Proteolysis of enteric cell villin by Entamoeba histolytica cysteine proteinases.
Invasive microorganisms efface enteric microvilli to establish intimate contact with the apical surface of enterocytes. To understand the molecular basis of this effacement in amebic colitis, we seeded Entamoeba histolytica trophozoites on top of differentiated human Caco-2 cell layers. Western blots of detergent lysates from such cocultures showed proteolysis of the actin-bundling protein villin within 1 min of direct contact of living trophozoites with enterocytes. Mixtures of separately prepared lysates excluded detergent colysis as the cause of villin proteolysis. Caspases were not responsible as evidenced by the lack of degradation of specific substrates and the failure of a specific caspase inhibitor to prevent villin proteolysis. A crucial role for amebic cysteine proteinases was shown by prevention of villin proteolysis and associated microvillar alterations through the treatment of trophozoites before coculture with synthetic inhibitors that completely blocked amebic cysteine proteinase activity on zymograms. Moreover, trophozoites of amebic strains pSA8 and SAW760 with strongly reduced cysteine proteinase activity showed a reduced proteolysis of villin in coculture with enteric cells. Salmonella typhimurium and enteropathogenic Escherichia coli disturb microvilli without villin proteolysis, indicating that the latter is not a consequence of the disturbance of microvilli. In conclusion, villin proteolysis is an early event in the molecular cross-talk between enterocytes and amebic trophozoites, causing a disturbance of microvilli.
Lauwaet
Tineke
T
Laboratory of Experimental Cancerology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium.
Oliveira
Maria José
MJ
Callewaert
Bert
B
De Bruyne
Georges
G
Saelens
Xavier
X
Ankri
Serge
S
Vandenabeele
Peter
P
Mirelman
David
D
Mareel
Marc
M
Leroy
Ancy
A
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002448:Cell Adhesion; D018920:Coculture Techniques; D015179:Colorectal Neoplasms; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D007413:Intestinal Mucosa / Q000469:parasitology*; D008840:Microfilament Proteins / Q000378:metabolism*; D008871:Microvilli / Q000469:parasitology*; D014407:Tumor Cells, Cultured; D014508:Urea / Q000378:metabolism
D002352:Carrier Proteins; D008840:Microfilament Proteins; C026615:villin; D014508:Urea; D003546:Cysteine Endopeptidases