title: Antisense inhibition of Entamoeba histolytica cysteine proteases inhibits colonic mucus degradation.
abstract: OBJECTIVE
  The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells.
  
  
  METHODS
  Cysteine protease-deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [(35)S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier.
  
  
  RESULTS
  Trophozoites with reduced cysteine protease activity were ineffective at degrading [(35)S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease-deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier.
  
  
  CONCLUSIONS
  These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis.
authors.affiliation: Institute of Parasitology of McGill University, Quebec, Canada.
authors.forename: Darcym
authors.initials: D
authors.lastname: Moncada
authors.forename: Kathy
authors.initials: K
authors.lastname: Keller
authors.forename: Serge
authors.initials: S
authors.lastname: Ankri
authors.forename: David
authors.initials: D
authors.lastname: Mirelman
authors.forename: Kris
authors.initials: K
authors.lastname: Chadee
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003106:Colon / Q000378:metabolism*; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004748:Entamoeba histolytica / Q000201:enzymology*; D006801:Humans; D009077:Mucins / Q000378:metabolism*; D016372:RNA, Antisense / Q000494:pharmacology*; D014162:Transfection
chemical_list: D015853:Cysteine Proteinase Inhibitors; D009077:Mucins; D016372:RNA, Antisense; D003546:Cysteine Endopeptidases