Entamoeba histolytica rhomboid protease 1 has a role in migration and motility as validated by two independent genetic approaches.

Rhomboid proteins represent a recently discovered family of intramembrane proteases present in a broad range of organisms and with increasing links to human diseases. The enteric parasite Entamoeba histolytica has evolved multiple mechanisms to adapt to the human host environment and establish infection. Our recent studies identified EhROM1 as a functional E. histolytica rhomboid protease with roles in adhesion to and phagocytosis of host cells. Since those studies were performed in a non-virulent strain, roles in parasite virulence could not be assessed. We focused this study on the comparison and validation of two genetic manipulation techniques: overexpression of a dominant-negative catalytic mutant of EhROM1 and knock down of EhROM1 using a RNAi-based silencing approach followed by functional studies of phenotypic analyses in virulent parasites. Both the EhROM1 catalytic mutant and parasites with EhROM1 downregulation were reduced in cytotoxicity, hemolytic activity, and directional and non-directional transwell migration. Importantly, the role for EhROM1 in cell migration mimics similar roles for rhomboid proteases from mammalian and apicomplexan systems. However, the EhROM1 catalytic mutant and EhROM1 downregulation parasites had different phenotypes for erythrophagocytosis, while complement resistance was not affected in either strain. In summary, in this study we genetically manipulated E. histolytica rhomboid protease EhROM1 by two different approaches and identified similarly attenuated phenotypes by both approaches, suggesting a novel role for EhROM1 in amebic motility.