EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica

Abstract

The highly conserved proteins of the 14-3-3 family are universal adaptors known to regulate an enormous range of cellular processes in eukaryotes. However, their biological functions remain largely uncharacterized in pathogenic protists comprising of several 14-3-3 protein isoforms. In this study, we report the role of 14-3-3 in coordinating cytoskeletal dynamics during phagocytosis in a professional phagocytic protist Entamoeba histolytica , the etiological agent of human amebiasis. There are three isoforms of 14-3-3 protein in amoeba and here we have investigated Eh14-3-3 Protein 3 (EhP3). Live and fixed cell imaging studies revealed the presence of this protein throughout the parasite phagocytosis process, with high rate of accumulation at the phagocytic cups and closed phagosomes. Conditional suppression of EhP3 expression caused significant defects in phagocytosis accompanied by extensive diminution of F-actin at the site of cup formation. Downregulated cells also exhibited defective recruitment of an F-actin stabilizing protein, EhCoactosin at the phagocytic cups. In addition, mass spectrometry based analysis further revealed a large group of EhP3-associated proteins, many of these proteins are known to regulate cytoskeletal architecture in E histolytica . The dynamics of these proteins may also be controlled by EhP3. Taken together, our findings strongly suggest that EhP3 is a novel and a key regulatory element of actin dynamics and phagocytosis in E . histolytica .

The actin cytoskeleton orchestra in Entamoeba histolytica

“…We have compiled a total of 11 different proteins, of which only one protein has been annotated as actophorin (C4LVG4). An earlier work published by Kumar et al described a unique coactosin (C4M4P4) that stabilized actin filaments rather than performing its regular severing action 19,73 . This role‐reversal in the mechanism of action of coactosin sheds light on the yet undiscovered roles of ADF/cofilin family proteins.…”

Comparative proteomic profiling of newly acquired, virulent and attenuated Neoparamoeba perurans proteins associated with amoebic gill disease

“…Pro lin (spot 582) and ADF-like-domain, an actin binding protein (ABP), were both found to be upregulated in the newly acquired culture. ABP's are recruited for the regulation of dynamic actin remodelling during phagocytosis in Entamoeba histolytica 48 , a key process relating to E. histolytica's pathogenesis. An allergic response to N. perurans has been postulated previously 49 and pro lin is commonly regarded as an allergen protein in many pollen and foods capable of inducing the production of IgE and IgM 50 .…”

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

“…For monitoring erythrophagocytosis, E. histolytica trophozoites were harvested in serum-free TYI-33 medium and RBCs were obtained in PBS, pH 7.4, via needle prick of the middle finger (Agarwal et al, 2019 ). The PKA inhibitor (H-89), recently identified EPAC inhibitor (ESI-09), and FK were each used at the optimal concentrations at which they have been used in previous studies in other parasites (Ydrenius et al, 2000 ; Kurokawa et al, 2011 ; Almahariq et al, 2013 ; Dawn et al, 2014 ; Jia et al, 2017 ).…”

“…It is a complex and multistep process that requires intensive cytoskeletal remodeling and initiation of several signaling events. A number of molecules such as actin filaments, actin-binding proteins, and myosins have been identified and characterized in controlling cytoskeletal dynamics and coordinating the process of phagocytosis in E. histolytica (Voigt and Guillen, 1999 ; Voigt et al, 1999 ; Kumar et al, 2014a ; Agarwal et al, 2019 ; Rath and Gourinath, 2020 ). The list also includes several novel proteins that have not been identified in mammalian and other systems such as transmembrane kinases and the serine-rich E. histolytica proteins (Boettner et al, 2008 ; Teixeira and Huston, 2008 ).…”