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

Asao Makioka, Masahiro Kumagai, Seiki Kobayashi, Tsutomu Takeuchi

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)977-987
Number of pages11
JournalParasitology Research
Volume105
Issue number4
DOIs
Publication statusPublished - 2009 Oct

Fingerprint

Entamoeba
excystation
Serine Proteases
serine proteinases
Phenylmethylsulfonyl Fluoride
Amoeba
Cysts
fluorides
Entamoeba histolytica
Serine Proteinase Inhibitors
Reverse Transcriptase Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Peptide Hydrolases
quantitative polymerase chain reaction
Trophozoites
proteinases
Cysteine Proteases
reverse transcriptase polymerase chain reaction
Enzymes
Gelatin

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

Cite this

Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens. / Makioka, Asao; Kumagai, Masahiro; Kobayashi, Seiki; Takeuchi, Tsutomu.

In: Parasitology Research, Vol. 105, No. 4, 10.2009, p. 977-987.

Research output: Contribution to journalArticle

Makioka, Asao ; Kumagai, Masahiro ; Kobayashi, Seiki ; Takeuchi, Tsutomu. / Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens. In: Parasitology Research. 2009 ; Vol. 105, No. 4. pp. 977-987.
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