1887

Microbiology Society logo

Volume 156, Issue 7

Hydrogen peroxide induces apoptosis-like death in trophozoites Free

Abstract

Programmed cell death (PCD) is an essential process in the growth and development of multicellular organisms. However, accumulating evidence indicates that unicellular eukaryotes can also undergo PCD with apoptosis-like features. This study demonstrates that after exposure to 0.8 mM HO for 9 h presents morphological and biochemical evidence of apoptosis-like death. Morphological characteristics of apoptosis-like death including DNA fragmentation, increased vacuolization, nuclear condensation and cell rounding were observed for HO-exposed trophozoites with preservation of membrane integrity. Biochemical alteration in ion fluxes is also a key feature in PCD, and HO-exposed trophozoites showed overproduction of reactive oxygen species, increased cytosolic Ca and decreased intracellular pH. Phosphatidylserine was also found to be expressed in the outer leaflet of the plasma membrane of the HO-treated trophozoites. Pretreatment with the cysteine protease inhibitor E-64d, the extracellular and intracellular Ca chelators EGTA and BAPTA/AM, and the Ca influx inhibitor verapamil prior to HO exposure abolished DNA fragmentation. The oxidatively stressed trophozoites also showed an increased calpain activity, indicating involvement of Ca-dependent calpain-like cysteine proteases in PCD of . A homogeneous caspase assay showed no significant caspase activity, and administration of caspase 1 inhibitor also did not prevent the death phenotype for the oxidatively stressed trophozoites, indicating a caspase-independent apoptosis-like death. Our observations clearly demonstrate that there is a distinct calpain-dependent but caspase-independent pathway for apoptosis-like death in oxidatively stressed trophozoites.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): [Ca2+]i, intracellular calcium , AMC, 7-amino-4-methylcoumarin, BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) , BCECF, 2,7-bis(2-carboxyethyl)-5-(and 6)-carboxyfluorescein , E-64d, (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester , eh29, thiol-dependent peroxidase , Fe-SOD, iron-containing superoxide dismutase , H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate , MDA, malondialdehyde , MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide , PCD, programmed cell death , PI, propidium iodide , PS, phosphatidylserine , ROS, reactive oxygen species , SOD, superoxide dismutase and TG, thapsigargin
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.034066-0
2010-07-01
2024-04-26
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/7/1926.html?itemId=/content/journal/micro/10.1099/mic.0.034066-0&mimeType=html&fmt=ahah

References

  1. Al-Olayan E. M., Williams G. T., Hurd H. 2002; Apoptosis in the malaria protozoan, Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito. Int J Parasitol 32:1133–1143
     [Google Scholar]
  2. Ameisen J. C. 1996; The origin of programmed cell death. Science 272:1278–1279
     [Google Scholar]
  3. Barbieri D., Troiano L., Grassilli E., Agnesini C., Cristofalo E. A., Monti D., Capri M., Cossarizza A., Franceschi C. 1992; Inhibition of apoptosis by zinc: a reappraisal. Biochem Biophys Res Commun 187:1256–1261
     [Google Scholar]
  4. Bruchhaus I., Tannich E. 1994; Induction of the iron-containing superxoide dismutase in Entamoeba histolytica by a superoxide anion-generating system or by iron chelation. Mol Biochem Parasitol 67:281–288
     [Google Scholar]
  5. Bruchhaus I., Richter S., Tannich E. 1997; Removal of hydrogen peroxide by the 29 kDa protein of Entamoeba histolytica. Biochem J 15:785–789
     [Google Scholar]
  6. Bruchhaus I., Richter S., Tannich E. 1998; Recombinant expression and biochemical characterization of an NADPH : flavin oxidoreductase from Entamoeba histolytica. Biochem J 330:1217–1221
     [Google Scholar]
  7. Bruchhaus I., Loftus B. J., Hall N., Tannich E. 2003; The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes, of which only a small subset is expressed during in vitro cultivation. Eukaryot Cell 2:501–509
     [Google Scholar]
  8. Chance B., Sies H., Boveris A. 1979; Hydroperoxide metabolism in mammalian organs. Physiol Rev 59:527–605
     [Google Scholar]
  9. Chávez Munguía B., Espinosa C. M., Castañón G., Martínez-Palomo A. 2000; Ultrastructural evidence of smooth endoplasmic reticulum and Golgi-like elements in Entamoeba histolytica and Entamoeba dispar. Arch Med Res 31:S165–S167
     [Google Scholar]
  10. Chose O., Noël C., Gerbod D., Brenner C., Viscogliosi E., Roseto A. 2002; A form of cell death with some features resembling apoptosis in the amitochondrial unicellular organism Trichomonas vaginalis. Exp Cell Res 276:32–39
     [Google Scholar]
  11. Chose O., Sarde C. O., Noel C., Gerbod D., Jimenez J. C., Brenner C., Capron M., Viscogliosi E., Roseto A. 2003; Cell death in protists without mitochondria. Ann N Y Acad Sci 1010:121–125
     [Google Scholar]
  12. Christensen S. T., Chemnitz J., Straarup E. M., Kristiansen K., Wheatley D. N., Rasmussenl L. 1998a; Staurosporine induced cell death in Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degeneration. Cell Biol Int 22:591–598
     [Google Scholar]
  13. Christensen S. T., Leick V., Rasmussen L., Wheatley D. N. 1998b; Signaling in unicellular eukaryotes. Int Rev Cytol 177:181–253
     [Google Scholar]
  14. Clark I. A., Hunt N. H., Cowden W. B. 1986; Oxygen derived free radicals in the pathogenesis of parasitic diseases. Adv Parasitol 25:1–73
     [Google Scholar]
  15. Clarke P. G. H. 1990; Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl 181:195–213
     [Google Scholar]
  16. Clement M. V., Pervaiz S. 1999; Reactive oxygen intermediates regulate cellular response to apoptotic stimuli: a hypothesis. Free Radic Res 30:247–252
     [Google Scholar]
  17. Collins R. J. B., Harmon V., Gobe G. C., Kerr J. F. R. 1992; Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int J Radiat Biol 61:451–453
     [Google Scholar]
  18. Cornillon S., Foa C., Davoust J., Buonavista N., Gross J. D., Golstein P. 1994; Programmed cell death in Dictyostelium. J Cell Sci 107:2691–2704
     [Google Scholar]
  19. Dale C., Welburn S. C., Maudlin I., Milligan P. J. M. 1995; The kinetics of maturation of trypanosome infections in tsetse. Parasitology 111:187–191
     [Google Scholar]
  20. Das M., Mukherjee S. B., Shaha C. 2001; Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes. J Cell Sci 114:2461–2469
     [Google Scholar]
  21. Debrabant A., Nakhasi H. 2003; Programmed cell death in trypanosomatids: is it an altruistic mechanism for survival of the fittest?. Kinetoplastid Biol Dis 2: 7
     [Google Scholar]
  22. Demaurex N., Frieden M., Arnaudeau S. 2003; ER calcium and ER chaperones: new players in apoptosis?. In Calreticulin, 2nd edn. pp 134–142 Edited by Eggleton P., Michalak M. Austin, TX: Eurekah;
     [Google Scholar]
  23. Diamond L. S., Harlow D. R., Cunnick C. C. 1978; A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72:431–432
     [Google Scholar]
  24. Duszenko M., Figarella K., Macleod E. T., Welburn S. C. 2006; Death of a trypanosome: a selfish altruism. Trends Parasitol 22:536–542
     [Google Scholar]
  25. El Hafidi M., Baños G. 1997; In vivo plasma lipid oxidation in sugar-induced rat hypertriglyceridemia and hypertension. Hypertension 30:624–628
     [Google Scholar]
  26. Evan G., Littlewood T. 1998; A matter of life and cell death. Science 281:1317–1322
     [Google Scholar]
  27. Facompre M., Goossens J. F., Bailly C. 2001; Apoptotic response of HL-60 human leukemia cells to the antitumor drug NB-506, a glycosylated indolocarbazole inhibitor of topoisomerase 1. Biochem Pharmacol 61:299–310
     [Google Scholar]
  28. Falcieri E., Martelli A. M., Bareggi R., Cataldi A., Cocco L. 1993; The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in MOLT-4 cells without concomitant DNA fragmentation. Biochem Biophys Res Commun 193:19–25
     [Google Scholar]
  29. Fraga C. G., Leibovitz B. E., Tappel A. L. 1987; Halogenated compounds as inducers of lipid peroxidation in tissue slices. Free Radic Biol Med 3:119–123
     [Google Scholar]
  30. Gatti R., Belletti S., Orlandini G., Bussolati O., Dall'Asta V., Gazzola G. C. 1998; Comparison of Annexin V and Calcein-AM as early vital markers of apoptosis in adherent cells by confocal laser microscopy. J Histochem Cytochem 46:895–900
     [Google Scholar]
  31. Ghadirian E., Somerfield S. D., Kongshavn P. A. L. 1986; Susceptibility of Entamoeba histolytica to oxidants. Infect Immun 51:263–267
     [Google Scholar]
  32. Ghosh A. S., Dutta S., Raha S. 2010; Hydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolytica. Parasitol Int Jan 15 (Epub ahead of print
    [Google Scholar]
  33. Greil R., Egle A., Villunger A. 1998; On the role and significance of Fas (Apo-1/CD95) ligand (FasL) expression in immune privileged tissues and cancer cells using multiple myeloma as a model. Leuk Lymphoma 31:477–490
     [Google Scholar]
  34. Grynkiewicz G., Poenie M., Tsien R. Y. 1985; A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
     [Google Scholar]
  35. Hesse F., Selzer P. M., Muhlstadt K., Duszenko M. 1995; A novel cultivation technique for long-term maintenance of bloodstream form trypanosomes in vitro. Mol Biochem Parasitol 70:157–166
     [Google Scholar]
  36. Hirata H., Hibasami H., Yoshida T., Morita A., Ohkaya S., Matsumoto M., Sasaki H., Uchida A. 1998; Differentiation and apoptosis without DNA fragmentation in cultured Schwann cells derived from wallerian-degenerated nerve. Apoptosis 3:353–360
     [Google Scholar]
  37. Howell D. M., Martz E. 1987; The degree of CTL-induced DNA solubilization is not determined by the human vs. mouse origin of the target cell. J Immunol 138:3695–3698
     [Google Scholar]
  38. Jacobson M. D., Weil M., Raff M. C. 1997; Programmed cell death in animal development. Cell 88:347–354
     [Google Scholar]
  39. Jiang S., Chow S. C., Nicotera P., Orrenius S. 1994; Intracellular Ca2+ signals activate apoptosis in thymocytes: studies using the Ca2+-ATPase inhibitor thapsigargin. Exp Cell Res 212:84–92
     [Google Scholar]
  40. Kohli V., Gao W., Camargo C. A., Clavien P. A. 1997; Calpain is a mediator of preservation reperfusion injury in rat liver transplantation. Proc Natl Acad Sci U S A 94:9354–9359
     [Google Scholar]
  41. Lee N., Bertholet S., Debrabant A., Muller J., Duncan R., Nakhasi H. L. 2002; Programmed cell death in the unicellular protozoan parasite Leishmania. Cell Death Differ 9:53–64
     [Google Scholar]
  42. Li J., Huang C. Y., Zheng R. L., Cui K. R., Li J. F. 2000; Hydrogen peroxide induces apoptosis in human hepatoma cells and alters cell redox status. Cell Biol Int 24:9–23
     [Google Scholar]
  43. Lipton S. A., Nicotera P. 1998; Calcium, free radicals and excitotoxins in neuronal apoptosis. Cell Calcium 23:165–171
     [Google Scholar]
  44. Liu X., Zou H., Slaughter C., Wang X. 1997; DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89:175–184
     [Google Scholar]
  45. Lo H., Reeves R. E. 1980; Purification and properties of NADPH : flavin oxidoreductase from Entamoeba histolytica. Mol Biochem Parasitol 2:23–30
     [Google Scholar]
  46. Madeo F., Frohlich E., Ligr M., Grey M., Sigrist S. J., Wolf D. H., Frohlich K. U. 1999; Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 145:757–767
     [Google Scholar]
  47. Mariante R. M., Guimaraes C. A., Linden R., Benchimol M. 2003; Hydrogen peroxide induces caspase activation and programmed cell death in the amitochondrial Tritrichomonas foetus. Histochem Cell Biol 120:129–141
     [Google Scholar]
  48. McCord J. M., Fridovich I. 1969; Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein. J Biol Chem 244:6049–6055
     [Google Scholar]
  49. Mesner P. W., Winters T. R., Green S. H. 1992; Nerve growth factor withdrawal-induced cell death in neuronal PC12 cells resembles that in sympathetic neurons. J Cell Biol 119:1669–1680
     [Google Scholar]
  50. Murray H. W., Aley S. B., Scott W. A. 1981; Susceptibility of Entamoeba histolytica to oxygen intermediates. Mol Biochem Parasitol 3:381–391
     [Google Scholar]
  51. Nasirudeen A. M., Yap E. H., Singh M., Kevin S. W. T. 2004; Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis. Microbiology 150:33–43
     [Google Scholar]
  52. Oberhammer F., Bursch W., Tiefenbacher R., Froschl G., Pavelka M., Purchio T., Schulte-Hermann R. 1993; Apoptosis is induced by transforming growth factor-beta 1 within 5 hours in regressing liver without significant fragmentation of the DNA. Hepatology 18:1238–1246
     [Google Scholar]
  53. Picot S., Burnod J., Bracchi V., Chumpitazi B. F., Ambroise-Thomas P. 1997; Apoptosis related to chloroquine sensitivity of the human malaria parasite Plasmodium falciparum. Trans R Soc Trop Med Hyg 91:590–591
     [Google Scholar]
  54. Ramos E., Olivos-Garcya A., Nequiz M., Saavedra E., Tello E., Saralegui A., Montfort I., Perez Tamayo R. 2007; Entamoeba histolytica: apoptosis induced in vitro by nitric oxide species. Exp Parasitol 116:257–265
     [Google Scholar]
  55. Ramos-Martínez E., Olivos-García A., Saavedra E., Nequiz M., Sánchez E. C., Tello E., El-Hafidi M., Saralegui A., Pineda E. other authors 2009; Entamoeba histolytica: oxygen resistance and virulence. Int J Parasitol 39:693–702
     [Google Scholar]
  56. Rao L., Perez D., White E. 1996; Lamin proteolysis facilitates nuclear events during apoptosis. J Cell Biol 135:1441–1455
     [Google Scholar]
  57. Ray S. K., Matzelle D. D., Wilford G. G., Hogan E. L., Banik N. L. 2001; Inhibition of calpain-mediated apoptosis by E-64 d reduced immediate early gene (IEG) expression and reactive astrogliosis in the lesion and penumbra following spinal cord injury in rats. Brain Res 916:115–126
     [Google Scholar]
  58. Robertson A. M. G., Thomson J. N. 1982; Morphology of programmed cell death in the ventral nerve cord of Caenorhabditis elegans larvae. J Embryol Exp Morphol 67:89–100
     [Google Scholar]
  59. Sahara S., Aoto M., Eguchi Y., Imamoto N., Moneda Y., Tsujimoto Y. 1999; Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation. Nature 401:168–173
     [Google Scholar]
  60. Sakahira H., Enari M., Nagata S. 1998; Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391:96–99
     [Google Scholar]
  61. Santi-Rocca J., Rigothier M. C., Guillen N. 2009; Host-microbe interactions and defense mechanisms in the development of amoebic liver abscesses. Clin Microbiol Rev 22:65–75
     [Google Scholar]
  62. Sen A., Chatterjee N. S., Akbar M. A., Nandi N., Das P. 2007; The 29-kilodalton thiol dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress. Eukaryot Cell 6:664–673
     [Google Scholar]
  63. Shaha C. 2006; Apoptosis in Leishmania species and its relevance to disease pathogenesis. Indian J Med Res 123:233–244
     [Google Scholar]
  64. Tagliarino C., Pink J. J., Dubyak G. R., Nieminen A. L., Boothman D. A. 2001; Calcium is a key signaling molecule in β-lapachone mediated cell death. J Biol Chem 276:19150–19159
     [Google Scholar]
  65. Thurman R. G., Ley H. G., Scholz R. 1972; Hepatic microsomal ethanol oxidation: hydrogen peroxide formation and the role of catalase. Eur J Biochem 25:420–430
     [Google Scholar]
  66. Tillack M., Biller L., Irmer H., Freitas M., Gomes M., Tannich E., Bruchhaus I. 2007; The Entamoeba histolytica genome: primary structure and expression of proteolytic enzymes. BMC Genomics 8:170
     [Google Scholar]
  67. Vardi A., Berman-Frank I., Rozenberg T., Hadas O., Kaplan A., Levine A. 1999; Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO2 limitation and oxidative stress. Curr Biol 9:1061–1064
     [Google Scholar]
  68. Vaux D. L., Haecker G., Strasser A. 1994; An evolutionary perspective on apoptosis. Cell 76:777–779
     [Google Scholar]
  69. Verma N. K., Dey S. C. 2004; Possible mechanism of miltefosine mediated death of Leishmania donovani. Antimicrob Agents Chemother 48:3010–3015
     [Google Scholar]
  70. Villalba J. D. A., Gomez C., Medel O., Sanchez V., Carrero J. C., Shibayama M., Ishiwara D. G. P. 2007; Programmed cell death in Entamoeba histolytica induced by the aminoglycoside G418. Microbiology 153:3852–3863
     [Google Scholar]
  71. Vollgraf U., Wegner M., Richter-Landsberg C. 1999; Activation of AP-1 and nuclear factor-kappa B transcription factor is involved in hydrogen peroxide induced apoptotic cell death of oligodendrocytes. J Neurochem 73:2501–2509
     [Google Scholar]
  72. Wanderley J. L., Benjamin A., Real F., Bonomo A., Moreira M. E., Barcinski M. A. 2005; Apoptotic mimicry: an altruistic behavior in host/ Leishmania interplay. Braz J Med Biol Res 38:807–812
     [Google Scholar]
  73. Welburn S. C. 1996; Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro. Cell Death Differ 3:229–236
     [Google Scholar]
  74. Welburn S. C., Barcinski M. A., Williams G. T. 1997; Programmed cell death in trypanosomatids. Parasitol Today 13:22–26
     [Google Scholar]
  75. WHO 1998 The World Health Report 1998 Life in the 21st Century: a Vision for All Geneva, Switzerland: World Health Organization;
  76. Wyllie A. H., Kerr J. F. R., Currie A. R. 1980; Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306
     [Google Scholar]
  77. Zhang Z., Wang L., Seydel K. B., Li E., Ankri S., Mirelman D., Stanley S. L. Jr 2000; Entamoeba histolytica cysteine proteinases with interleukin-1 beta converting enzyme (ICE) activity cause intestinal inflammation and tissue damage in amoebiasis. Mol Microbiol 37:542–548
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.034066-0
Loading
Hydrogen peroxide induces apoptosis-like death in Entamoeba histolytica trophozoites
Microbiology 156, 1926 (2010); https://doi.org/10.1099/mic.0.034066-0
/content/journal/micro/10.1099/mic.0.034066-0
/content/journal/micro/10.1099/mic.0.034066-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error