The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cultivation of Fungal Strains
2.2. Stress Treatments
2.3. RNA Sequencing (RNASeq)
2.4. Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) Assay
2.5. Gene Set Enrichment Analysis
- (i)
- antioxidant enzyme genes—this gene group includes genes encoding functionally verified and/or putative antioxidant enzymes including the catalase (GOID: 4096), superoxide dismutase (GOID: 4784), glutaredoxin (GOID: 6749), thioredoxin (GOIDs: 8379 and 51920) and peroxidase (GOID: 4601) GO terms;
- (ii)
- iron metabolism-related genes—genes involved in iron acquisition by C. albicans were collected according to Fourie et al. [22];
- (iii)
- zinc and copper homeostasis genes—genes involved in zinc and copper acquisition by C. albicans were collected according to Gerwien et al. [23];
- (iv)
- metabolic pathway-related genes—this group contains all genes related to the TCA, ethanol fermentation, glycogen metabolism, and ergosterol biosynthesis biochemical pathways according to the pathway database (http://pathway.candidagenome.org, accessed on 5 July 2021).
2.6. Determination of Virulence Attributes
Biofilm Development and Metabolic Activity-Based Susceptibility Testing
2.7. Assays of Redox Homeostasis and Antioxidant Enzyme Activities
2.8. Assay of Glucose Consumption, Ethanol Production, and Iron, Zinc and Copper Contents of C. albicans Cells
2.9. Statistical Analysis
3. Results
3.1. BM and MSB Suppressed Growth and Metabolic Activity of Biofilm Synergistically
3.2. Genome-Wide Transcriptional Changes Confirmed the Interaction between BM and MSB Treatments
3.3. Gene Ontology Analysis of the Transcriptome Changes
3.3.1. The CaPPZ1 Gene Deletion Has a Moderate Transcriptomic Effect
3.3.2. The Cellular Response to the Oxidative Stress Was More Extensive in the Cappz1 Knock Out Strain
3.3.3. Combination of CaPPZ1 Gene Deletion and MSB Stress Led to a Global Reprogramming of Gene Expression in BM Pre-Treated Cells
3.4. Testing of the Predicted Consequences of Transcriptome Changes
3.4.1. BM Did Not Influence the Growth, Extracellular Phospholipase and Proteinase Production and Hypha Formation of C. albicans
3.4.2. BM Pre-Treatment Enhanced the Oxidative Stress and the Stress Response Induced by MSB
3.4.3. Deletion of CaPPZ1 and MSB-Induced Oxidative Stress Reduced Glucose Consumption, Ethanol Production and Influenced Metal Ion Content of C. albicans
4. Discussion
4.1. Phenotypes and Transcriptional Changes Attributed to CaPPZ1 Gene Deletion
4.2. Transcriptional Changes Related to MSB Exposures
4.3. Combined Effects of CaPPZ1 Deletion and MSB Treatment
4.4. Synergistic Effects of BM Exposure and MSB Treatment on the Phosphatase Deletion Mutant
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strains | Median (Range) MIC Values | Interaction | ||||
---|---|---|---|---|---|---|
MIC Alone | MIC in Combination | Median (Range) FICI | Type of Interaction | |||
BM (mM) | MSB (mM) | BM (mM) | MSB (mM) | |||
QMY23 (WT) | >4 a | 0.25 | 2 (2–4) | 0.06 (0.03–0.06) | 0.49 (0.49–1) | Synergy |
cappz1 (KO) | >4 a | 0.25 | 0.125 (0.125–0.5) | 0.06 (0.008–0.06) | 0.49 (0.302–0.49) | Synergy |
Significantly Enriched Gene Groups (Number of Genes Associated with the Group) | Number of Up- and Down-Regulated Genes (with Corrected p-Value) a | ||||||
---|---|---|---|---|---|---|---|
KO vs. WT | KO + MSB vs. WT + MSB | KO + BM + MSB vs. WT + BM + MSB | WT + MSB vs. WT | WT + BM + MSB vs. WT | KO + MSB vs. KO | KO + BM + MSB vs. KO | |
Transmembrane transport b (365) | 46(3 × 10−2) | 103(3 × 10−5) | 27(9.9 × 10−7) | ||||
Plasma membrane b (509) | 32 (1 × 10−5) | 132 (1.3 × 10−5) | 28 (6.6 × 10−5) | ||||
Carbohydrate transport b (34) | 11(9 × 10−3) | 20(7.4 × 10−5) | 6(2.6 × 10−2) | ||||
Oxidoreductase activity b (429) | 27 (2 × 10−4) | 64(2.9 × 10−7) | 127 (3.9 × 10−9) | 21 (5.3 × 10−12) 8 (5 × 10−3) | 60(7.24 × 10−17) | 110 (5.6 × 10−17) | 165(9.7 × 10−22) |
Response to oxidative stress b (153) | 27(4 × 10−3) | 12 (7.2 × 10−8) | 26(1.1 × 10−7) | 43 (2.5 × 10−6) | 54 (1 × 10−3) | ||
Antioxidant activity c (58) | 13(1.1 × 10−4) | 17 (1.3 × 10−2) | 6(2.5 × 10−6) | 16 (3.4 × 10−10) | 23 (8.9 × 10−9) | 26(2.8 × 10−6) | |
Peroxisome b (70) | 18(1 × 10−4) | 38 (2.6 × 10−10) | 23 (2.0 × 10−4) | 35 (5.0 × 10−7) | |||
RNA metabolic process b (776) | 92 (2.5 × 10−6) | 231 (5.4 × 10−22) | 252 (7 × 10−49) | 325 (4.1 × 10−56) | |||
Ribosome b (194) | 76 (4.6 × 10−39) | 131 (7.8 × 10−58) | 73(4.7 × 10−16) | 95(6.3 × 10−20) | |||
RNA transport b (114) | 47 (1.4 × 10−7) | 37 (8.4 × 10−5) | 51 (1.6 × 10−7) | ||||
Alpha-amino acid metabolism b (132) | 9 (8 × 10−3) | 21(2.3 × 10−5) | 41(2.1 × 10−7) | 51 (5.2 × 10−5) | |||
Branched-chain amino acid biosynthesis b (12) | 4 (3 × 10−3) | 6(2 × 10−3) | 9(1 × 10−3) | 10(4 × 10−3) | |||
Glutamate metabolic process b (11) | 2(3 × 10−2) | ||||||
Mitochondrion b (649) | 145 (1.5 × 10−9) | 201 (2.2 × 10−13) | |||||
Iron homeostasis-related genes c (48) | 11 (7.8 × 10−9) | 7(4 × 10−2) | 5(2 × 10−7) | 9 (2.4 × 10−8) | 19(8 × 10−7) | 18(4 × 10−3) | |
Glycolytic process b (17) | 7(1.6 × 10−7) | 14 (2.1 × 10−8) | 16 (4.4 × 10−8) | ||||
Gluconeogenesis b (9) | 5 (9.3 × 10−6) | ||||||
Ethanol fermentation pathway c (8) | 4 (3 × 10−2) | 4(7 × 10−3) | 7 (4.6 × 10−5) | ||||
Glycogen biosynthesis pathway c (7) | 6(2.6 × 10−5) | 4(7 × 10−3) | 5(7 × 10−3) | ||||
Fatty acid catabolic process b (18) | 15 (2.3 × 10−6) | 12(2 × 10−2) | |||||
Ergosterol biosynthesis pathway c (20) | 8 (1.9 × 10−5) | 7 (3 × 10−2) |
Virulence Attributes | QMY23 (WT) | cappz1 (KO) | ||
---|---|---|---|---|
Untreated | +BM | Untreated | +BM | |
Extracellular proteinase activity (Pz values) a | 0.72 ± 0.05 | 0.71 ± 0.06 | 0.74 ± 0.04 | 0.73 ± 0.05 |
Secreted phospholipase activity (Pz values) a | 0.48 ± 0.06 | 0.44 ± 0.07 | 0.45 ± 0.06 | 0.43± 0.08 |
Hypha formation (%) b | ||||
7 days | 15.0 ± 3.1 | 17.5 ± 1.8 | 9.7 ± 2.4 c | 11.1 ± 2.1 c |
10 days | 31.3 ± 3.3 | 33.2 ± 2.5 | 24.5 ± 2.1 c | 25.1 ± 2.9 c |
Oxidative Stress Related Parameters | QMY23 (WT) | cappz1 (KO) | ||||||
---|---|---|---|---|---|---|---|---|
Untreated | +BM | +MSB | +BM + MSB | Untreated | +BM | +MSB | +BM + MSB | |
glutathione reductase (mkat/kg protein) | 1.8 ± 0.4 | 1.8 ± 0.45 | 2.5 ± 0.2 c | 3.2 ± 0.6 c | 2.3 ± 0.15 b | 2.1 ± 0.15 | 3.3 ± 0.25 b,c | 5.0 ±0.85 b,c,d |
glutathione peroxidase (mkat/kg protein) | 0.17 ± 0.02 | 0.19 ± 0.01 | 0.22 ± 0.02 c | 0.26 ± 0.03 c | 0.22 ± 0.04 b | 0.21 ± 0.02 | 0.34 ± 0.07 b,c,e | 0.44 ± 0.05 b,c,d |
catalase (kat/kg protein) | 0.44 ± 0.07 | 0.46 ± 0.06 | 0.65 ± 0.08 c | 0.83 ± 0.17 c | 0.6 ± 0.11 | 0.59 ± 0.10 | 0.9 ± 0.17 b,c | 1.7 ± 0.47 b,c,d |
superoxide dismutase (mU/mg protein) | 52 ± 2.3 | 50.8 ± 1.4 | 56.9 ± 3.3 | 59.4 ± 3.1 c | 56.6 ± 2.3 | 54.5 ± 2.4 | 65.3 ± 4.5 c | 69.5 ± 7.3 c |
DCF (nmol DCF/OD640) a | 13.4 ± 3.1 | 12.5 ± 3.3 | 21.5 ± 2.0 c | 26.5 ± 2.6 c,d | 33.1 ± 2.3 b | 32.8 ± 3.0 b | 60.3 ± 8.7 b,c,e | 95.2 ± 11.9 b,c,d |
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Jakab, Á.; Emri, T.; Csillag, K.; Szabó, A.; Nagy, F.; Baranyai, E.; Sajtos, Z.; Géczi, D.; Antal, K.; Kovács, R.; et al. The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure. J. Fungi 2021, 7, 540. https://doi.org/10.3390/jof7070540
Jakab Á, Emri T, Csillag K, Szabó A, Nagy F, Baranyai E, Sajtos Z, Géczi D, Antal K, Kovács R, et al. The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure. Journal of Fungi. 2021; 7(7):540. https://doi.org/10.3390/jof7070540
Chicago/Turabian StyleJakab, Ágnes, Tamás Emri, Kinga Csillag, Anita Szabó, Fruzsina Nagy, Edina Baranyai, Zsófi Sajtos, Dóra Géczi, Károly Antal, Renátó Kovács, and et al. 2021. "The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure" Journal of Fungi 7, no. 7: 540. https://doi.org/10.3390/jof7070540
APA StyleJakab, Á., Emri, T., Csillag, K., Szabó, A., Nagy, F., Baranyai, E., Sajtos, Z., Géczi, D., Antal, K., Kovács, R., Szabó, K., Dombrádi, V., & Pócsi, I. (2021). The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure. Journal of Fungi, 7(7), 540. https://doi.org/10.3390/jof7070540