title: Luminal bacteria and proteases together decrease adherence of Entamoeba histolytica trophozoites to Chinese hamster ovary epithelial cells: a novel host defence against an enteric pathogen.
abstract: BACKGROUND
  Factors that prevent colonic mucosal invasion by pathogenic Entamoeba histolytica are not understood. A key initial step in pathogenesis of injury induced by amoeba is adherence to target cells mediated by a surface glycoprotein lectin on E histolytica. Mucin degrading bacteria normally present in the colon lumen produce glycosidases that degrade soluble or cell surface glycoconjugates.
  
  
  OBJECTIVE
  To determine whether glycosidases produced by mucin degrading bacteria, alone or in combination with proteases present in colon lumen, can decrease E histolytica adherence to target epithelial cells by degrading E histolytica adherence lectin.
  
  
  METHODS
  The effects of exposure of E histolytica trophozoites strains HM1:IMSS and 200:NIH to faecal culture supernatant fluids, culture supernatant preparations of mucin degrading bacteria, and luminal proteases on their adherence to Chinese hamster ovary (CHO) cells were determined. The amount of surface adherence lectin on E histolytica trophozoites before and after treatment with glycosidases and proteases was determined by immunofluorescence. The effect of glycosidases and proteases on purified E histolytica lectin was determined by gel electrophoresis.
  
  
  RESULTS
  Incubation of E histolytica with culture supernatant preparations or proteases alone did not modify their CHO cell adherence. However, 24 hour incubation of trophozoites with culture supernatant preparations together with pancreatic proteases decreased CHO cell adherence of HM1:IMSS strain by 71.1% (p < 0.001) and of 200: NIH strain by 95% (p < 0.05). Incubation of trophozoites for 24 hours with faecal extracts which contain bacterial and host hydrolases decreased the adherence of the HM1:IMSS strain by 69.2% (p < 0.01) and of the 200: NIH strain by 83.0%. Reduction of trophozoite adherence to CHO cells by hydrolases was promoted by 7.5 mM cycloheximide, and was reversible on incubation in an enzyme free medium. Decrease in CHO cell adherence of trophozoites was associated with decreased lectin on trophozoites as determined by immunofluorescence using a monoclonal antibody to the lectin. Purified lectin was degraded by the mixture of faecal culture supernant preparations and proteases, but not by either alone.
  
  
  CONCLUSIONS
  Mucin degrading bacterial glycosidases and colonic luminal proteases together, but not alone, degrade the key adherence lectin on E histolytica trophozoites resulting in decreased epithelial cell adherence. These in vitro findings suggest a potential novel host defence mechanism in the human colon wherein the invasiveness of a pathogen could be curtailed by the combined actions of bacterial and host hydrolases. This mechanism may be responsible for preventing mucosal invasion by pathogenic E histolytica.
authors.affiliation: Department of Medicine, Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106, USA.
authors.forename: E P
authors.initials: EP
authors.lastname: Variyam
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001419:Bacteria / Q000378:metabolism; D016466:CHO Cells; D002448:Cell Adhesion / Q000187:drug effects; D003106:Colon / Q000382:microbiology / Q000469:parasitology*; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004748:Entamoeba histolytica / Q000502:physiology*; D004749:Entamoebiasis / Q000517:prevention & control*; D005455:Fluorescent Antibody Technique; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006790:Host-Parasite Interactions; D037102:Lectins / Q000032:analysis / Q000378:metabolism; D009077:Mucins / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D037102:Lectins; D009077:Mucins; D003513:Cycloheximide; D006026:Glycoside Hydrolases; D010450:Endopeptidases