Advertisement
  • Loading metrics

Ferroptosis and microbial pathogenesis

Ferroptosis and microbial pathogenesis

  • Qing Shen, 
  • Naweed I. Naqvi
PLOS
x

Introduction

Precisely controlled cell death plays a key role in development and disease in eukaryotes. Ferroptosis is a newly defined form of iron-dependent cell death best known for its role in tumor suppression in mammalian cells [13]. To date, ferroptosis or ferroptosis-like cell death has been observed in pathogen-challenged rice and tobacco leaves [46], heat-stressed Arabidopsis roots [7], and in the sleeping sickness causal parasite Trypanosoma brucei [8], but not in any microbial system (e.g., bacteria, archaea, or fungi) as yet. Recently, the occurrence of ferroptosis was confirmed during pathogenic development in the rice-blast fungus Magnaporthe oryzae, and the contribution of such regulated cell death to virulence of this rice pathogen was highlighted [5]. Collectively, these findings set forth a new area of research in microbial pathogenesis and molecular host–microbe interactions; and suggest novel strategies for pathogen control based on modulating ferroptotic death and/or iron homeostasis.

What is ferroptosis?

Ferroptosis is a regulated mode of cell demise driven by iron-dependent peroxidation of membrane lipids [13,9]. Such cell death can be induced upon failure of the lipid peroxide reducing system that involves glutathione peroxidase 4 (GPX4) [10,11], which enzymatically converts phospholipid hydroperoxides to nontoxic lipid alcohols using glutathione (GSH) as a cosubstrate [12]. Accordingly, either GSH depletion caused by buthionine sulfoximine (BSO) or Erastin treatment, or GPX4 inactivation through gene deletion or pharmacological inhibition, leads to lethal accumulation of lipid peroxides in cellular membranes and results in strong induction of ferroptosis [10,11]. Such death, however, can be blocked by lipophilic antioxidants such as liproxstatin-1 (Lip-1) and ferrostatin-1 (Fer-1), which were selected from chemico-genetic screens and established as specific inhibitors of ferroptosis [10,13]. Like exogenous lipophilic antioxidants, endogenous ones such as the reduced Coenzyme Q10 (CoQ10) and Tetrahydrobiopterin (BH4) are capable of potently suppressing ferroptosis as membrane radical-trappers, and thus contribute to a GPX4-independent lipid peroxide detoxification mechanism [1416].

In addition to accumulation of lethal levels of lipid peroxides, ferroptosis is characterized by its strict iron dependency as well. Ferroptotic death can be prevented through iron chelation [13], whereas iron supplementation enhances or directly activates such type of cell mortality [13,17]. Ferroptosis sensitivity is modulated by the import, storage, and export of iron [18,19]; and iron uptake via the transferrin receptor serves as a specific marker for ferroptosis [20]. Furthermore, initiation of ferroptosis is enabled by iron-dependent metabolic enzymes, such as NADPH oxidase (Nox) and lipoxygenase, that lead to peroxidation of membrane lipids [1,13]. For instance, the membrane spanning Nox enzymes have 2 iron-containing hemes, which are noncovalently bound to it and participate in the electron transfer from NADPH to O2 and the consequent superoxide production [21]. Superoxides generated subsequently interact with membrane lipids and trigger cell death once GPX4 and the requisite antioxidant systems are inactivated.

What function does ferroptosis serve in M. oryzae development?

Ferroptosis contributes to the programmed cell death of the 3-celled asexual spores, also known as conidia, in M. oryzae (Fig 1) [5], which causes blast disease in several important cereal crops such as rice and wheat, and adversely impacts global agriculture [22]. Pathogenic life cycle of M. oryzae starts when the 3-celled conidium germinates on the leaf surface, and produces a polarized germ tube, which forms a specialized infection structure called the appressorium, which breaches the leaf epidermis using enormous turgor and a thin but rigid penetration peg [22]. Bulbous fungal hyphae differentiated from penetration pegs then resume filamentous growth within the plant cells and finally kill them and produce more conidia using the host-derived nutrients [22]. During appressorium maturation, the 3 conidial cells transport their cellular contents to the developing appressorium and then degrade their nuclei and undergo a specific autophagic cell death [22,23].

Such programmed death can be suppressed by iron chelators or the ferroptosis inhibitor Lip-1, and this death suppression is invariably accompanied by a dramatic decrease in lipid peroxide levels in the plasma membrane [5]. Lack of Nox activity through genetic or pharmacological inhibition simulates iron chelation or Lip-1–based inhibition in terms of suppressing conidial death and the associated lipid peroxidation [5]. Conversely, iron supplementation or GSH depletion via BSO drives peroxidation of membrane lipids and advances conidial ferroptosis [5]. These typical characteristics confirmed the occurrence of ferroptosis in the 3 conidial cells in a highly controlled and sequential manner: Ferroptosis initiates first in the terminal conidial cell distal to the appressorium, and then sequentially spreads to the middle and proximal cells (Fig 1) [5]. Such precise execution of conidial death may be attributable to the wave-like nature of ferroptosis propagation [24]. In mammalian cell populations or tissues, ferroptosis spreads as a wave in response to iron supplementation or GSH depletion, but not GPX4 inactivation [24,25]. Such unique propagation suggests a cell–cell communication that delivers ferroptosis trigger(s), which is supported by the dynamic spread of conidial ferroptosis in M. oryzae too. The abundance of iron increases considerably within the terminal conidial cell before it undergoes ferroptosis [5]. Such iron accumulation followed by cell death appears subsequently in the middle and proximal cells following the same chronology or sequence of conidial death [5], thus implying iron as a propagation trigger that fine-tunes and controls the crucial conidial ferroptosis in rice blast.

thumbnail
Fig 1. Ferroptosis occurs sequentially in the 3-celled conidium and is essential for pathogenesis in Magnaporthe oryzae.

In M. oryzae, ferroptosis initiates first in the terminal conidial cell distal to the infection structure (appressorium). Within this cell, lipid peroxides are generated via the iron-dependent NADPH oxidase activity that accrues in the plasma membrane and trigger cell death as assessed by nuclear and cellular degradation. The reduced GSH-dependent GPX function acts as a negative regulator of such death-inducing lipid peroxides. Ferroptosis subsequently spreads to the middle and proximal conidial cells and the germ tube prior to appressorium maturation. The ferroptosis-enabling iron is acquired from intracellular source(s) in M. oryzae and is transported via autophagy, although the nature of such iron source is still unclear. The ferroptosis inhibitor Lip-1 is a lipophilic antioxidant that acts as a potent suppressor of conidial ferroptosis in rice blast. Nuclei and lipid peroxides are indicated as blue circles and orange dashes, respectively, in the conidial cell(s) undergoing ferroptosis. GPX, glutathione peroxidase; GSH, glutathione; Lip-1, liproxstatin-1; ROS, reactive oxygen species.

https://doi.org/10.1371/journal.ppat.1009298.g001

What is the role of ferroptosis/iron homeostasis in microbial pathogenesis?

Regarding this question, what is known is that ferroptotic conidial death during appressorium development determines proper pathogenesis of M. oryzae [5]. When ferroptosis is subverted in the conidium through iron chelation or Lip-1–based inhibition or Nox inactivation, M. oryzae is unable to colonize rice cells and fails to cause the typical blast disease lesions. In contrast, an additional supply of iron boosts the conidial cell death and also increases the ability of M. oryzae to infect the rice plants.

So how does conidial ferroptosis impact the infection ability of M. oryzae? One possibility could be that successful ferroptosis occurs within a limited time period/window, when the nutrients stored in the conidium can still support its life activities, and guarantees proper development of the infection structure/appressorium. This is supported by the observation that smaller or immature appressoria (unable to penetrate the host by implication) are inevitably produced upon disruption of conidial ferroptosis [5]. Further investigation is required to unveil the link between conidial ferroptosis and the proper morphogenesis or formation of a functional appressorium.

To date, it is unclear whether ferroptosis occurs in microbial pathogens other than M. oryzae. However, the ability to take up iron and maintain iron homeostasis is essential for full virulence of an array of microbial pathogens, with hosts ranging from plants to humans [26,27]. For example, the human pathogenic fungus Candida albicans employs a high-affinity iron permease system [28] and utilizes siderophores produced by other microbes for iron acquisition [2931]. C. albicans also uses a series of transporters that deliver heme-iron across the cell wall, and then takes it up through endocytosis, to ensure proper iron uptake during growth and colonization within the host, thus enabling a strong infection capability [31]. In line with the iron requirement, Nox function and lipoxygenase activities, which are sources of lipid peroxidation in mammals, have been reported in fungi and bacteria too [3234]. Thus, it will be interesting to investigate whether iron-dependent ferroptosis serves as an evolutionary conserved mechanism widely involved in microbial pathogenesis.

What is the source of iron in ferroptosis?

The availability of iron from external host-derived sources or growth medium is extremely limited during pathogenic differentiation prior to host penetration in M. oryzae. As such, it is most likely the internally stored iron that enables and supports ferroptosis in the rice blast fungus. Presently, the nature of such internal source(s) and the type of iron involved is unclear. In mammalian cells, internal iron is stored as ferric ion in the form of ferritin complexes, and a selective form of autophagy referred to as ferritinophagy is responsible for ferritin degradation, thus releasing free iron for ferroptosis [35]. Although, M. oryzae lacks ferritin-like complexes, autophagy is still involved in fine-tuning ferroptosis likely through the trafficking and/or distribution of iron (Fig 1) [5]. It remains to be seen whether such regulation of intracellular ferric ions is indicative of the potential intrinsic stores (endoplasmic reticulum, mitochondria, and/or vacuoles) and sinks for iron in microbial pathogens that affect plants and animals. A key issue that needs to be resolved in the near future is what triggers and executes the release of ferroptosis-enabling iron in the pathogen. Lastly, it would be interesting to address whether the host plays a role in directly or indirectly regulating ferroptosis in the microbial pathogen, for instance, by modulating iron availability at the crucial stages of pathogenic development therein.

Acknowledgments

We thank the Fungal Pathobiology Group (Temasek Life Sciences Laboratory, Singapore), and Yi Zhen Deng (South China Agricultural University) for helpful discussions and suggestions. Our apologies to the authors whose ferroptosis-related research could not be cited here due to space constraints.

References

  1. 1. Stockwell BR, Jiang X. The Chemistry and Biology of Ferroptosis. Cell Chemical Biology. 2020;27(4):365–75. pmid:32294465
  2. 2. Zou Y, Schreiber SL. Progress in Understanding Ferroptosis and Challenges in Its Targeting for Therapeutic Benefit. Cell Chemical Biology. 2020;27(4):463–71. pmid:32302583
  3. 3. Anandhan A, Dodson M, Schmidlin CJ, Liu P, Zhang DD. Breakdown of an Ironclad Defense System: The Critical Role of NRF2 in Mediating Ferroptosis. Cell Chemical Biology. 2020;27(4):436–47. pmid:32275864
  4. 4. Dangol S, Chen Y, Hwang BK, Jwa N-S. Iron- and Reactive Oxygen Species-Dependent Ferroptotic Cell Death in Rice-Magnaporthe oryzae Interactions. Plant Cell. 2019;31(1):189–209. pmid:30563847
  5. 5. Shen Q, Liang M, Yang F, Deng YZ, Naqvi NI. Ferroptosis contributes to developmental cell death in rice blast. New Phytol. 2020;227(6):1831–46. pmid:32367535
  6. 6. Macharia M, Das PP, Naqvi NI, Wong S-M. iTRAQ-based quantitative proteomics reveals a ferroptosis-like programmed cell death in plants infected by a highly virulent tobacco mosaic virus mutant 24A+UPD. Phytopathology Research. 2020;2 (1):1.
  7. 7. Distéfano AM, Martin MV, Córdoba JP, Bellido AM, D’Ippólito S, Colman SL, et al. Heat stress induces ferroptosis-like cell death in plants. J Cell Biol 2017;216(2):463–76. pmid:28100685
  8. 8. Bogacz M, Krauth-Siegel RL. Tryparedoxin peroxidase-deficiency commits trypanosomes to ferroptosis-type cell death. Elife. 2018;7:e37503. pmid:30047863
  9. 9. Armenta DA, Dixon SJ. Investigating Nonapoptotic Cell Death Using Chemical Biology Approaches. Cell Chemical Biology. 2020;27(4):376–86. pmid:32220334
  10. 10. Friedmann Angeli JP, Schneider M, Proneth B, Tyurina YY, Tyurin VA, Hammond VJ, et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol. 2014;16(12):1180–91. pmid:25402683
  11. 11. Yang Wan S, SriRamaratnam R, Welsch Matthew E, Shimada K, Skouta R, Viswanathan Vasanthi S, et al. Regulation of Ferroptotic Cancer Cell Death by GPX4. Cell. 2014;156(1):317–31. pmid:24439385
  12. 12. Ursini F, Maiorino M, Valente M, Ferri L, Gregolin C. Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides. Biochimica et Biophysica Acta (BBA)—Lipids and Lipid Metabolism. 1982;710(2):197–211. pmid:7066358
  13. 13. Dixon Scott J, Lemberg Kathryn M, Lamprecht Michael R, Skouta R, Zaitsev Eleina M, Gleason Caroline E, et al. Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death. Cell. 2012;149(5):1060–72. pmid:22632970
  14. 14. Bersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature. 2019;575(7784):688–92. pmid:31634900
  15. 15. Doll S, Freitas FP, Shah R, Aldrovandi M, da Silva MC, Ingold I, et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature. 2019;575(7784):693–8. pmid:31634899
  16. 16. Kraft VAN, Bezjian CT, Pfeiffer S, Ringelstetter L, Müller C, Zandkarimi F, et al. GTP Cyclohydrolase 1/Tetrahydrobiopterin Counteract Ferroptosis through Lipid Remodeling. ACS Central Science. 2020;6(1):41–53. pmid:31989025
  17. 17. Huang K-J, Wei Y-H, Chiu Y-C, Wu S-R, Shieh D-B. Assessment of zero-valent iron-based nanotherapeutics for ferroptosis induction and resensitization strategy in cancer cells. Biomater Sci. 2019;7(4):1311–22. pmid:30734774
  18. 18. Stockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 2017;171(2):273–85. pmid:28985560
  19. 19. Brown CW, Amante JJ, Chhoy P, Elaimy AL, Liu H, Zhu LJ, et al. Prominin2 Drives Ferroptosis Resistance by Stimulating Iron Export. Dev Cell. 2019;51(5):575–86.e4. pmid:31735663
  20. 20. Feng H, Schorpp K, Jin J, Yozwiak CE, Hoffstrom BG, Decker AM, et al. Transferrin Receptor Is a Specific Ferroptosis Marker. Cell Rep. 2020;30(10):3411–23.e7. pmid:32160546
  21. 21. Magnani F, Mattevi A. Structure and mechanisms of ROS generation by NADPH oxidases. Curr Opin Struct Biol. 2019;59:91–7. pmid:31051297
  22. 22. Fernandez J, Orth K. Rise of a Cereal Killer: The Biology of Magnaporthe oryzae Biotrophic Growth. Trends Microbiol. 2018;26(7):582–97. pmid:29395728
  23. 23. Veneault-Fourrey C, Barooah M, Egan M, Wakley G, Talbot NJ. Autophagic Fungal Cell Death Is Necessary for Infection by the Rice Blast Fungus. Science. 2006;312(5773):580–3. pmid:16645096
  24. 24. Riegman M, Sagie L, Galed C, Levin T, Steinberg N, Dixon SJ, et al. Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture. Nat Cell Biol. 2020;22(9):1042–8. pmid:32868903
  25. 25. Linkermann A, Skouta R, Himmerkus N, Mulay SR, Dewitz C, De Zen F, et al. Synchronized renal tubular cell death involves ferroptosis. Proc Natl Acad Sci. 2014;111(47):16836–41. pmid:25385600
  26. 26. Cassat James E, Skaar EP. Iron in Infection and Immunity. Cell Host Microbe. 2013;13(5):509–19. pmid:23684303
  27. 27. Verbon EH, Trapet PL, Stringlis IA, Kruijs S, Bakker PAHM, Pieterse CMJ. Iron and Immunity. Annu Rev Phytopathol. 2017;55(1):355–75. pmid:28598721
  28. 28. Ramanan N, Wang Y. A High-Affinity Iron Permease Essential for Candida albicans Virulence. Science. 2000;288(5468):1062–4. pmid:10807578
  29. 29. Ardon O, Bussey H, Philpott C, Ward DM, Davis-Kaplan S, Verroneau S, et al. Identification of a Candida albicans Ferrichrome Transporter and Its Characterization by Expression in Saccharomyces cerevisiae. J Biol Chem. 2001;276(46):43049–55. pmid:11562378
  30. 30. Heymann P, Gerads M, Schaller M, Dromer F, Winkelmann G, Ernst JF. The Siderophore Iron Transporter of Candida albicans (Sit1p/Arn1p) Mediates Uptake of Ferrichrome-Type Siderophores and Is Required for Epithelial Invasion. Infect Immun. 2002;70 (9):5246–55. pmid:12183576
  31. 31. Fourie R, Kuloyo OO, Mochochoko BM, Albertyn J, Pohl CH. Iron at the Centre of Candida albicans Interactions. Front Cell Infect Microbiol. 2018;8(185). pmid:29922600
  32. 32. Horn T, Adel S, Schumann R, Sur S, Kakularam KR, Polamarasetty A, et al. Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling. Prog Lipid Res. 2015;57:13–39. pmid:25435097
  33. 33. Bedard K, Lardy B, Krause K-H. NOX family NADPH oxidases: Not just in mammals. Biochimie. 2007;89(9):1107–12. pmid:17400358
  34. 34. Hajjar C, Cherrier MV, Dias Mirandela G, Petit-Hartlein I, Stasia MJ. Fontecilla-Camps JC, et al. The NOX Family of Proteins Is Also Present in Bacteria mBio. 2017;8(6):e01487–17. pmid:29114025
  35. 35. Liu J, Kuang F, Kroemer G, Klionsky DJ, Kang R, Tang D. Autophagy-Dependent Ferroptosis: Machinery and Regulation. Cell Chemical Biology. 2020;27(4):420–35. pmid:32160513