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. 1988 Jun;56(6):1485–1491. doi: 10.1128/iai.56.6.1485-1491.1988

Phorbol esters specifically enhance the cytolytic activity of Entamoeba histolytica.

C S Weikel 1, C F Murphy 1, E Orozco 1, J I Ravdin 1
PMCID: PMC259425  PMID: 2897334

Abstract

Entamoeba histolytica causes invasive amebiasis by lysis of host tissue and inflammatory cells. The in vitro cytolysis of target Chinese hamster ovary (CHO) cells by axenic E. histolytica trophozoites (strain HM1:IMSS) is a calcium- and phospholipase A-dependent event initiated by the binding to the target cell of the galactose-inhibitable surface lectin of the parasite. We utilized phorbol esters as a probe to determine whether an amebic protein kinase C has a role in the cytolytic event. The addition of phorbol 12-myristate 13-acetate (PMA) at 10(-6) or 10(-7) M resulted in a greater than twofold enhancement of amebic killing of target CHO cells over 30 min (P less than 0.01). Prior exposure of only the amebae, but not the CHO cells, to PMA produced a similar effect (P less than 0.01). The inactive analog 4-alpha-phorbol had no effect on amebic killing of CHO cells. The PMA-mediated enhancement of amebic cytolysis persisted for up to 60 min after a 5-min exposure; however, after a 30-min exposure to PMA (10(-6) M) there was no augmentation of amebic killing of CHO cells. PMA (10(-6) M) did not promote adherence of parasites to CHO cells but did enhance amebic cytolysis of previously adherent target cells (P less than 0.01). Sphingosine, a specific inhibitor of protein kinase C, abolished both the PMA-stimulated and the basal cytolytic activity of E. histolytica. PMA enhanced CHO cell cytolysis by the less virulent wild-type strain H-303:NIH (P less than or equal to 0.02) but did not augment the activity of the less virulent strain H-200:NIH or two avirulent clones of HM1 (L6 and C919). In summary, these experiments with the phorbol esters and sphingosine as probes to modulate the activity of protein kinase C indicate participation of a parasite protein kinase C in the cytolytic activity of virulent, axenic E. histolytica trophozoites and thus in the pathogenesis of amebiasis.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aley S. B., Scott W. A., Cohn Z. A. Plasma membrane of Entamoeba histolytica. J Exp Med. 1980 Aug 1;152(2):391–404. doi: 10.1084/jem.152.2.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blumberg P. M. In vitro studies on the mode of action of the phorbol esters, potent tumor promoters: part 1. Crit Rev Toxicol. 1980 Dec;8(2):153–197. doi: 10.3109/10408448009037493. [DOI] [PubMed] [Google Scholar]
  3. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  4. Chadee K., Petri W. A., Jr, Innes D. J., Ravdin J. I. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica. J Clin Invest. 1987 Nov;80(5):1245–1254. doi: 10.1172/JCI113199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dawson R. M., Irvine R. F., Bray J., Quinn P. J. Long-chain unsaturated diacylglycerols cause a perturbation in the structure of phospholipid bilayers rendering them susceptible to phospholipase attack. Biochem Biophys Res Commun. 1984 Dec 14;125(2):836–842. doi: 10.1016/0006-291x(84)90615-6. [DOI] [PubMed] [Google Scholar]
  6. Garrison J. C., Johnsen D. E., Campanile C. P. Evidence for the role of phosphorylase kinase, protein kinase C, and other Ca2+-sensitive protein kinases in the response of hepatocytes to angiotensin II and vasopressin. J Biol Chem. 1984 Mar 10;259(5):3283–3292. [PubMed] [Google Scholar]
  7. Guerrant R. L., Brush J., Ravdin J. I., Sullivan J. A., Mandell G. L. Interaction between Entamoeba histolytica and human polymorphonuclear neutrophils. J Infect Dis. 1981 Jan;143(1):83–93. doi: 10.1093/infdis/143.1.83. [DOI] [PubMed] [Google Scholar]
  8. Hannun Y. A., Loomis C. R., Merrill A. H., Jr, Bell R. M. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J Biol Chem. 1986 Sep 25;261(27):12604–12609. [PubMed] [Google Scholar]
  9. Hansson A., Serhan C. N., Haeggström J., Ingelman-Sundberg M., Samuelsson B. Activation of protein kinase C by lipoxin A and other eicosanoids. Intracellular action of oxygenation products of arachidonic acid. Biochem Biophys Res Commun. 1986 Feb 13;134(3):1215–1222. doi: 10.1016/0006-291x(86)90380-3. [DOI] [PubMed] [Google Scholar]
  10. Kiss Z., Steinberg R. A. Phorbol ester-mediated protein phosphorylations in S49 mouse lymphoma cells. Cancer Res. 1985 Jun;45(6):2732–2740. [PubMed] [Google Scholar]
  11. Kuo J. F., Andersson R. G., Wise B. C., Mackerlova L., Salomonsson I., Brackett N. L., Katoh N., Shoji M., Wrenn R. W. Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7039–7043. doi: 10.1073/pnas.77.12.7039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Long-Krug S. A., Fischer K. J., Hysmith R. M., Ravdin J. I. Phospholipase A enzymes of Entamoeba histolytica: description and subcellular localization. J Infect Dis. 1985 Sep;152(3):536–541. doi: 10.1093/infdis/152.3.536. [DOI] [PubMed] [Google Scholar]
  13. Lynch E. C., Rosenberg I. M., Gitler C. An ion-channel forming protein produced by Entamoeba histolytica. EMBO J. 1982;1(7):801–804. doi: 10.1002/j.1460-2075.1982.tb01250.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mattern C. F., Keister D. B., Caspar P. A. Experimental amebiasis. III. A rapid in vitro assay for virulence of Entamoeba histolytica. Am J Trop Med Hyg. 1978 Sep;27(5):882–887. [PubMed] [Google Scholar]
  15. Mattern C. F., Keister D. B. Experimental amebiasis. II. Hepatic amebiasis in the newborn hamster. Am J Trop Med Hyg. 1977 May;26(3):402–411. [PubMed] [Google Scholar]
  16. McPhail L. C., Clayton C. C., Snyderman R. A potential second messenger role for unsaturated fatty acids: activation of Ca2+-dependent protein kinase. Science. 1984 May 11;224(4649):622–625. doi: 10.1126/science.6231726. [DOI] [PubMed] [Google Scholar]
  17. Merrill A. H., Jr, Sereni A. M., Stevens V. L., Hannun Y. A., Bell R. M., Kinkade J. M., Jr Inhibition of phorbol ester-dependent differentiation of human promyelocytic leukemic (HL-60) cells by sphinganine and other long-chain bases. J Biol Chem. 1986 Sep 25;261(27):12610–12615. [PubMed] [Google Scholar]
  18. Murakami K., Chan S. Y., Routtenberg A. Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids. J Biol Chem. 1986 Nov 25;261(33):15424–15429. [PubMed] [Google Scholar]
  19. Niedel J. E., Kuhn L. J., Vandenbark G. R. Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci U S A. 1983 Jan;80(1):36–40. doi: 10.1073/pnas.80.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  21. O'Brien T. G., Diamond L. A cell culture bioassay to analyze metabolism of phorbol diester tumor promoters. Cancer Res. 1978 Aug;38(8):2567–2572. [PubMed] [Google Scholar]
  22. Orozco E., Guarneros G., Martinez-Palomo A., Sánchez T. Entamoeba histolytica. Phagocytosis as a virulence factor. J Exp Med. 1983 Nov 1;158(5):1511–1521. doi: 10.1084/jem.158.5.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Orozco E., Rodriquez M. A., Murphy C. F., Salata R. A., Petri W. A., Smith R. D., Ravdin J. I. Entamoeba histolytica: cytopathogenicity and lectin activity of avirulent mutants. Exp Parasitol. 1987 Apr;63(2):157–165. doi: 10.1016/0014-4894(87)90157-3. [DOI] [PubMed] [Google Scholar]
  24. Petri W. A., Jr, Smith R. D., Schlesinger P. H., Murphy C. F., Ravdin J. I. Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica. J Clin Invest. 1987 Nov;80(5):1238–1244. doi: 10.1172/JCI113198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ravdin J. I., Croft B. Y., Guerrant R. L. Cytopathogenic mechanisms of Entamoeba histolytica. J Exp Med. 1980 Aug 1;152(2):377–390. doi: 10.1084/jem.152.2.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ravdin J. I., Guerrant R. L. Role of adherence in cytopathogenic mechanisms of Entamoeba histolytica. Study with mammalian tissue culture cells and human erythrocytes. J Clin Invest. 1981 Nov;68(5):1305–1313. doi: 10.1172/JCI110377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ravdin J. I., John J. E., Johnston L. I., Innes D. J., Guerrant R. L. Adherence of Entamoeba histolytica trophozoites to rat and human colonic mucosa. Infect Immun. 1985 May;48(2):292–297. doi: 10.1128/iai.48.2.292-297.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ravdin J. I., Murphy C. F., Guerrant R. L., Long-Krug S. A. Effect of antagonists of calcium and phospholipase A on the cytopathogenicity of Entamoeba histolytica. J Infect Dis. 1985 Sep;152(3):542–549. doi: 10.1093/infdis/152.3.542. [DOI] [PubMed] [Google Scholar]
  29. Ravdin J. I., Murphy C. F., Salata R. A., Guerrant R. L., Hewlett E. L. N-Acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica. I. Partial purification and relation to amoebic virulence in vitro. J Infect Dis. 1985 May;151(5):804–815. doi: 10.1093/infdis/151.5.804. [DOI] [PubMed] [Google Scholar]
  30. Ravdin J. I., Murphy C. F., Schlesinger P. H. The cellular regulation of vesicle exocytosis by Entamoeba histolytica. J Protozool. 1988 Feb;35(1):159–163. doi: 10.1111/j.1550-7408.1988.tb04096.x. [DOI] [PubMed] [Google Scholar]
  31. Ravdin J. I. Pathogenesis of disease caused by Entamoeba histolytica: studies of adherence, secreted toxins, and contact-dependent cytolysis. Rev Infect Dis. 1986 Mar-Apr;8(2):247–260. doi: 10.1093/clinids/8.2.247. [DOI] [PubMed] [Google Scholar]
  32. Ravdin J. I., Schlesinger P. H., Murphy C. F., Gluzman I. Y., Krogstad D. J. Acid intracellular vesicles and the cytolysis of mammalian target cells by Entamoeba histolytica trophozoites. J Protozool. 1986 Nov;33(4):478–486. doi: 10.1111/j.1550-7408.1986.tb05646.x. [DOI] [PubMed] [Google Scholar]
  33. Ravdin J. I., Sperelakis N., Guerrant R. L. Effect of ion channel inhibitors on the cytopathogenicity of Entamoeba histolytica. J Infect Dis. 1982 Sep;146(3):335–340. doi: 10.1093/infdis/146.3.335. [DOI] [PubMed] [Google Scholar]
  34. Rodríguez M. A., Orozco E. Isolation and characterization of phagocytosis- and virulence-deficient mutants of Entamoeba histolytica. J Infect Dis. 1986 Jul;154(1):27–32. doi: 10.1093/infdis/154.1.27. [DOI] [PubMed] [Google Scholar]
  35. Salata R. A., Ravdin J. I. The interaction of human neutrophils and Entamoeba histolytica increases cytopathogenicity for liver cell monolayers. J Infect Dis. 1986 Jul;154(1):19–26. doi: 10.1093/infdis/154.1.19. [DOI] [PubMed] [Google Scholar]
  36. Sando J. J., Young M. C. Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells: requirement for calcium, magnesium, and phospholipids. Proc Natl Acad Sci U S A. 1983 May;80(9):2642–2646. doi: 10.1073/pnas.80.9.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shoyab M., Warren T. C., Todaro G. J. Phorbol-12,13-diester 12-ester hydrolase may prevent tumour promotion by phorbol diesters in skin. Nature. 1982 Jan 14;295(5845):152–154. doi: 10.1038/295152a0. [DOI] [PubMed] [Google Scholar]
  38. Weikel C. S., Sando J. J., Guerrant R. L. Stimulation of porcine jejunal ion secretion in vivo by protein kinase-C activators. J Clin Invest. 1985 Dec;76(6):2430–2435. doi: 10.1172/JCI112258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wilson E., Olcott M. C., Bell R. M., Merrill A. H., Jr, Lambeth J. D. Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst. J Biol Chem. 1986 Sep 25;261(27):12616–12623. [PubMed] [Google Scholar]
  40. Young J. D., Young T. M., Lu L. P., Unkeless J. C., Cohn Z. A. Characterization of a membrane pore-forming protein from Entamoeba histolytica. J Exp Med. 1982 Dec 1;156(6):1677–1690. doi: 10.1084/jem.156.6.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]

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