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.

Background: PlaC is a GDSL enzyme and the major GCAT secreted by Legionella pneumophila. Results: The zinc metalloproteinase ProA processes and activates PlaC. Deletion of regions within a disulfide loop increased GCAT activity. Conclusion: Inhibitory disulfide loop reduction/deletion by ProA activates PlaC GCAT. Significance: Here we recognized the postexport GCAT activation mechanism essential for modification of typical eukaryotic sterols.

The protozoan parasite, Entamoeba histolytica, invades the host colon causing significant tissue destruction and inflammation. Upon host infection, the parasite is confronted with reactive oxygen and nitrogen species (ROS/RNS) that cause large-scale changes in gene expression profiles, which likely support the parasite’s adaptation to the host environment. We have previously identified oxidative and nitrosative stress responsive genes using whole-genome expression profiling. Functional studies on two such genes are now reported and demonstrate that they have roles in parasite virulence. EHI_056680 encodes a small hypothetical protein named E. histolytica stress-induced adhesion factor (EhSIAF); EHI_188210 encodes a putative phospholipid transporting P-type ATPase/flippase (EhPTPA). Over-expression of each protein in E. histolytica trophozoites enhanced parasite survival in response to oxidative stress. Exposure to oxidative and nitrosative stress did not affect the localization of EhSIAF or EhPTPA but markedly increased EhPTPA protein levels. Interestingly, over-expression of each gene resulted in parasites with increased adherence to healthy mammalian cells, but increased adherence to apoptotic cells was noted only in EhSIAF over-expressing parasites. However, despite having increased adherence to both healthy and apoptotic host cells, EhSIAF-over-expressing parasites were reduced in their ability to destroy mammalian cell monolayers, raising the intriguing possibility that EhSIAF over-expression caused signaling defects or resulted in a dominant negative phenotype. Over-expression of EhSIAF and EhPTPA also resulted in decreased motility in a transwell motility assay. Thus, we have confirmed that two genes that are upregulated by ROS confer increased resistance to oxidative stress and have identified an unexpected role of EhSIAF and EhPTPA in host cell adherence and a role of EhSIAF in parasite virulence. Our data imply that stress response genes may play multi-factorial roles in amoebic pathogenesis.

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.

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