Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1993 Apr;91(4):1532–1540. doi: 10.1172/JCI116359

Cloning of a virulence factor of Entamoeba histolytica. Pathogenic strains possess a unique cysteine proteinase gene.

S Reed 1, J Bouvier 1, A S Pollack 1, J C Engel 1, M Brown 1, K Hirata 1, X Que 1, A Eakin 1, P Hagblom 1, F Gillin 1, et al.
PMCID: PMC288129  PMID: 8473498

Abstract

Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined.

Full text

PDF
1539

Images in this article

1534Image
on p.1534
1534Image
on p.1534
1534Image
on p.1534
1535Image
on p.1535
1535Image
on p.1535
1535Image
on p.1535
1536Image
on p.1536
1536Image
on p.1536
1537Image
on p.1537
1537Image
on p.1537
1537Image
on p.1537

Selected References

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

  1. Barrett A. J., Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. doi: 10.1016/s0076-6879(81)80043-2. [DOI] [PubMed] [Google Scholar]
  2. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  3. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Diamond L. S., Harlow D. R., Cunnick C. C. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg. 1978;72(4):431–432. doi: 10.1016/0035-9203(78)90144-x. [DOI] [PubMed] [Google Scholar]
  5. Eakin A. E., Bouvier J., Sakanari J. A., Craik C. S., McKerrow J. H. Amplification and sequencing of genomic DNA fragments encoding cysteine proteases from protozoan parasites. Mol Biochem Parasitol. 1990 Feb;39(1):1–8. doi: 10.1016/0166-6851(90)90002-4. [DOI] [PubMed] [Google Scholar]
  6. Eakin A. E., Mills A. A., Harth G., McKerrow J. H., Craik C. S. The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi. J Biol Chem. 1992 Apr 15;267(11):7411–7420. [PubMed] [Google Scholar]
  7. Edman U., Meraz M. A., Rausser S., Agabian N., Meza I. Characterization of an immuno-dominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica. J Exp Med. 1990 Sep 1;172(3):879–888. doi: 10.1084/jem.172.3.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edman U., Meza I., Agabian N. Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica. Proc Natl Acad Sci U S A. 1987 May;84(9):3024–3028. doi: 10.1073/pnas.84.9.3024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garfinkel L. I., Giladi M., Huber M., Gitler C., Mirelman D., Revel M., Rozenblatt S. DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect Immun. 1989 Mar;57(3):926–931. doi: 10.1128/iai.57.3.926-931.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gitler C., Calef E., Rosenberg I. Cytopathogenicity of Entamoeba histolytica. Philos Trans R Soc Lond B Biol Sci. 1984 Nov 13;307(1131):73–85. doi: 10.1098/rstb.1984.0110. [DOI] [PubMed] [Google Scholar]
  11. Huber M., Garfinkel L., Gitler C., Mirelman D., Revel M., Rozenblatt S. Entamoeba histolytica: cloning and characterization of actin cDNA. Mol Biochem Parasitol. 1987 Jul;24(3):227–235. doi: 10.1016/0166-6851(87)90154-x. [DOI] [PubMed] [Google Scholar]
  12. Keene W. E., Hidalgo M. E., Orozco E., McKerrow J. H. Entamoeba histolytica: correlation of the cytopathic effect of virulent trophozoites with secretion of a cysteine proteinase. Exp Parasitol. 1990 Aug;71(2):199–206. doi: 10.1016/0014-4894(90)90022-5. [DOI] [PubMed] [Google Scholar]
  13. Keene W. E., Petitt M. G., Allen S., McKerrow J. H. The major neutral proteinase of Entamoeba histolytica. J Exp Med. 1986 Mar 1;163(3):536–549. doi: 10.1084/jem.163.3.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leippe M., Ebel S., Schoenberger O. L., Horstmann R. D., Müller-Eberhard H. J. Pore-forming peptide of pathogenic Entamoeba histolytica. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7659–7663. doi: 10.1073/pnas.88.17.7659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Luaces A. L., Barrett A. J. Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica. Biochem J. 1988 Mar 15;250(3):903–909. doi: 10.1042/bj2500903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  17. Mirelman D., Bracha R., Wexler A., Chayen A. Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization. Infect Immun. 1986 Dec;54(3):827–832. doi: 10.1128/iai.54.3.827-832.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mort J. S., Leduc M., Recklies A. D. A latent thiol proteinase from ascitic fluid of patients with neoplasia. Biochim Biophys Acta. 1981 Dec 15;662(2):173–180. doi: 10.1016/0005-2744(81)90027-9. [DOI] [PubMed] [Google Scholar]
  19. Orozco E. Pathogenesis in amebiasis. Infect Agents Dis. 1992 Feb;1(1):19–21. [PubMed] [Google Scholar]
  20. 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]
  21. Que X., Reed S. L. Nucleotide sequence of a small subunit ribosomal RNA (16S-like rRNA) gene from Entamoeba histolytica: differentiation of pathogenic from nonpathogenic isolates. Nucleic Acids Res. 1991 Oct 11;19(19):5438–5438. doi: 10.1093/nar/19.19.5438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Reed S. L., Keene W. E., McKerrow J. H., Gigli I. Cleavage of C3 by a neutral cysteine proteinase of Entamoeba histolytica. J Immunol. 1989 Jul 1;143(1):189–195. [PubMed] [Google Scholar]
  24. Reed S. L., Keene W. E., McKerrow J. H. Thiol proteinase expression and pathogenicity of Entamoeba histolytica. J Clin Microbiol. 1989 Dec;27(12):2772–2777. doi: 10.1128/jcm.27.12.2772-2777.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Robinson G. L. The laboratory diagnosis of human parasitic amoebae. Trans R Soc Trop Med Hyg. 1968;62(2):285–294. doi: 10.1016/0035-9203(68)90170-3. [DOI] [PubMed] [Google Scholar]
  26. Sargeaunt P. G. The reliability of Entamoeba histolytica zymodemes in clinical diagnosis. Parasitol Today. 1987 Feb;3(2):40–37. doi: 10.1016/0169-4758(87)90211-0. [DOI] [PubMed] [Google Scholar]
  27. Sargeaunt P. G., Williams J. E., Grene J. D. The differentiation of invasive and non-invasive Entamoeba histolytica by isoenzyme electrophoresis. Trans R Soc Trop Med Hyg. 1978;72(5):519–521. doi: 10.1016/0035-9203(78)90174-8. [DOI] [PubMed] [Google Scholar]
  28. Scholze H., Werries E. Cysteine proteinase of Entamoeba histolytica. I. Partial purification and action on different enzymes. Mol Biochem Parasitol. 1986 Jan;18(1):103–112. doi: 10.1016/0166-6851(86)90054-x. [DOI] [PubMed] [Google Scholar]
  29. Sloane B. F., Dunn J. R., Honn K. V. Lysosomal cathepsin B: correlation with metastatic potential. Science. 1981 Jun 5;212(4499):1151–1153. doi: 10.1126/science.7233209. [DOI] [PubMed] [Google Scholar]
  30. Tannich E., Horstmann R. D., Knobloch J., Arnold H. H. Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5118–5122. doi: 10.1073/pnas.86.13.5118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tannich E., Scholze H., Nickel R., Horstmann R. D. Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression. J Biol Chem. 1991 Mar 15;266(8):4798–4803. [PubMed] [Google Scholar]
  32. Torian B. E., Flores B. M., Stroeher V. L., Hagen F. S., Stamm W. E. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6358–6362. doi: 10.1073/pnas.87.16.6358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Troen B. R., Ascherman D., Atlas D., Gottesman M. M. Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation. J Biol Chem. 1988 Jan 5;263(1):254–261. [PubMed] [Google Scholar]
  34. 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]
  35. de Meester F., Shaw E., Scholze H., Stolarsky T., Mirelman D. Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica. Infect Immun. 1990 May;58(5):1396–1401. doi: 10.1128/iai.58.5.1396-1401.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES