Proteomics of Paracoccidioides lutzii: Overview of Changes Triggered by Nitrogen Catabolite Repression

Abstract

Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MS^E proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, β-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection.

Keywords: NCR-proline; Paracoccidioides; metabolic reprogramming; nitrogen starvation; proteomics.

Figure 1

Functional classification of proteins regulated in P. lutzii identified by nanoUPLC-MS^E. (a) Two hundred sixty-nine up-regulated proteins with their percentage (%) in each biological category. (b) Sixty-nine down-regulated proteins with their percentage (%) in each biological category. Biological processes involving the differentially expressed proteins were obtained using the Uniprot databases (http://www.uniprot.org/, accessed on 27 September 2022).

Figure 2

General overview of P. lutzii metabolism under NCR conditions. The metabolic pathways were determined based on the molecules identified by proteomic analysis in NCR-proline conditions. In red and blue, the molecules that were up-regulated and down-regulated, respectively, in NCR conditions are shown. Abbreviations: 6-PGDH: 6-phosphogluconate dehydrogenase; ACAT: acetyl-CoA acyltransferase; ACD: acyl-CoA dehydrogenase; ADH: alcohol dehydrogenase; ALA: alanine; ALD: aldolase; AREA: transcription factor AreA; ASP: asparagine; CAR: carnitine O-acetyltransferase; CIS: cysteine; COFA: odd-chain fatty acids; CS: citrate synthase; CytC: cytochrome C; DHDH: dihydrolipoyl dehydrogenase; ECFA: even-chain fatty acids; FMD: formamidase; G6PDH: glucose-6-phosphate 1-dehydrogenase; GAP: general amino acid permease; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GC: glyoxylate cycle; GLI: Glycine; GLU: glutamate; HSP70: heat shock protein 70; HSP88: heat shock protein Hsp88; HSP90: heat shock protein Hsp90; HX: hexokinase; ICL: isocitrate lyase; IDH: isocitrate dehydrogenase; ISO: isoleucine; MCC: methylcitrate cycle; MCD: 2-methylcitrate dehydratase; MDH: malate dehydrogenase; MET: methionine; MS: malate synthase; OXA: oxaloacetate; PC: pyruvate carboxylase; PDC: pyruvate decarboxylase; PDH: pyruvate dehydrogenase; PEP: phosphoenolpyruvate; PER: peroxidase; PGK: phosphoglycerate kinase; PRO: proline; PRX: mitochondrial peroxiredoxin; RPPK: ribose-phosphate pyrophosphokinase; SDH: succinate dehydrogenase; SER: serine; SOD: superoxide dismutase; SSB1: heat shock protein SSB1; THR: threonine; TRX: thioredoxin. TYR: tyrosine UGM: UDP-galactopyranose mutase; URE: urease; VAL: valine.

Figure 3

Ethanol measurement in NCR conditions. A total 2 × 10⁸ cells/mL were analyzed at 48 h under NCR conditions. Ethanol concentration was determined using an enzymatic detection kit (UV test for ethanol). The data are expressed as the mean ± standard deviation of biological triplicates analyzed in independent experiments and using the Student’s t-test, with p ≤ 0.01 (**) considered as statistically significant. Prol: proline; Glut: glutamine.

Figure 4

Cell wall remodeling in P. lutzii under proline conditions. Yeast cells were collected after 48 h of culture under NCR conditions, stained with aniline blue (AB) and calcofluor white (CFW) and visualized using an Axiocam MRc-Scope A1 fluorescence microscope. (a) Cells stained with AB in NCR-glutamine and NCR-proline conditions. (b) Fluorescence intensity of AB (in pixels) for yeast cells under NCR conditions. (c) Cells stained with CFW under NCR-glutamine and NCR-proline conditions. (d) Fluorescence intensity of CFW (in pixels) for yeast cells under NCR conditions was determined using AxioVision Software, determining the standard error in each analysis. Statistical comparisons were performed using Student’s t-test, with p ≤ 0.0001 (****), considered statistically significant. Prol: proline; Glut: glutamine; Magnification 40×.

Figure 5

Transcriptional level of NCR-related genes under NCR conditions. The expression of NCR-related genes in P. lutzii yeast cells under NCR conditions was assessed through RT-qPCR, using relative quantification. The tubulin gene (PAAG_03031) served as the endogenous control, and the transcriptional levels of the genes coding for γ-glutamyl transpeptidase (ggt), formamidase (fmd), arginase (arg) and urease (ure) were analyzed. Data are expressed as the mean ± standard deviation of triplicates of independent experiments. Statistical analysis was performed through the Student’s t-test, demonstrating values of p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***), considered as statistically significant. P: proline; G: glutamine.

Figure 6

Enzymatic activity of enzymes related to cell rescue and virulence under NCR conditions. The assays were performed using soluble proteins obtained under NCR conditions. (a) The enzymatic activity of gamma-glutamyltransferase (γ-GT or GGT) was assessed using 4 μg and 12 μg of soluble proteins from NCR-proline and NCR-glutamine conditions. GGT specific activity (U/L) was calculated by measuring the number of nmol of ρNA released per liter of sample in the incubation time (min). (b) GST activity was measured using a total of 10 μg of soluble proteins obtained in NCR-proline and NCR-glutamine conditions. Student’s t-test was applied for statistical analysis, showing values of p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****), considered as statistically significant. Prol: proline; Glut: glutamine.

Figure 7

Enzymatic activity of formamidase in NCR conditions. The assays were performed with soluble proteins obtained in NCR conditions. Formamidase enzymatic activity in 10 μg of soluble proteins under NCR-proline and NCR-glutamine conditions; Student’s t-test was applied for statistical analysis showing values of p ≤ 0.05 (*), considered as statistically significant. Glut: glutamine; Prol: proline.

Figure 8

Survival of P. lutzii cells within macrophages. Prior to the ex vivo assays, P. lutzii yeast cells were cultured under NCR conditions for 48 h, followed by incubation with macrophages at a ratio of 1:5 (macrophages to yeast cells). The cells were then incubated for 12 and 24 h at 36 °C and in 5% CO₂. The viable cell count was determined via quantification of colony-forming units (CFUs). The mean value and standard deviation were calculated from triplicates, and statistical analysis was conducted using Student’s t-test, showing values of p ≤ 0.05 (*) and p ≤ 0.01 (**), considered as statistically significant. BHI: brain heart infusion; Prol: proline; Glut: glutamine.