Glutathione System in pathogenic fungi: Comparison
View Latest Version
Please note this is a comparison between Version 2 by Dean Liu and Version 1 by Monsicha Pongpom.

The glutathione system has been recognized as one of the most important intracellular antioxidant systems; however, the contribution of this system in pathogenesis has been neglected, especially in human fungal pathogens. This paper provides, for the first time, data collection on the role of the glutathione system in virulence of pathogenic fungi. Several recent biological aspects of glutathione-related roles were also added into this report. Our review highlights the intertwined role of the glutathione system and virulence, and emphasizes the need for more molecular research, especially in dimorphic fungi. 

  • glutathione
  • metabolism
  • morphogenesis

1. Introduction

The integration of metabolism with virulence has become a new paradigm in the host–fungal pathogen interaction. In addition to virulence attributes, the term “fitness attributes” has been defined as the cellular functions that are required to support microbial growth and survival [1]. The fitness attributes include the metabolic capacity to assimilate host nutrients, resistance to host-imposed stress, tolerance to elevated host temperature, and the construction of a robust cell wall [2]. The inactivation of fitness attributes will diminish the ability of a fungus to obtain nutrients or combat environmental stressors and hence, will attenuate its ability to grow, express virulence factors, and ultimately cause infection. The metabolism provides a platform for generating the precursors and energy required for growth, antioxidant production and cell wall remodeling. Moreover, metabolic adaptation can modulate the expression of virulence factors and immunogenicity. Thus, the metabolism impacts fungal pathogenicity through both virulence and fitness attributes and is indispensable for a fungal pathogen to colonize and infect a host successfully.
Fungi can infect multiple sites on the human body and cause both superficial and life-threatening infections. As one and a half million people are killed by pathogenic fungi every year, fungal pathogens are known as a “hidden killer” [3]. The most common culprits of human fungal infection come from four major fungal groups: Candida species, Cryptococcus neoformans, Aspergillus fumigatus and thermal dimorphic fungi [4]. Important human fungal pathogens belonging to the thermal dimorphic fungus group are Talaromyces marneffei, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Blastomyces dermatitidis and Sporothrix schenckii. During infection, the host’s innate immune cells, such as macrophages or neutrophils, commonly phagocytize and destroy the fungal cells by generating reactive oxygen species (ROS) and reactive nitrogen species (RNS) [5]. The host macrophages have been reported to produce up to 14 mM hydrogen peroxide and up to 57 uM nitric oxide in response to fungal infections [6,7][6][7]. To cope with these host-imposed stressors, fungal pathogens possess antioxidant defense systems with both enzymatic and non-enzymatic mechanisms [8]. In addition, several fungi can switch their morphology to protect themselves from the human immune system.
Morphological plasticity is one of the main virulence attributes in pathogenic fungi. Cell differentiation and development contribute to diverse morphological changes, including germination, conidiation, morphological switching between yeast and mold forms, and even autolysis. Fungal species generally undergo a morphological transformation during host colonization by responding to specific environmental cues. For example, thermal dimorphic fungi switch morphology from a multicellular mold in environmental niches to a yeast form in warm-blooded hosts due to temperature changes [9,10,11][9][10][11]. Another example is Candida albicans, which can be a commensal organism of the human microbiome while also being the most prevalent human fungal pathogen. At least nine distinct cell shapes have already been found in this species. C. albicans changes morphotypes when it inhabits different host niches or when it changes between being a commensal or pathogenic organism [12]. In A. fumigatus, a ubiquitous pathogenic mold, germination of conidia and hyphal growth occurs during an invasive infection of human lungs, while conidiation is strictly inhibited [13,14,15][13][14][15]. The dysregulation of these morphology pathways consistently attenuates the virulence of the pathogenic fungi in animal studies [15,16,17,18][15][16][17][18]. Overall, cell differentiation and development are critical for fungal morphogenesis and pathogenicity.

 

2. Glutathione Systems

Glutathione (L-γ-glutamylcysteinylglycine) is a crucial metabolite in eukaryotes and plays a major role in protecting cells against oxidative damage [19,20][19][20]. Glutathione directly scavenges diverse oxidants, such as superoxide anion, hydroxyl radical, nitric oxide and carbon radicals and is also a cofactor for various antioxidant enzymes, including glutathione peroxidases and glutathione S-transferase [21,22][21][22]. There are two states of glutathione in the cells: reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG) (Figure 1). Importantly, GSH is a major tissue antioxidant, while GSSG is accumulated when cells are exposed to increased levels of oxidative stress. Thus, increased ratios of GSSG to GSH are indicative of oxidative stress, and cells tightly maintain levels of reduced glutathione through the balance of its synthesis and reduction.
Ijms 23 10645 g001 550
Figure 1. The glutathione pathway in Saccharomyces cerevisiae. The synthesis of glutathione (GSH) is a two-step process catalyzed by the enzyme γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2) in the cytoplasm. GSH plays a crucial role in the protection of macromolecules from stressors, especially oxidants. The reduced form (GSH) directly scavenges diverse oxidants and xenobiotics via the action of glutathione peroxidase (GPX1-3) and glutathione S-transferase (GST) and becomes oxidized (GSSH). Regeneration of oxidized GSSG to reduced GSH is catalyzed by glutathione reductase (GLR1) (requires NADPH).
The GSH/GSSH pathway is composed of five enzymes, which will be the major focus of this review paper. The first two enzymes are involved with glutathione de novo biosynthesis via two ATP-dependent steps: the first step is catalyzed by γ-glutamylcysteine synthetase (GSH1), while the second step is catalyzed by glutathione synthetase (GSH2) (Figure 1). Next, glutathione peroxidase and glutathione S-transferase catalyze the production of GSSH. Glutathione peroxidase is a major enzyme in the defense against hydrogen peroxide (GPx: 2GSH + ROOH → GSSG + H2O + ROH, EC 1.11.1.9). Glutathione S-transferase is involved with the detoxification of many xenobiotic compounds by catalyzing the conjugation of substrates to GSH (GST: GSH + RX → GS-X + RH, EC 2.5.1.18), which can then be eliminated from the cells via glutathione conjugate pumps. Lastly, glutathione reductase catalyzes the regeneration of GSH (GSSG + NADPH + H+ → 2GSH + NADP+, EC 1.6.4.2).

3. Glutathione and Role in Oxidative Stress Protection

Molecular mechanisms elucidating how the glutathione system plays a crucial role in the fungal stress response are primarily obtained from model fungi, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans, or common human fungal pathogens, such as C. albicans, A. fumigatus and C. neoformans. In thermal dimorphic fungi, however, an understanding of the glutathione system is lacking because glutathione genetic studies have not been extensively explored, as seen in model fungi and other human fungal pathogens; the role of the glutathione system is mostly inferred from gene or protein expression analyses. Therefore, wresearchers summarized the experimentally verified functions of each homologous glutathione gene from diverse fungal species (Table 1). This summarized table will be beneficial in predicting the gene functions for other less studied fungi, such as thermal dimorphic fungi or other non-model fungi.

3.1. γ-Glutamylcysteine Synthetase and Glutathione Synthetase

The deletion of the genes encoding glutathione synthetic enzymes (GSH1 or GSH2 genes or their homologs) resulted in fungal mutants that were glutathione auxotrophs and had an increased sensitivity to various oxidants. Interestingly, the deletion of gshA in A. fumigatus also impaired cellular iron sensing, indicating crosstalk between glutathione biosynthesis and fungal iron homeostasis [23]. Likewise, the deletion of glutathione synthetase (GSH2) from C. neoformans resulted in glutathione auxotrophy under iron starvation-induced stress [24]. Indeed, glutathione is proposed to be involved with iron metabolism by its requirement in the Fe–S cluster assembly [25,26][25][26]. Importantly, the GSH1 and GSH2 genes are essential in diverse fungal species. A study in H. capsulatum reported that the deletion of the GSH1 or GSH2 genes caused non-viable mutants, indicating that both the GSH1 and GSH2 genes are essential. Consistent with the result from H. capsulatum, gshA (γ-glutamylcysteine synthetase gene) from Aspergillus oryzae was also reported to be an essential gene, while the role of the glutathione synthetase gene (GSH2 homolog) in cellular viability has not been characterized in this fungal species [27]. In Candida glabrata, GSH1, but not GSH2, was an essential gene. These results suggest that the role of the GSH1 and GSH2 genes differs from various species in cell viability, and glutathione synthesis has an essential role in iron homeostasis in many fungi.

3.2. Glutathione Reductase

In addition, glutathione reductase is required for resistance to oxidative stress because the deletion of the glutathione reductase gene commonly results in fungal mutants that are sensitive to various stressors. The details of the growth and stress response defects in deletion mutants are different according to the species. For example, in S. pombe yeast, glutathione reductase is indispensable for growth, as the pgr1Δ strain was not viable due to the accumulation of GSSG [39,60][28][29]. In S. cerevisiae and C. albicans, the grl1Δ mutant was viable and only showed growth defects under oxidative stress [37,38,40][30][31][32]. In C. neoformans, the gr1Δ mutant grew normally under normal conditions and was sensitive to only nitric oxide stress but not to peroxide stress [41][33]. In A. nidulans, the grlAΔ mutant exhibited growth defects, even under normal conditions, yet was still viable [42,61][34][35]. The grlAΔ strain of A. nidulans was also defective in its growth under high temperatures. Overall, these results suggest that glutathione reductase functions differently among fungal species.

3.3. Glutathione Peroxidase

Furthermore, glutathione peroxidase plays a crucial role in protecting fungi against oxidative stress since the absence of glutathione peroxidase gene(s) results in fungal strains that are not able to cope with various oxidants, especially peroxides. Nonetheless, distinct cellular responses to oxidative stress could be observed among fungal species. For example, the gpx3Δ mutants from S. cerevisiae and C. albicans were highly sensitive to H2O2, while the gpx1Δ and gpx2Δ mutants from C. neoformans were not sensitive to H2O2 but were sensitive to other peroxides. Furthermore, the gpx1Δ and gpx2Δ mutants from S. cerevisiae and C. albicans did not show any defects in response to oxidative stress, and hence, GPX3 is proposed to be the main gene encoding for glutathione peroxidase in these fungi. In T. marneffei, the glutathione peroxidase gene (gpx1; the homolog of the glutathione peroxidase HYR1 gene from S. cerevisiae) was isolated as one of the antigenic proteins [51][36]. The expression levels of the T. marneffei gpx1 gene were high in the pathogenic yeast form and were relatively unchanged in the conidia or mold forms. These results imply that glutathione peroxidase contributes to immunological response during T. marneffei infection and plays an important role in the pathogenic yeast phase. Collectively, these results suggest that glutathione peroxidase is required for the general oxidative stress defense mechanisms yet could respond distinctly to the different stressors, depending on the fungal species.

3.4. Glutathione S-Transferase

Glutathione S-transferase is involved in the resistance to xenobiotics because this enzyme can detoxify a broad range of harmful substances. In addition, glutathione S-transferase can also protect the cells against oxidative stress as it possesses GSH-dependent peroxidase activity. Accordingly, the glutathione S-transferase gene deletion mutants from a wide range of fungi became sensitive to both xenobiotics and various stressors. As seen in the case of other glutathione-related genes, there are differences in the glutathione S-transferase function among individual fungal species. In S. pombe, the gst1Δgst2Δ and gst3Δ mutants were sensitive to the antifungal drug fluconazole, suggesting the role of glutathione S-transferase in mediating drug resistance. In C. albicans, the GST2 gene was additionally induced under nitrogen starvation [56][37]. In S. cerevisiae, the gtt1Δ, gtt2Δ, and gtt1Δgtt2Δ mutants showed an increased sensitivity to heat shock or exhibited growth defects at high temperatures. In A. nidulans, the gstAΔ mutant was sensitive to heavy metals [57][38]. Taken together, glutathione S-transferase is an important enzyme that protects fungal cells from diverse types of substances and stressors.

References

  1. Brown, A. Integration of metabolism with virulence in Candida albicans. In Fungal Genomics; Springer: Berlin/Heidelberg, Germany, 2006; pp. 185–203.
  2. Brown, A.J.; Brown, G.D.; Netea, M.G.; Gow, N.A. Metabolism impacts upon Candida immunogenicity and pathogenicity at multiple levels. Trends Microbiol. 2014, 22, 614–622.
  3. Brown, G.D.; Denning, D.W.; Gow, N.A.; Levitz, S.M.; Netea, M.G.; White, T.C. Hidden killers: Human fungal infections. Sci. Transl. Med. 2012, 4, 165rv13.
  4. Kim, J.Y. Human fungal pathogens: Why should we learn? J. Microbiol. 2016, 54, 145–148.
  5. Gilbert, A.S.; Wheeler, R.T.; May, R.C. Fungal pathogens: Survival and replication within macrophages. Cold Spring Harb. Perspect. Med. 2014, 5, a019661.
  6. Gross, N.T.; Nessa, K.; Camner, P.; Jarstrand, C. Production of nitric oxide by rat alveolar macrophages stimulated by Cryptococcus neoformans or Aspergillus fumigatus. Med. Mycol. 1999, 37, 151–157.
  7. Hidalgo, H.A.; Helmke, R.J.; German, V.F.; Mangos, J.A. Pneumocystis carinii induces an oxidative burst in alveolar macrophages. Infect. Immun. 1992, 60, 1–7.
  8. Irato, P.; Santovito, G. Enzymatic and Non-Enzymatic Molecules with Antioxidant Function. Antioxidants 2021, 10, 579.
  9. Edwards, J.A.; Chen, C.; Kemski, M.M.; Hu, J.; Mitchell, T.K.; Rappleye, C.A. Histoplasma yeast and mycelial transcriptomes reveal pathogenic-phase and lineage-specific gene expression profiles. BMC Genom. 2013, 14, 695.
  10. Nemecek, J.C.; Wüthrich, M.; Klein, B.S. Global control of dimorphism and virulence in fungi. Science 2006, 312, 583–588.
  11. Beyhan, S.; Gutierrez, M.; Voorhies, M.; Sil, A. A temperature-responsive network links cell shape and virulence traits in a primary fungal pathogen. PLoS Biol. 2013, 11, e1001614.
  12. Noble, S.M.; Gianetti, B.A.; Witchley, J.N. Candida albicans cell-type switching and functional plasticity in the mammalian host. Nat. Rev. Microbiol. 2017, 15, 96–108.
  13. Tochigi, N.; Okubo, Y.; Ando, T.; Wakayama, M.; Shinozaki, M.; Gocho, K.; Hata, Y.; Ishiwatari, T.; Nemoto, T.; Shibuya, K. Histopathological implications of Aspergillus infection in lung. Mediat. Inflamm. 2013, 2013, 809798.
  14. Mah, J.H.; Yu, J.H. Upstream and downstream regulation of asexual development in Aspergillus fumigatus. Eukaryot. Cell 2006, 5, 1585–1595.
  15. Stewart, J.I.P.; Fava, V.M.; Kerkaert, J.D.; Subramanian, A.S.; Gravelat, F.N.; Lehoux, M.; Howell, P.L.; Cramer, R.A.; Sheppard, D.C. Reducing Aspergillus fumigatus virulence through targeted dysregulation of the conidiation pathway. mBio 2020, 11, e03202-19.
  16. Boyce, K.J.; Andrianopoulos, A. Fungal dimorphism: The switch from hyphae to yeast is a specialized morphogenetic adaptation allowing colonization of a host. FEMS Microbiol. Rev. 2015, 39, 797–811.
  17. Lo, H.J.; Köhler, J.R.; DiDomenico, B.; Loebenberg, D.; Cacciapuoti, A.; Fink, G.R. Nonfilamentous C. albicans mutants are avirulent. Cell 1997, 90, 939–949.
  18. Thompson, D.S.; Carlisle, P.L.; Kadosh, D. Coevolution of morphology and virulence in Candida species. Eukaryot. Cell 2011, 10, 1173–1182.
  19. Carlos, I.Z.; Silva Monnazzi, L.G.; Falcão, D.P.; Machado de Medeiros, B.M. TNF-alpha, H2O2 and NO response of peritoneal macrophages to Yersinia enterocolitica O:3 derivatives. Microbes Infect. 2004, 6, 207–212.
  20. Penninckx, M.J. An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res. 2002, 2, 295–305.
  21. Grant, C.M. Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions. Mol. Microbiol. 2001, 39, 533–541.
  22. Pizzorno, J. Glutathione! Integr. Med. (Encinitas) 2014, 13, 8–12.
  23. Misslinger, M.; Lechner, B.E.; Bacher, K.; Haas, H. Iron-sensing is governed by mitochondrial, not by cytosolic iron-sulfur cluster biogenesis in Aspergillus fumigatus. Metallomics 2018, 10, 1687–1700.
  24. Attarian, R. Analysis of the Roles of a Monothiol Glutaredoxin and Glutathione Synthetase in the Virulence of the AIDS-Associated Pathogen Cryptococcus neoformans. Ph.D. Thesis, University of British Columbia, Vancouver, BC, Canada, 2016.
  25. Kumar, C.; Igbaria, A.; D’Autreaux, B.; Planson, A.G.; Junot, C.; Godat, E.; Bachhawat, A.K.; Delaunay-Moisan, A.; Toledano, M.B. Glutathione revisited: A vital function in iron metabolism and ancillary role in thiol-redox control. EMBO J. 2011, 30, 2044–2056.
  26. Berndt, C.; Lillig, C.H. Glutathione, glutaredoxins, and iron. Antioxid. Redox Signal. 2017, 27, 1235–1251.
  27. Hattori, R.; Tada, S.; Matsushita-Morita, M.; Suzuki, S.; Kusumoto, K.-I. Gamma-glutamylcysteine synthetase gene homolog (gshA) isiImportant in glutathione homeostasis in Aspergillus oryzae. Jpn. Agric. Res.Q. JARQ 2018, 52, 301–305.
  28. Lee, J.; Dawes, I.W.; Roe, J.H. Isolation, expression, and regulation of the pgr1(+) gene encoding glutathione reductase absolutely required for the growth of Schizosaccharomyces pombe. J. Biol. Chem. 1997, 272, 23042–23049.
  29. Song, J.Y.; Cha, J.; Lee, J.; Roe, J.H. Glutathione reductase and a mitochondrial thioredoxin play overlapping roles in maintaining iron-sulfur enzymes in fission yeast. Eukaryot. Cell 2006, 5, 1857–1865.
  30. Collinson, L.P.; Dawes, I.W. Isolation, characterization and overexpression of the yeast gene, GLR1, encoding glutathione reductase. Gene 1995, 156, 123–127.
  31. Grant, C.M.; Collinson, L.P.; Roe, J.H.; Dawes, I.W. Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation. Mol. Microbiol. 1996, 21, 171–179.
  32. Tillmann, A.T.; Strijbis, K.; Cameron, G.; Radmaneshfar, E.; Thiel, M.; Munro, C.A.; MacCallum, D.M.; Distel, B.; Gow, N.A.; Brown, A.J. Contribution of Fdh3 and Glr1 to Glutathione Redox State, Stress adaptation and virulence in Candida albicans. PLoS ONE 2015, 10, e0126940.
  33. Missall, T.A.; Pusateri, M.E.; Donlin, M.J.; Chambers, K.T.; Corbett, J.A.; Lodge, J.K. Posttranslational, translational, and transcriptional responses to nitric oxide stress in Cryptococcus neoformans: Implications for virulence. Eukaryot. Cell 2006, 5, 518–529.
  34. Sato, I.; Shimizu, M.; Hoshino, T.; Takaya, N. The glutathione system of Aspergillus nidulans involves a fungus-specific glutathione S-transferase. J. Biol. Chem. 2009, 284, 8042–8053.
  35. Bakti, F.; Király, A.; Orosz, E.; Miskei, M.; Emri, T.; Leiter, É.; Pócsi, I. Study on the glutathione metabolism of the filamentous fungus Aspergillus nidulans. Acta Microbiol. Immunol. Hung. 2017, 64, 255–272.
  36. Pongpom, M.; Vanittanakom, N. Stress adaptation in Talaromyces marneffei. Chiang Mai Med. J. 2016, 55, 23–30.
  37. Lee, S.H.; Chung, S.C.; Shin, J.; Oh, K.B. GST2 is required for nitrogen starvation-induced filamentous growth in Candida albicans. J. Microbiol. Biotechnol. 2014, 24, 1207–1215.
  38. Fraser, J.A.; Davis, M.A.; Hynes, M.J. A gene from Aspergillus nidulans with similarity to URE2 of Saccharomyces cerevisiae encodes a glutathione S-transferase which contributes to heavy metal and xenobiotic resistance. Appl. Environ. Microbiol. 2002, 68, 2802–2808.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
Video Production Service
Feedback