Trogocytosis by Entamoeba histolytica mediates acquisition and display of human cell membrane proteins and evasion of lysis by human serum

Abstract

Entamoeba histolytica is the protozoan parasite responsible for amoebiasis. We previously showed that E. histolytica kills human cells through a mechanism that we termed trogocytosis (trogo-: nibble), due to its resemblance to trogocytosis in other organisms. In parasites, trogocytosis is used to kill host cells. In multicellular organisms, trogocytosis is used for cell-cell interactions in the immune system, in the central nervous system, and during development. Thus, nibbling is an emerging theme in cell-cell interactions both within and between species, and it is relevant to host-pathogen interactions in many different contexts. When trogocytosis occurs between mammalian immune cells, cell membrane proteins from the nibbled cell can be acquired and displayed by the recipient cell. In this study, we tested the hypothesis that through trogocytosis of human cells, amoebae acquire and display human cell membrane proteins. Here we demonstrate for the first time that through trogocytosis, E. histolytica acquires and displays human cell membrane proteins and that this leads to protection from lysis by human serum. Protection from human serum only occurs after amoebae have undergone trogocytosis of live cells, but not phagocytosis of dead cells. Likewise, mutant amoebae that exhibit a phagocytosis defect, but are unaltered in their capacity to perform trogocytosis, are nevertheless protected from human serum. Our studies are the first to demonstrate that amoebae can display human cell membrane proteins and suggest that acquisition and display of membrane proteins is a general feature of trogocytosis that is not restricted to trogocytosis between mammalian immune cells. These studies have major implications for interactions between E. histolytica and the immune system and also reveal a novel strategy for immune evasion by a pathogen. Since other microbial eukaryotes use trogocytosis for cell killing, our findings may apply to the pathogenesis of other infections.

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

“…All of these processes require coordinated regulation of signal transduction, cytoskeletal reorganization, and vesicular trafficking (Aguilar‐Rojas, Olivo‐Marin, & Guillen, 2016; Babuta, Bhattacharya, & Bhattacharya, 2020; Nozaki & Nakada‐Tsukui, 2006). Recent studies indicate in particular that trogo‐ and phagocytosis play a pivotal role in disease manifestations and host immune evasion (Miller, Suleiman, & Ralston, 2019; Nakada‐Tsukui & Nozaki, 2020; Ralston et al, 2014; Somlata, Nakada‐Tsukui, & Nozaki, 2017). It has been shown that both amebic trogo‐ and phagocytosis are energy dependent and require Gal/GalNAc lectin, protein and lipid kinases such as C2‐domain‐containing protein kinase (EhC2PK), atypical kinase (EhAK1), and phosphoinositide‐3‐kinase (PI3K), phosphatidylinositide‐binding proteins such as FYVE domain containing protein 4 (Nakada‐Tsukui, Okada, Mitra, & Nozaki, 2009) and SNXs (Watanabe, Nakada‐Tsukui, & Nozaki, 2020), and proteins involved in actin rearrangement such as calcium binding proteins, ArpA2/3, Rho/Rac, EhP3, and EhFP10 (Agarwal et al, 2019; Babuta et al, 2020; Babuta, Kumar, Gourinath, Bhattacharya, & Bhattacharya, 2018; Babuta, Mansuri, Bhattacharya, & Bhattacharya, 2015; Bharadwaj et al, 2018; Bharadwaj, Arya, Shahid Mansuri, Bhattacharya, & Bhattacharya, 2017; Gautam, Ali, Bhattacharya, & Gourinath, 2019; Nakada‐Tsukui et al, 2009; Powell et al, 2006; Ralston et al, 2014; Somlata, Bhattacharya, & Bhattacharya, 2011; Watanabe et al, 2020).…”

Trogocytosis between Non-Immune Cells for Cell Clearance, and among Immune-Related Cells for Modulating Immune Responses and Autoimmunity

“…In addition, the adherence of Eh to host cells could mediate multiple cell cytotoxicity including promotion of cell death via phagocytosis, apoptosis, and trogocytosis those would play crucial roles in immune invasion [ 31 ]. Miller et al [ 4 ] further confirmed that amoebic trogocytosis by acquisition and displaying human cell membrane proteins could facilitate escape from immune lysis by human serum.…”

“…Partial phagocytosis, cell nibbling or cannibalization is also different names for trogocytosis [ 2 ]. This cellular growth behavior was first observed among amoebas [ 3 , 4 ] and then between cells of the immune system [ 5 ].…”

Direct and high-throughput assays for human cell killing through trogocytosis by Entamoeba histolytica

“…This plasmid, or a corresponding vector control, was transfected into amoebae using Attractene transfection reagent (Qiagen), and then stable transfectants were selected and maintained with Geneticin at 6 µg/mL (Invitrogen). Clonal lines were obtained by limiting dilution, and silencing was confirmed using RT-PCR (Miller, 2019). A single clonal line was used for experiments.…”

“…To enable quantitative cell death measurements, we previously developed an assay using imaging flow cytometry (Ralston, 2014). In this assay, amoebae and human cells are fluorescently labeled, allowing for trogocytosis to be directly measured, and Live/Dead Violet is used to stain dead (permeable) cells (Ralston, 2014, Gilmartin, 2017, Miller, 2019. This assay allows for automated analysis of thousands of images per sample, but is limited in practicality since imaging flow cytometers are not widely available.…”