Expression analysis of Entamoeba invadens profilins in encystation and excystation

Abstract

Cell motility by actin cytoskeleton is essential for differentiation processes of excystation and encystation of Entamoeba. We recently studied an actin depolymerizing factor (ADF)/cofilin (Cfl) of Entamoeba invadens (Ei), and demonstrated its contribution to the encystation and excystation of E. invadens through actin cytoskeletal reorganization. Profilin is also an actin-binding protein but its function is different from that of Cfl in actin assembly. This study investigated E. invadens profilins in relation to encystation and excystation which were induced in axenic culture systems. A homology search of the E. invadens genome database and molecular cloning identified four profilins of the parasite named EiPFN1, EiPFN2, EiPFN3, and EiPFN4. There were also multiple genes of profilin in Entamoeba histolytica (Eh) and Entamoeba dispar (Ed), each of which had three profilins. A search for conserved domains revealed that these profilins of Entamoeba had actin, phosphoinositide, and poly-proline binding sites. Phylogenetic analysis revealed that EiPFN3 and EiPFN4 formed the same clades including EhPFN3 and EdPFN3, and EhPFN2 and EdPFN2, respectively, while EiPFN1 and EiPFN2 were separated from EhPFN1 and EdPFN1. Rabbit anti-EiPFN1 serum reacted with recombinant EiPFN3 and EiPFN4 but not EiPFN2, and also reacted with EiPFN in lysates of cysts and trophozoites. Immunofluorescence staining with this antiserum showed co-localization of EiPFN with actin beneath the cell membrane through the life stages and also showed cytoplasmic localization. Both proteins proved to be rich in pseudopodia of trophozoites. Real-time RT-PCR showed that the mRNA level of EiPFN1 and EiPFN4 in trophozoites was comparable but that of EiPFN2 and EiPFN3 was very low. During encystation, the mRNA expression of EiPFN1 and EiPFN4 increased remarkably in the early phase much higher than that of EiPFN2 and EiPFN3. Then, the expression of all four PFNs sharply decreased in the later phase. This was in contrast to the sharp decrease in the mRNA level of EiCfl-2 during encystation in our previous study. In cysts, EiPFN1 was most abundantly expressed and EiPFN4 was at a lower level, while the expressions of EiPFN2 and EiPFN3 were virtually absent. Following the induction of excystation, mRNA levels of EiPFN1, EiPFN2, and EiPFN4 in cysts 5 h after induction were significantly higher than those in cysts before induction, while that of EiPFN3 was slightly higher than before induction. The mRNAs of EiPFN1 increased most extensively when the excystation was induced in the presence of cytochalasin D. Small interfering RNA (siRNA) to EiPFN1 inhibited both encystation and excystation but not growth. These findings demonstrate different expression of EiPFNs and the contribution of EiPFN to the encystation and excystation.

Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

“…However, other proteins that appeared to be important in E. invadens development were not detected in our study. For example, the E. histolytica homologue of one of the cysteine proteases EiCP-B9 [35] , or profilins (such as EiPFN1 and EiPFN4; [36] ) that are expressed in the cyst stage of E. invadens , were not detected in our proteome study. These discrepancies could be due to the fact that we were only able to identify a fraction of all cyst-specific proteins.…”

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

“…The expression levels of profilin (an actin‐binding protein) (Makioka et al. ), actophorin (a depolymerization factor of actin) and generation factor (a polymerization factor of actin) (Bouyer et al. ) were reported to be regulated in the encystment process of A. castellanii or E. invadens .…”

“…lamblia (Castillo-Romero et al 2009). The expression levels of profilin (an actin-binding protein) (Makioka et al 2012), actophorin (a depolymerization factor of actin) and generation factor (a polymerization factor of actin) (Bouyer et al 2009) were reported to be regulated in the encystment process of A. castellanii or E. invadens. It has thus been suggested that actin is necessary for encystment and that it plays a role in the maintenance of the cells' shape.…”

The actin cytoskeleton orchestra in Entamoeba histolytica

“…Entamoeba histolytica encodes three profilins with their identity in the range of 22%‐32% only (Figure S1). 68 Further investigations may help us justify the presence of three profilins in amoeba.…”