Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (17 results)

  • Article
  • Peer Reviewed
UC Davis Previously Published Works (2015)
Entamoeba histolytica was named 'histolytica' (from histo-, 'tissue'; lytic-, 'dissolving') for its ability to destroy host tissues. Direct killing of host cells by the amoebae is likely to be the driving factor that underlies tissue destruction, but the mechanism was unclear. We recently showed that, after attaching to host cells, amoebae bite off and ingest distinct host cell fragments, and that this contributes to cell killing. We review this process, termed 'amoebic trogocytosis' (trogo-, 'nibble'), and how this process interplays with phagocytosis, or whole cell ingestion, in this organism. 'Nibbling' processes have been described in other microbes and in multicellular organisms. The discovery of amoebic trogocytosis in E. histolytica may also shed light on an evolutionarily conserved process for intercellular exchange.
    Cover page: Chew on this: amoebic trogocytosis and host cell killing by Entamoeba histolytica
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2015)
    Entamoeba histolytica is a diarrheal pathogen with the ability to cause profound host tissue damage. This organism possesses contact-dependent cell killing activity, which is likely to be a major contributor to tissue damage. E. histolytica trophozoites were recently shown to ingest fragments of living human cells. It was demonstrated that this process, termed amoebic trogocytosis, contributes to cell killing. Recent advances in ex vivo and 3-D cell culture approaches have shed light on mechanisms for tissue destruction by E. histolytica, allowing amoebic trogocytosis to be placed in the context of additional host and pathogen mediators of tissue damage. In addition to its relevance to pathogenesis of amoebiasis, an appreciation is emerging that intercellular nibbling occurs in many organisms, from protozoa to mammals.
      Cover page: Taking a bite: Amoebic trogocytosis in Entamoeba histolytica and beyond
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2020)
      Entamoeba histolytica is the causative agent of amoebiasis. Pathogenesis is associated with profound damage to human tissues. We previously showed that amoebae kill human cells through trogocytosis. Trogocytosis is likely to underlie tissue damage during infection, although the mechanism is still unknown. Trogocytosis is difficult to assay quantitatively, which makes it difficult to study. Here, we developed two new, complementary assays to measure trogocytosis by quantifying human cell death. One assay uses CellTiterGlo, a luminescent readout for ATP, as a proxy for cell death. We found that the CellTiterGlo could be used to detect death of human cells after co-incubation with amoebae, and that it was sensitive to inhibition of actin or the amoeba surface Gal/GalNAc lectin, two conditions that are known to inhibit amoebic trogocytosis. The other assay uses two fluorescent nuclear stains to directly differentiate live and dead human cells by microscopy, and is also sensitive to inhibition of amoebic trogocytosis through interference with actin. Both assays are simple and inexpensive, can be used with suspension and adherent human cell types, and are amenable to high-throughput approaches. These new assays are tools to improve understanding of trogocytosis and amoebiasis pathogenesis.
        Cover page: Direct and high-throughput assays for human cell killing through trogocytosis by Entamoeba histolytica
        • Thesis
        • Peer Reviewed
        UC Davis Electronic Theses and Dissertations (2021)

        Trogocytosis is an endocytic process that is present in many eukaryotes including multicellular organisms and unicellular microbes. Paradoxically, it can be used as a benign form of cell-cell interaction, and it can also be used as a mechanism for cell-mediated killing. Trogocytosis is characterized by ingestion of small pieces of living cells. Additionally, in mammalian immune cells, and in the pathogen Entamoeba histolytica, trogocytosis results in the transfer of membrane proteins from one cell to another. Here, we give a broad overview of trogocytosis, its functions, and proposed molecular mechanisms in different cell types. These studies also reveal trogocytosis as a novel immune evasion mechanism employed by E. histolytica, the parasite responsible for the diarrheal disease amoebiasis. We demonstrate that E. histolytica displays human proteins after performing trogocytosis on human cells, and that this leads to protection from complement lysis. Trophozoites (amoebae) are protected from complement lysis following trogocytosis of live cells but not phagocytosis of dead cells. Amoebic trogocytosis leads to decreased deposition of the complement protein C3b. Furthermore, amoebae acquire and display human CD59 and CD46, which are known negative regulators of the complement pathway. While deletion of one or two complement regulatory proteins from human cells was not sufficient to alter conferred protection, amoebae mutants that exogenously expressed human CD46 or CD55 were protected from lysis. Thus, surface display of an individual negative regulator of complement is sufficient to protect amoebae from lysis. These findings indicate that amoebae likely acquire multiple redundant complement regulators from trogocytosis of human cells which leads to robust protection from complement lysis. Finally, we developed an assay to quantify trogocytosis in mammalian immune cells and generated mutant amoebae trogocytosis and phagocytosis defects. These tools can be used for future study of this important eukaryotic process.

          Cover page: Defining the Role of Trogocytosis in Complement Evasion by Entamoeba histolytica and Tools for Future Study
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2020)

          Background

          Entamoeba histolytica kills human cells by ingesting fragments of live cells until the cell eventually dies, a process termed amebic trogocytosis. In a previous study, we showed that acidified amebic lysosomes are required for both amebic trogocytosis and phagocytosis, as well as cell killing.

          Methods

          Amebic cysteine proteases (CPs) were inhibited using an irreversible inhibitor, E-64d.

          Results

          Interfering with amebic CPs decreased amebic trogocytosis and amebic cytotoxicity but did not impair phagocytosis.

          Conclusions

          We show that amebic CPs are required for amebic trogocytosis and cell killing but not phagocytosis. These data suggest that amebic CPs play a distinct role in amebic trogocytosis and cell killing.
            Cover page: Inhibition of Amebic Cysteine Proteases Blocks Amebic Trogocytosis but Not Phagocytosis
            • Thesis
            • Peer Reviewed
            UC Davis Electronic Theses and Dissertations (2022)

            Entamoeba histolytica is a pathogen of global importance, and is responsible for the diarrheal disease amoebiasis. Infection is characterized by massive tissue damage, which is poorly understood and thought to be driven by the contact-dependent cell-killing activity of E. histolytica. E. histolytica performs two main forms of ingestion of human cells: trogocytosis and phagocytosis. Trogocytosis involves taking bites of live host cells and contributes to host cell killing, whereas phagocytosis involves swallowing dead host cells whole. In this body of work, we investigated this distinctions between phagocytosis and trogocytosis, and developed several genetic tools to enable greater study of this parasite. We investigated the connection between host cell attachment and ingestion type by knocking down proteins implicated in attachment using trigger-induced RNA interference. In this process we found that trigger-silenced mutants should be analyzed immediately before phenotypes are lost. We demonstrated for the first time that amoebae acquire and display host cell membrane proteins through trogocytosis but not phagocytosis, allowing them to survive in human serum. To test how host cell deformability influences the type of ingestion performed by amoebae, we designed and optimized an ingestion assay capable of distinguishing between whole host cells, membrane bites, and external host cells. Using this assay, we found that host cells with cytoskeletal proteins knocked down were trogocytosed less by amoebae. We provided practical instruction on how to grow and genetically manipulate E. histolytica in a laboratory research environment. We also constructed the first E. histolytica RNAi knockdown mutant library, and used it to identify and characterize novel genes involved in growth. Finally, we made great progress in establishing CRISPR interference in E. histolytica, discovering that increasing antibiotic concentration affects gene expression, and that a common expression promoter is insufficient for expression of dCas9. Overall, this body of work represents a significant contribution to our understanding of the pathogenesis and cell biology of E. histolytica.

              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2022)
              Entamoeba histolytica is a parasitic protozoan and the causative agent of amoebiasis in humans. Amoebiasis has a high incidence of disease, resulting in ∼67,900 deaths per year, and it poses a tremendous burden of morbidity and mortality in children. Despite its importance, E. histolytica is an understudied parasite. These protocols describe the in vitro growth, maintenance, cryopreservation, genetic manipulation, and cloning of axenic E. histolytica trophozoites. There has been significant progress in genetic manipulation of this organism over the past decade, and these protocols outline the ways in which these advances can be implemented. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culturing E. histolytica trophozoites Support Protocol 1: Preparation of TYI-S-33 medium Support Protocol 2: Lot testing of Biosate peptone and adult bovine serum for TYI-S-33 medium Basic Protocol 2: Cryopreservation of E. histolytica trophozoites Support Protocol 3: Preparation of cryoprotectant solutions Basic Protocol 3: Transfection of E. histolytica trophozoites with Attractene reagent Basic Protocol 4: Creating clonal lines using limiting dilution Basic Protocol 5: Knockdown of one to two genes with trigger-induced RNA interference Support Protocol 4: Evaluation of RNA interference knockdown with reverse transcriptase PCR Basic Protocol 6: E. histolytica growth curves.
                Cover page: Growth and Genetic Manipulation of Entamoeba histolytica
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2019)
                We previously showed that Entamoeba histolytica kills human cells through a mechanism that we termed trogocytosis ("trogo-" means "nibble"), due to its resemblance to trogocytosis in other organisms. In microbial eukaryotes like E. histolytica, trogocytosis is used to kill host cells. In multicellular eukaryotes, trogocytosis is used for cell killing and cell-cell communication in a variety of contexts. Thus, nibbling is an emerging theme in cell-cell interactions both within and between species. When trogocytosis occurs between mammalian immune cells, cell membrane proteins from the nibbled cell are acquired and displayed by the recipient cell. In this study, we tested the hypothesis that through trogocytosis, amoebae acquire and display human cell membrane proteins. We demonstrate that E. histolytica acquires and displays human cell membrane proteins through trogocytosis and that this leads to protection from lysis by human serum. Protection from human serum occurs only after amoebae have undergone trogocytosis of live cells but not phagocytosis of dead cells. Likewise, mutant amoebae defective in phagocytosis, but unaltered in their capacity to perform trogocytosis, are protected from human serum. Our studies are the first to reveal that amoebae can display human cell membrane proteins and suggest that the acquisition and display of membrane proteins is a general feature of trogocytosis. 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.IMPORTANCEEntamoeba histolytica causes amoebiasis, a potentially fatal diarrheal disease. Abscesses in organs such as the liver can occur when amoebae are able to breach the intestinal wall and travel through the bloodstream to other areas of the body. Therefore, understanding how E. histolytica evades immune detection is of great interest. Here, we demonstrate for the first time that E. histolytica acquires and displays human cell membrane proteins by taking "bites" of human cell material in a process named trogocytosis ("trogo-" means "nibble"), and that this allows amoebae to survive in human serum. Display of acquired proteins through trogocytosis has been previously characterized only in mammalian immune cells. Our study suggests that this is a more general feature of trogocytosis not restricted to immune cells and broadens our knowledge of eukaryotic biology. These findings also reveal a novel strategy for immune evasion by a pathogen and may apply to the pathogenesis of other infections.
                  Cover page: Trogocytosis by Entamoeba histolytica Mediates Acquisition and Display of Human Cell Membrane Proteins and Evasion of Lysis by Human Serum
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2014)
                  Entamoeba histolytica is the causative agent of amoebiasis, a potentially fatal diarrhoeal disease in the developing world. The parasite was named "histolytica" for its ability to destroy host tissues, which is probably driven by direct killing of human cells. The mechanism of human cell killing has been unclear, although the accepted model was that the parasites use secreted toxic effectors to kill cells before ingestion. Here we report the discovery that amoebae kill by ingesting distinct pieces of living human cells, resulting in intracellular calcium elevation and eventual cell death. After cell killing, amoebae detach and cease ingestion. Ingestion of human cell fragments is required for cell killing, and also contributes to invasion of intestinal tissue. The internalization of fragments of living human cells is reminiscent of trogocytosis (from Greek trogo, nibble) observed between immune cells, but amoebic trogocytosis differs because it results in death. The ingestion of live cell material and the rejection of corpses illuminate a stark contrast to the established model of dead cell clearance in multicellular organisms. These findings change the model for tissue destruction in amoebiasis and suggest an ancient origin of trogocytosis as a form of intercellular exchange.
                    Cover page: Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2014)
                    The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream-form motility mutant in 427-derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time-course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.
                      Cover page: Trypanosome flagellum motility and infection
                      Top