Navarro-García F, Chávez-Dueñas L, Tsutsumi V, Posadas del Río F, López-Revilla R
Department of Cell Biology, CINVESTAV-IPN, Mexico DF, Mexico.
Exp Parasitol. 1995 May;80(3):361-72. doi: 10.1006/expr.1995.1048.
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.
我们将低毒力的溶组织内阿米巴HM1株与通过仓鼠肝传代从HM1衍生而来的高毒力1659克隆的肠毒性和半胱氨酸蛋白酶（CP）进行了比较。在安装于尤斯灌流小室中的0.28平方厘米肠段上测定50,000个冻融滋养体的肠毒性；通过明胶 - 十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和一种CP特异性底物苄氧羰基 - L - 精氨酸 - L - 精氨酰 - 对硝基苯胺测定Nonidet - P40变形虫裂解物的CP活性。用变形虫裂解物处理沙鼠盲肠段会导致其电生理特性（电位差、短路电流和跨膜电阻）立即下降，1659裂解物导致的下降速率明显快于HM1裂解物。非免疫和免疫抗阿米巴人血清以及CP特异性抑制剂E - 64（反式环氧琥珀酰 - L - 亮氨酰胺基（4 - 胍基）丁烷）可防止电生理特性下降。在HM1中活性低于1659滋养体的明胶酶，在含有10 mM对羟基汞苯甲酸（pHMB）的裂解物中保存得更好，以防止自身蛋白水解：在不含pHMB的裂解物中，HM1样品中几乎未观察到明胶酶条带，而在1659样品中可见强烈的30K、35K、44K和75K条带；在含有pHMB的裂解物中，仅发现53K和75K条带，1659样品中的条带强度更高，75K条带在HM1样品中几乎不可见。1659裂解物中的总体CP活性比HM1裂解物高17倍，被E - 64抑制（平均抑制剂量，20 microM），在HM1裂解物中被2 - 巯基乙醇（ME）刺激3.7倍，在1659裂解物中被刺激2.4倍，并且在含有pHMB的裂解物中被ME重新激活。HM1裂解物中的大多数CP活性在15,600g下沉淀，但在1659上清液中占主导。因此，溶组织内阿米巴毒力增加与体外肠毒性以及两种CP（53K和75K）的显著增加相关，表明这些蛋白酶是重要的致病因素。
