title: Entamoeba histolytica: increase of enterotoxicity and of 53- and 75-kDa cysteine proteinases in a clone of higher virulence.
abstract: We compared the enterotoxicity and cysteine proteinases (CP) of the low-virulence Entamoeba histolytica HM1 strain with the highly virulent 1659 clone, derived from HM1 by hamster liver passages. Enterotoxicity of 50,000 freeze-thawed trophozoites was determined on 0.28-cm2 intestinal segments mounted in Ussing chambers; CP activity of Nonidet-P40 amebal lysates was assayed by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and carbobenzoxy-L-arginine-L-arginyl-p-nitroaniline, a CP-specific substrate. Treatment of gerbil cecum segments with amebal lysates caused an immediate fall of their electrophysiologic properties (potential difference, short-circuit current, and transmural resistance) whose decay rates were clearly faster with 1659 than with HM1 lysates. Nonimmune and immune antiamebic human sera and the CP-specific inhibitor E-64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane) prevented the fall of the electrophysiologic properties. Gelatinases, less active in HM1 than in 1659 trophozoites, were better preserved in lysates containing 10 mM p-hydroxymercuribenzoate (pHMB) to prevent autoproteolysis: in lysates without pHMB nearly no gelatinase bands were observed in HM1 samples, whereas intense 30K, 35K, 44K, and 75K bands were seen in 1659 samples; in lysates with pHMB only 53K and 75K bands were found that were much more intense in 1659 samples, 75K being barely visible in HM1 samples. The overall CP activity was 17 times higher in 1659 than in HM1 lysates, was inhibited by E-64 (mean inhibitory dose, 20 microM), was stimulated by 2-mercaptoethanol (ME) 3.7 times in HM1 and 2.4 times in 1659 lysates, and was reactivated by ME in lysates containing pHMB. Most of the CP activity in HM1 lysates sedimented at 15,600g but predominated in 1659 supernatants. The increase of E. histolytica virulence thus correlates with a remarkable increase both of in vitro enterotoxicity and of two CPs (53K and 75K), suggesting that these proteinases are significant pathogenicity factors.
authors.affiliation: Department of Cell Biology, CINVESTAV-IPN, Mexico DF, Mexico.
authors.forename: F
authors.initials: F
authors.lastname: Navarro-García
authors.forename: L
authors.initials: L
authors.lastname: Chávez-Dueñas
authors.forename: V
authors.initials: V
authors.lastname: Tsutsumi
authors.forename: F
authors.initials: F
authors.lastname: Posadas del Río
authors.forename: R
authors.initials: R
authors.lastname: López-Revilla
mesh_terms: D000818:Animals; D002432:Cecum / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004404:Dysentery, Amebic / Q000469:parasitology*; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D004748:Entamoeba histolytica / Q000201:enzymology / Q000378:metabolism / Q000472:pathogenicity*; D004768:Enterotoxins / Q000378:metabolism* / Q000633:toxicity; D018093:Gelatinases / Q000378:metabolism; D005849:Gerbillinae; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D066298:In Vitro Techniques; D008099:Liver / Q000473:pathology; D008101:Liver Abscess, Amebic / Q000276:immunology / Q000469:parasitology*; D008297:Male; D011817:Rabbits; D014774:Virulence
chemical_list: D004768:Enterotoxins; D007106:Immune Sera; D003546:Cysteine Endopeptidases; D018093:Gelatinases