Novel regulatory roles of PtdIns(4,5)P₂generating enzyme EhPIPKI in actin dynamics and phagocytosis ofEntamoeba histolytica

Abstract

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin-dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P 2 as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G-actin unlike PIPKs from other organisms. PtdIns(4,5)P 2 , the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP-tagged PH-domain from PLCδ, which specifically binds PtdIns(4,5)P 2 in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.

Two StAR-related lipid transfer proteins play specific roles in endocytosis, exocytosis, and motility in the parasitic protist Entamoeba histolytica

“…Such isotype-specific and tempo-spatial recruitment of EhLTP1 and EhLTP3 was similar to that of AGC kinases 2 and 1, respectively [19], which may infer potential cross talk between the proteins via PIPs. The localization of EhLTP1 and EhLTP3, shown in this study, is similar with that of PtdIns(4,5)P 2 and their synthetic enzyme, EhPIPKI [20,38]. This is consistent with the fact that both EhLTP1 and 3 have PtdIns4P and PtdIns(4,5)P 2 transport activities.…”

“…PtdIns4P and PtdIns(4,5)P 2 )] to EhPIPKI and PtdIns 3-kinase, respectively (S9 Fig), as suggested as a role of LTPs in lipid metabolism [2]. The localization and dynamics of PIs including PtdIns4P [20,39], PtdIns3P [35], PtdIns(4,5)P 2 [38], PtdIns (3,4,5)P 3 [19,37] in E. histolytica during trogo-and phagocytosis were well studied through their binding effectors. It has been shown that EhFP4 interacts with PtdIns4P, EhSNX1 and 2 interact with PtdIns3P, and EhAGCK1 and 2 interact with PtdIns(3,4,5)P 3 .…”

“…The interplay between PtdIns 3-kinase and PTEN regulates the levels of PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3, which act on downstream effectors, including the Rac-GTPase, Arf-GTPases, Akt kinase (PKB) and pleckstrin homology (PH) domain proteins (PhdB, PhdG) have been shown to regulate the motility by controlling the actin cytoskeleton in Dictyostelium and mammals [44]. Similarly, EhPIPKI, PtdIns 3-kinase, and their corresponding products, PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 , respectively, regulate the actin dynamics, and control motility of Entamoeba trophozoites [37,38]. The intracellular levels of PtdIns(4,5)P 2 inversely correlates with motility [45], while PtdIns(3,4,5)P 3 , localized on extending pseudopods positively regulates the migrating ability of Entamoeba trophozoites [37].…”

Uncovering the Cyclic AMP Signaling Pathway of the Protozoan Parasite Entamoeba histolytica and Understanding Its Role in Phagocytosis

“…Calcium (Ca 2+ ) has evolved as one of the key second messengers in this pathogen regulating phagocytosis at multiple levels. The pathogen encodes several novel Ca 2+ binding proteins (CaBPs), PIP 2 , IP 3 , IP 4 , and P-type Ca 2+ -ATPases, for Ca 2+ regulation and homeostasis (Sahoo et al, 2004;Loftus et al, 2005;Jain et al, 2008;Nozaki and Bhattacharya, 2014;Sharma et al, 2019). The calcium binding protein 1 (EhCABP1) has been identified as the central indispensable molecule recruited at the early phagocytic cups.…”

“…The kinase EhAK1 along with other cytoskeletal proteins such as actin branching complex Arp 2/3, Myosin1B, and the calcium binding protein 3 (EhCABP3) are known to regulate the formation and progression of the phagocytic cup toward its closure (Aslam et al, 2012;Babuta et al, 2018). CaBPs (EhCaBP1, EhCaBP3, and EhCaBP5) have been shown to play a crucial role in establishment of phagocytosis in E. histolytica (Sahoo et al, 2004;Jain et al, 2008;Aslam et al, 2012;Kumar et al, 2014b;Nozaki and Bhattacharya, 2014;Sharma et al, 2019). However, relative to Ca 2+ signaling, the knowledge of another crucial second messenger cAMP and its signaling pathway is very limited in E. histolytica.…”