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Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens

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An Erratum to this article was published on 16 July 2009

Abstract

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.

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Acknowledgments

We thank N. Watanabe and J. Watanabe for their valuable discussions with us, L. S. Diamond for supplying the E. invadens, K. Hiranuka and T. Katayama for their search of databases, and T. Yamashita and T. Tadano for their technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports and Technology of Japan.

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Authors and Affiliations

  1. Department of Tropical Medicine, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 105-8461, Japan

    Asao Makioka & Masahiro Kumagai

  2. Department of Tropical Medicine and Parasitology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan

    Seiki Kobayashi & Tsutomu Takeuchi

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  1. Asao Makioka
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  2. Masahiro Kumagai
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  3. Seiki Kobayashi
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  4. Tsutomu Takeuchi
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Correspondence to Asao Makioka.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s00436-009-1564-0

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Makioka, A., Kumagai, M., Kobayashi, S. et al. Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens . Parasitol Res 105, 977–987 (2009). https://doi.org/10.1007/s00436-009-1478-x

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