Department of Biological Sciences, Clemson University, Clemson, South Carolina, United States of America.
Eukaryotic Pathogens Innovations Center (EPIC), Clemson University, Clemson, South Carolina, United States of America.
PLoS One. 2020 Mar 5;15(3):e0219870. doi: 10.1371/journal.pone.0219870. eCollection 2020.
Entamoeba histolytica is a food- and waterborne parasite that causes amebic dysentery and amoebic liver abscesses. Adhesion is one of the most important virulence functions as it facilitates motility, colonization of host, destruction of host tissue, and uptake of nutrients by the parasite. The parasite cell surface adhesin, the Gal/GalNAc lectin, facilitates parasite-host interaction by binding to galactose or N-acetylgalactosamine residues on host components. It is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), whereas Hgl and Lgl transiently associate with rafts. When all three subunits are localized to rafts, galactose-sensitive adhesion is enhanced. Thus, submembrane location may regulate the function of this adhesion. Rhomboid proteases are a conserved family of intramembrane proteases that also participate in the regulation of parasite-host interactions. In E. histolytica, one rhomboid protease, EhROM1, cleaves Hgl as a substrate, and knockdown of its expression inhibits parasite-host interactions. Since rhomboid proteases are found within membranes, it is not surprising that lipid composition regulates their activity and enzyme-substrate binding. Given the importance of the lipid environment for both rhomboid proteases and the Gal/GalNAc lectin, we sought to gain insight into the relationship between rhomboid proteases and submembrane location of the lectin in E. histolytica. We demonstrated that EhROM1, itself, is enriched in highly buoyant triton-insoluble membranes reminiscent of rafts. Reducing rhomboid protease activity, either pharmacologically or genetically, correlated with an enrichment of Hgl and Lgl in rafts. In a mutant cell line with reduced EhROM1 expression, there was also a significant augmentation of the level of all three Gal/GalNAc subunits on the cell surface and an increase in the molecular weight of Hgl and Lgl. Overall, the study provides insight into the molecular mechanisms governing parasite-host adhesion for this pathogen.
溶组织内阿米巴是一种食源性和水源性寄生虫，可引起阿米巴痢疾和肝脓肿。黏附是最重要的毒力功能之一，因为它促进了寄生虫的运动、定植于宿主、破坏宿主组织和摄取营养物质。寄生虫细胞表面黏附素，即半乳糖/ N-乙酰半乳糖胺凝集素，通过与宿主成分上的半乳糖或 N-乙酰半乳糖胺残基结合，促进寄生虫-宿主相互作用。它由重（Hgl）、中（Igl）和轻（Lgl）亚基组成。Igl 组成型定位于脂筏（富含胆固醇的膜域），而 Hgl 和 Lgl 则与脂筏短暂结合。当所有三个亚基都定位于脂筏时，半乳糖敏感性黏附增强。因此，亚膜定位可能调节这种黏附的功能。
孤蛋白水解酶是一个保守的跨膜蛋白酶家族，也参与寄生虫-宿主相互作用的调节。在溶组织内阿米巴中，一种孤蛋白水解酶 EhROM1 作为底物切割 Hgl，其表达的敲低抑制寄生虫-宿主相互作用。由于孤蛋白水解酶位于膜内，因此脂质组成调节其活性和酶-底物结合并不奇怪。鉴于脂质环境对孤蛋白水解酶和半乳糖/ N-乙酰半乳糖胺凝集素的重要性，我们试图深入了解 EhROM1 与凝集素在溶组织内阿米巴中的亚膜定位之间的关系。
我们证明，EhROM1 本身富含高度浮力的 Triton 不溶性膜，类似于脂筏。药理学或遗传学上降低孤蛋白水解酶的活性与 Hgl 和 Lgl 在脂筏中的富集相关。在 EhROM1 表达降低的突变细胞系中，细胞表面所有三种半乳糖/ N-乙酰半乳糖胺亚基的水平也显著增加，Hgl 和 Lgl 的分子量增加。总体而言，该研究提供了对这种病原体寄生虫-宿主黏附的分子机制的深入了解。
