Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis

Abstract

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

Nucleus-localized Antisense Small RNAs with 5′-Polyphosphate Termini Regulate Long Term Transcriptional Gene Silencing in Entamoeba histolytica G3 Strain

“…The G3 parasites were a gift from Dr. David Mirelman, and G3-based secondary gene silenced parasites were generated by transfection of G3 parasites with psAP-2/5ЈSTIRP plasmid or psAP-2/3ЈSTIRP plasmid (see "Plasmid Construction for EhSTIRP Gene Silencing"). EhROM(KD) parasites were generated as described previously (26), with separate cultures having been maintained with or without drug at 2 g/ml G418. The N-terminal Myc-tagged EhAGO2-2 parasites were previously generated (20) and have been maintained at 24 g/ml G418.…”

“…A single cloned parasite from this transfectant line is named G3 (24). Importantly, it was later found that a second unrelated gene of interest could be transcriptionally silenced by transfecting G3 with a plasmid in which a second gene was cloned directly after the ap-a promoter fragment (23,25,26). The resulting secondary gene silencing is also inheritable and can be maintained after removal of the plasmid.…”

New insights into parasite rhomboid proteases

“…In resting conditions, EhROM1 is located both at the parasite surface and in internal punctate structures but upon erythrophagocytosis it relocates to internal vesicles [68]. EhROM1 knockdown leads to a defect in parasite adhesion to healthy cells as well as a generalized phagocytosis defect that are attributed to independent functions of the rhomboid protease, perhaps related to different substrates [69]. Although EhROM1 can cleave in vitro the parasite adhesin Gal/GalNAc lectin, there was no change in the expression or localization of this protein in cells depleted of the rhomboid [69].…”

“…In vivo: unknown Entamoeba histolytica EhROM1 Parasite surface and internal punctuate structures (resting conditions) Internal vesicles (erythrophagocytosis) [68] In vitro: EhGal/GalNAc lectin [68] Parasite adhesion to healthy cells Phagocytosis [69] In vivo: unknown Phylogenetic analysis of Kinetoplastidiae ROMs. The tree is based on neighbour-joining-distance analysis.…”

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

“…Rhomboid proteases are known virulence factors that function in adhesion and phagocytosis of the host cells and thus have a well-studied role in host-parasite interactions (46)(47)(48). By fusing 152-Trigger to full-length myc-tagged ROM1 (1,008-bp coding region), the 152-Trigger-EiROM construct was generated (Fig.…”