Glutamic acid-lysine (EK) rich motif of RabD2 self-associates and regulates pathogenesis through multivesicular bodies pathway in E. histolytica

Abstract

Entamoeba histolytica, an enteric pathogen, causes disease by adhering to and destroying the host tissues. The interactions between the parasite and host tissue enable rewiring of the gene expression and global membrane trafficking in the parasite. A fine balance between cargoes/receptors endocytosis and exocytosis is required to establish infection in the host. Multivesicular bodies (MVBs) act as sorting platforms, delivering cargoes/receptors to lysosomes for degradation or secreting their content through plasma membrane fusion. Some of the small GTPases are known to control MVB biogenesis in various organisms. However, the functional contribution of Rab GTPases in MVB biogenesis is poorly studied in E. histolytica. Here, we identified a novel atypical protein RabD2, with an N-terminal glutamic acid-lysine rich motif and a C-terminal conserved Rab domain. Our biochemical and cell biological assays provide evidence that RabD2 self-associates, and this interaction is controlled by the N-terminal EK-rich motif and the GTPase activity mutants (in a nucleotide-specific manner). RabD2 localizes on the surface of MVBs and controls their biogenesis. In line with these findings, overexpression of RabD2 upregulates global ubiquitination, directing the down regulation of the heavy chain of GalNAc lectin, ultimately leading to decreased adherence of E. histolytica trophozoites to host cells. Thus, amoebic RabD2 is a new class of Rab protein that forms high-ordered self-association variants and regulates the pathogenicity of E. histolytica through the biogenesis of MVBs. Author SummaryAmoebiasis is an enteral infection caused by Entamoeba histolytica that primarily remains asymptomatic but can eventually result in systemic complications like amoebic dysentery, liver abscess, and pulmonary effusions. Recent studies showed that Entamoeba ubiquitin is a robust antigen and linked with the invasive amoebiasis patient samples. Here, we identified a novel RabD2 that self-associates via its glutamic acid-lysine rich motif that causes high ubiquitination levels and biogenesis of multivesicular bodies. We uncovered for the first time that RabD2-mediated ubiquitin-dependent pathway is involved in the down regulation of the notable antigenic marker heavy chain of galactose-N-acetylgalactosamine lectin and thereby controls the parasite adherence to host cells. Further studies on crosstalk between parasite ubiquitination and antigenic receptors downregulation provide insights into how parasites use these strategies to establish the infection in the host intestine.