Entamoeba histolytica forms
chitin-walled
cysts during encystation process, where formation of the
cyst wall needs not only
chitin synthase but also
chitinase. During excystation, quadruplet amoebae emerge from the
chitin-walled
cysts by dissolving the wall, so that
chitinase may be necessary for excystation process as well. There is, however, no report on
chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied
chitinase mRNA expression in those processes. Although expression of three E. invadens
chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another
enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and
mRNA expression of these four
chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four
chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four
chitinases sharply decreased in the later phase. In
cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation,
mRNA levels of EiChit1 and EiChit4 in
cysts 5 h after induction were significantly lower than those in
cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of
cytochalasin D. These data demonstrate different structures and expressions of four
chitinases in the differentiation of E. invadens.