
(A) The expression of fructose-1,6-biphosphatase, isocitrate lyase and 3-ketoacyl-CoA thiolase genes in Pb01 yeast cells grown in MMcM medium with 4% (carbon source) or absence of glucose (carbon starvation) were analyzed. The cells were incubated at 36°C for several time intervals. The data were normalized using the constitutive gene encoding the 60S ribosomal L34 gene as the endogenous control and are presented as relative expression in comparison to the experimental control cells value set at 1. Data are expressed as the mean ± standard deviation of the triplicates of independent experiments. *, significantly different from the control, at a p value of ≤0.05. (B) Proteins (50 µg) of Pb01 yeast cells were incubated at 36°C in MMcM medium with 4% or 0% of glucose and the abundance of PbIcl was analyzed by western blotting. The proteins were fractionated by one-dimensional gel electrophoresis. The proteins were blotted onto a nitrocellulose membrane and the ∼60 kDa protein species was detected by using the rabbit polyclonal antibody anti-PbIcl [43]. Densitometric analysis of immunoblotting bands was performed using the software AphaEaseFC.
Figure 2.
Growth of Paracoccidioides under carbon starvation.
A total of 5.107 cells/50 mL of Paracoccidioides yeast cells were incubated in minimal medium (MVM) with carbon (4% glucose) or not (0% glucose) up to 72 h. At time points 0, 24, 48 and 72 h, cells were collected, killed by heat, and lyophilized to determine the cell dry weight. Data are expressed as the mean ± standard deviation of the triplicates of independent experiments.*, significantly different from the carbon condition, at a p-value of ≤0.05.
Figure 3.
Overview of metabolic responses of Paracoccidioides to carbon starvation.
The figure summarizes the data from transcriptome and proteomic analyses and suggests the mechanism and the first flow of carbon used by this fungus to overcome the carbon starvation stress. HXK: hexokinase; PGM: phosphoglucomutase; PFK: 6-phosphofructokinase; FBPase: fructose-1,6-biphosphatase; ALD: fructose-bisphosphate aldolase; TPI: triosephosphate isomerase; PGK: phosphoglycerate kinase; ENO: enolase; PEPCK: phosphoenolpyruvate carboxykinase; PYC: pyruvate carboxylase; PDC: pyruvate decarboxylase; ADH: alcohol dehydrogenase; ALDH: aldehyde dehydrogenase; ACD: acyl-CoA dehydrogenase; ECH: enoyl-CoA hydratase; THIO: 3-ketoacyl-CoA thiolase; PDH: pyruvate dehydrogenase; ICL: isocitrate lyase; MLS: malate synthase; IDH: isocitrate dehydrogenase; OGDC: 2-oxoglutarate dehydrogenase E1; SUCLA: succinyl-CoA ligase; FUM: fumarate hydratase (fumarase) and MDH: malate dehydrogenase. Enzymes were colored according to their differences in expression and labeled to indicate whether the data were obtained from transcriptome or proteomics. Italic, bold, and underlined labels indicate that the data were obtained from transcriptome, proteome, or both, respectively. Green or red indicate up- or down-regulated proteins, respectively. The numbers 1, 2, 3, and 4 indicate up-regulated amino acids involved in pyruvate, oxaloacetate, succinate, and acetyl-CoA production, respectively. 1) pyruvate production: tryptophan and cysteine. 2) oxaloacetate production: phenylalanine, glutamate and tyrosine. 3) succinate production: threonine. 4) acetyl-CoA production: threonine, tryptophan, tyrosine and leucine. Italic, bold, and underlined labels indicate that the amino acids accumulations were obtained from transcriptome, proteome, or both analyzes, respectively. OXA: oxaloacetate; e−: released electrons from enzymatic reaction.
Figure 4.
Ethanol detection in Paracoccidioides, Pb01 yeast cells, under carbon starvation.
The concentration of ethanol (g/L) in Paracoccidioides yeast cells under carbon or carbon-starved conditions was determined. A total of 106 cells were used for each sample, and the ethanol compound was quantified using the enzymatic detection kit (UV-test for ethanol, RBiopharm, Darmstadt, Germany). Data are expressed as the mean ± standard deviation of the biological triplicates of independent experiments. Student's t-test was used.*, significantly different from the carbon condition, at a p-value of ≤0.05.
Figure 5.
Isocitrate lyase activity assay.
The activity was determined by measuring the formation of glyoxylate as its phenylhydrazone derivative in each condition. A total of 50 µg of each total protein extracts of Paracoccidioides under carbon and carbon-starvation (0% glucose) conditions for 48 h in MMcM medium was used. The specific activities were determined as the amount of enzyme required to form 1 µmol of glyoxylate-phenylhydrazone per minute per mg of total protein and are represented as U.mg−1. Errors bars represent standard deviation from three biological replicates while * represents p≤0.05.
Table 1.
The most abundant up-regulated proteins of Paracoccidioides (Pb01) yeast cells under carbon starvation detected using NanoUPLC-MSE.
Table 2.
The most abundant down-regulated proteins of Paracoccidioides (Pb01) yeast cells under carbon starvation detected using NanoUPLC-MSE.
Figure 6.
Number of transcripts and proteins related to metabolism and energy subcategories regulated in Paracoccidioides, Pb01, under carbon starvation.
The number of transcripts and proteins, in percentage (%), regulated in Paracoccidioides, Pb01, under carbon starvation was calculated based on number of transcripts/proteins in each category shown in Figures S3 and S9, panels A. (A) Metabolism. The subcategories were represented by: amino acid; nitrogen/sulfur; C- compound and carbohydrates; lipid/fatty acid, and isoprenoid; purin nucleotide/nucleoside/nucleobase; secondary and phosphate metabolism. (B) Energy. The subcategories were represented by glycolysis/gluconeogenesis; TCA cycle; electron transport and membrane associated energy; ethanol production; pentose phosphate pathway and glyoxylate cycle. Black and gray bars indicate genes and proteins, respectively.
Figure 7.
Expression of Paracoccidioides fbp, icl and thio genes and susceptibility of yeast cells to macrophages killing during infection.
(A) Pb01 yeast cells were grown without (yeast cells) and with macrophages (yeast cells-macrophages) for 24 h in RPMI medium, and the relative expression of genes fbp (fructose-1,6-biphosphatase), icl (isocitrate lyase), and thio (3-ketoacyl-CoA thiolase) was determined. The data were normalized using the constitutive gene encoding the 60S ribosomal L34 gene as the endogenous control and are presented as relative expression in comparison to the experimental control cells value set at 1. (B) Pb01 yeast cells were previously grown in MMcM medium with carbon (4% of glucose) or absence of glucose (carbon starvation) up to 48 h and then were incubated with macrophages at a 1∶2.5 macrophages: yeast ratio, for both conditions. As demonstrated, the number of viable cells was determined by quantifying the number of colony forming units/mL (CFUs/mL) during infection from culture supernatant (non-internalized cells removed by aspiration prior to macrophages lysis) and after internalization. Data are expressed as the mean ± standard deviation of the biological triplicates of independent experiments. Student's t-test was used. *, significantly different from the control, at a p-value of ≤0.05.