Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens
Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens
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DOI：
10.1007/s00436-009-1478-x
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发表时间：
2009-10-01
影响因子：
2
通讯作者：
Takeuchi, Tsutomu
中科院分区：
文献类型：
--
作者：
Makioka, Asao;Kumagai, Masahiro;Takeuchi, Tsutomu
虽然半胱氨酸蛋白酶参与溶组织内阿米巴的致病和分化的功能已被证实，但丝氨酸蛋白酶的功能知之甚少。我们使用丝氨酸蛋白酶特异性抑制剂研究了丝氨酸蛋白酶在阿米巴脱囊和后囊发育中的作用。以侵袭内阿米巴IP-1株作为E.溶组织剂四种丝氨酸蛋白酶抑制剂，苯甲磺酰氟（PMSF），4-（2-氨乙基）苯磺酰氟盐酸盐，3，4-二氯异香豆素，和N-甲苯磺酰基-苯氯甲基酮，以剂量依赖的方式减少后囊阿米巴原虫的数量，没有显示出对包囊的细胞毒性。PMSF抑制不仅增加，但也确定了从四核到单核阿米巴的细胞核数目的变化后囊阿米巴的发展。PMSF还能抑制胞囊裂解液中的蛋白酶活性，使蛋白酶在明胶-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的条带减弱。从大肠杆菌数据库中检索到3个丝氨酸蛋白酶家族S28（3种）、S9（2种）和S26（1种）。入侵。系统发育分析表明，来自丝氨酸蛋白酶家族的阿米巴酶形成了与其他生物不同的分支。这些丝氨酸蛋白酶在包囊中的表达水平5小时后，通过实时逆转录聚合酶链反应（RT-PCR）评估的诱导excystation高于观察到的诱导前通过实时RT-PCR测定;增加一种类型的S9（命名为S9-3）的表达是最高的。从包囊到滋养体，S9酶的表达也比其他家族的丝氨酸蛋白酶高。因此，结果表明，内阿米巴利用其丝氨酸蛋白酶在脱囊和后囊发育，这导致成功的感染。
Although the functions of cysteine proteases involved in the pathogenicity and differentiation of Entamoeba histolytica have been demonstrated, little is known about the functions of serine proteases. We examined the involvement of serine proteases in amoebic excystation and metacystic development using inhibitors specific for serine proteases. Entamoeba invadens IP-1 strain was used as the model of excystation and metacystic development of E. histolytica. Four serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl) bezensulfonylfluoride hydrochloride, 3, 4-dichloroisocoumarin, and N-tosyl-phe-chloromethylketone, decreased the number of metacystic amoebae in a dose-dependent manner, without showing cytotoxicity to cysts. PMSF inhibited not only the increase but also the development of metacystic amoebae as determined by the change of nucleus number from four- to one-nucleate amoebae. The protease activity in cyst lysates was also inhibited by PMSF and the band of protease on gelatin sodium dodecyl sulfate polyacrylamide gel electrophoresis was weaker than controls when treated with PMSF. Three serine protease families, S28 (three types), S9 (two), and S26 (one) were retrieved from the database of E. invadens. Phylogenetic analysis revealed that amebic enzymes from the serine protease families formed different clades from those from other organisms. The expression levels of these serine proteases in cysts 5 h after the induction of excystation as assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) were higher than those observed prior to induction assayed by real-time RT-PCR; the increase in one type of S9 (named S9-3) expression was the highest. The expression of S9 enzymes also increased from cysts to trophozoites higher than the other family serine proteases. Thus, the results show that Entamoeba uses their serine proteases in the excystation and metacystic development, which leads to successful infection.