Two isotypes of phosphatidylinositol 3‐phosphate ‐ binding sorting nexins play distinct roles in trogocytosis in Entamoeba histolytica

Abstract

Phosphatidylinositol phosphates (PIPs) function as important second messengers in many cellular events. In the human intestinal protist Entamoeba histolytica, where phagocytosis/trogocytosis plays an indispensable role in proliferation and pathophysiology during infection, various PIPs are involved in multiple steps of phago/trogocytosis. PI3-phosphate (PI3P) plays a pivotal role in the biogenesis of phagosome/trogosomes via recruitment of PI3P effectors. Because no known PI3P downstream effectors are conserved in E. histolytica, we exploited a unique method to identify the proteins PI3P dependently recruited to phagosomes. We rationalised that overexpression of PI3P-binding GFP-HrsFYVE competes for PI3P on phagosomal membranes and results in dissociation of PI3P effectors from phagosomes. EhVps26 and EhVps35, but not sorting nexins (SNXs), of the retromer complex were detected from phagosomes only without GFP-HrsFYVE overexpression. Two potential SNXs, EhSNX1 and EhSNX2, identified in the genome, possess only phox homology domain and specifically bound to PI3P, but retromer components, EhVps26 and EhVps35, did not bind to PI3P. Live and immunofluorescence imaging showed that EhSNX1 was recruited to the trogocytic cup and tunnel-like structures, and subsequently, EhSNX2 was recruited to trogosomes. Furthermore, EhSNX1, but not EhSNX2, specifically bound to Arp2/3 and EhVps26, which were localised to the tunnel-like structures and the trogosomes, respectively. EhSNX2 gene silencing increased trogocytosis, suggesting that EhSNX2 plays an inhibitory role in trogocytosis.

Dynamism of PI4-Phosphate during Interactions with Human Erythrocytes in Entamoeba histolytica

“…A similar dependence on the prey was previously demonstrated for PI3P recruitment to phagosomes. GFP-HrsFYVE, a PI3P bioprobe, was recruited to phagosomes that contained serum-coated beads, but not to those containing non-coated beads in E. histolytica [ 3 , 6 ]. Furthermore, it was demonstrated that the trogocytosis of CHO was enhanced while the phagocytosis of non-coated beads was repressed by HrsGFP-FYVE overexpression [ 3 ], suggesting that the role of PI3P may differ in live cell trogocytosis and bead phagocytosis.…”

“…The significance of PIPs, particularly PI3P and PI(3,4,5)P 3 , in the trogo- and phagocytosis of E. histolytica has been well documented [ 3 , 4 , 5 ]. It has been shown that PI3P, monitored by Hrs-FYVE (Fab-1, YGL023, Vps27, EEA1) and sorting nexin 1 (SNX1) as its biosensor, is recruited to the bottom of the trogo- and phagocytic cups, and subsequently to trogo- and phagosomes [ 3 , 6 ]. On the other hand, the E. histolytica FYVE domain containing protein 4 (FP4), which binds to PI4P, has been shown to be recruited to the neck of the closing trogocytic cup and closed (mature) trogosomes [ 3 ].…”

Rab7D small GTPase is involved in phago‐, trogocytosis and cytoskeletal reorganization in the enteric protozoan Entamoeba histolytica

“…While effectors and binding proteins for EhRab7D remain elusive, the effector of EhRab7A was previously demonstrated (Nakada-Tsukui et al, 2005). Retromer complex, the EhRab7A effector, is assumed to be involved in the forward transport of hydrolytic enzymes or their cargo receptors to trogosomes and phagosomes or in the transport in a reverse direction in E. histolytica (Nakada-Tsukui et al, 2005;Picazarri et al, 2015;Watanabe et al, 2020 Figure S2). This inter-switch region was reported to be an interface between Rab7 and Rab interacting lysosomal protein (RILP) (Cantalupo, Alifano, Roberti, Bruni, & Bucci, 2001;Harrison, Bucci, Vieira, Schroer, & Grinstein, 2003;Jordens et al, 2001), which serves as a bridge between microtubule Arp2/3 complex (Pollitt & Insall, 2009;Rougerie, Miskolci, & Cox, 2013).…”

Biting Off What Can Be Chewed: Trogocytosis in Health, Infection, and Disease

“…The EhAGCK1 knockdown mutants used in the study were generated using an approach that affects the expression of off-target genes (77). It has been suggested that E. histolytica phosphatidylinositol 3-phosphate (PI3P) membrane glycerophospholipid-binding proteins such as Vsps and SNXs might be involved in trogocytosis and phagocytosis (78). Further work is still needed to determine which mechanisms are specific to E. histolytica trogocytosis.…”