Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth.

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

Inhibition of Amebic Cysteine Proteases Blocks Amebic Trogocytosis but Not Phagocytosis.

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.

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

Trogocytosis is part of an emerging, exciting theme of cell-cell interactions both within and between species, and it is relevant to host-pathogen interactions in many different contexts. Trogocytosis is a process in which one cell physically extracts and ingests "bites" of cellular material from another cell. It was first described in eukaryotic microbes, where it was uncovered as a mechanism by which amoebae kill cells. Trogocytosis is potentially a fundamental form of eukaryotic cell-cell interaction, since it also occurs in multicellular organisms, where it has functions in the immune system, in the central nervous system, and during development. There are numerous scenarios in which trogocytosis occurs and an ever-evolving list of functions associated with this process. Many aspects of trogocytosis are relevant to microbial pathogenesis. It was recently discovered that immune cells perform trogocytosis to kill Trichomonas vaginalis parasites. Additionally, through trogocytosis, Entamoeba histolytica acquires and displays human cell membrane proteins, enabling immune evasion. Intracellular bacteria seem to exploit host cell trogocytosis, since they can use it to spread from cell to cell. Thus, a picture is emerging in which trogocytosis plays critical roles in normal physiology, infection, and disease.

Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion.

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.

Mouse infection and pathogenesis by Trypanosoma brucei motility mutants.

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.

The Trypanosoma brucei flagellum: moving parasites in new directions.

African trypanosomes are devastating human and animal pathogens. Trypanosoma brucei rhodesiense and T. b. gambiense subspecies cause the fatal human disease known as African sleeping sickness. It is estimated that several hundred thousand new infections occur annually and the disease is fatal if untreated. T. brucei is transmitted by the tsetse fly and alternates between bloodstream-form and insect-form life cycle stages that are adapted to survive in the mammalian host and the insect vector, respectively. The importance of the flagellum for parasite motility and attachment to the tsetse fly salivary gland epithelium has been appreciated for many years. Recent studies have revealed both conserved and novel features of T. brucei flagellum structure and composition, as well as surprising new functions that are outlined here. These discoveries are important from the standpoint of understanding trypanosome biology and identifying novel drug targets, as well as for advancing our understanding of fundamental aspects of eukaryotic flagellum structure and function.

Sialic acid-dependent attachment of mucins from three mouse strains to Entamoeba histolytica.

Mouse strain-specific differences in the carbohydrate composition of intestinal mucins were hypothesized to account for strain-dependent susceptibility to Entamoeba histolytica. To test this hypothesis, intestinal mucins from susceptible and resistant inbred strains of mice were analyzed for their O-glycan content and for their ability to inhibit amoebic adherence to (GalNAc)12-27-HSA neo-glycoproteins. The results showed that the colorectal mucin O-glycan of susceptible CBA mice was lower in sialic acid content than that of resistant C57BL/6 and BALB/c mice. Mucins from CBA mice were more potent inhibitors of E. histolytica adherence to neo-glycoproteins than were mucins from C57BL/6 or BALB/c mice. Consistent with the role of terminal Gal/GalNAc as a receptor for amoebic adherence, sialidase treatment of C57BL/6 and BALB/c colorectal mucins increased their ability to inhibit E. histolytica adherence to the neo-glycoproteins. These results provide evidence of mouse strain-specific differences in the sialic acids content of mucin O-glycans. These dissimilarities likely contribute to the differential susceptibility of the three mouse strains to E. histolytica infection.

Structure-function analysis of dynein light chain 1 identifies viable motility mutants in bloodstream-form Trypanosoma brucei.

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.

Growth and Genetic Manipulation of Entamoeba histolytica.

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.

Entamoeba histolytica Develops Resistance to Complement Deposition and Lysis after Acquisition of Human Complement-Regulatory Proteins through Trogocytosis.

Entamoeba histolytica is the cause of amoebiasis. The trophozoite (amoeba) form of this parasite is capable of invading the intestine and can disseminate through the bloodstream to other organs. The mechanisms that allow amoebae to evade complement deposition during dissemination have not been well characterized. We previously discovered a novel complement-evasion mechanism employed by E. histolytica. E. histolytica ingests small bites of living human cells in a process termed trogocytosis. We demonstrated that amoebae were protected from lysis by human serum following trogocytosis of human cells and that amoebae acquired and displayed human membrane proteins from the cells they ingested. Here, we aimed to define how amoebae are protected from complement lysis after performing trogocytosis. We found that amoebae were protected from complement lysis after ingestion of both human Jurkat T cells and red blood cells and that the level of protection correlated with the amount of material ingested. Trogocytosis of human cells led to a reduction in deposition of C3b on the surface of amoebae. We asked whether display of human complement regulators is involved in amoebic protection, and found that CD59 was displayed by amoebae after trogocytosis. Deletion of a single complement-regulatory protein, CD59 or CD46, from Jurkat cells was not sufficient to alter amoebic protection from lysis, suggesting that multiple, redundant complement regulators mediate amoebic protection. However, exogeneous expression of CD46 or CD55 in amoebae was sufficient to confer protection from lysis. These studies shed light on a novel strategy for immune evasion by a pathogen. IMPORTANCE Entamoeba histolytica is the cause of amoebiasis, a diarrheal disease of global importance. While infection is often asymptomatic, the trophozoite (amoeba) form of this parasite is capable of invading and ulcerating the intestine and can disseminate through the bloodstream to other organs. Understanding how E. histolytica evades the complement system during dissemination is of great interest. Here, we demonstrate for the first time that amoebae that have performed trogocytosis (nibbling of human cells) resist deposition of the complement protein C3b. Amoebae that have performed trogocytosis display the complement-regulatory protein CD59. Overall, our studies suggest that acquisition and display of multiple, redundant complement regulators is involved in amoebic protection from complement lysis. These findings shed light on a novel strategy for immune evasion by a pathogen. Since other parasites use trogocytosis for cell killing, our findings may apply to the pathogenesis of other infections.

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

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.

Trogocytosis by Entamoeba histolytica Mediates Acquisition and Display of Human Cell Membrane Proteins and Evasion of Lysis by Human Serum.

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.

Trypanin, a component of the flagellar Dynein regulatory complex, is essential in bloodstream form African trypanosomes.

The Trypanosoma brucei flagellum is a multifunctional organelle with critical roles in motility, cellular morphogenesis, and cell division. Although motility is thought to be important throughout the trypanosome lifecycle, most studies of flagellum structure and function have been restricted to the procyclic lifecycle stage, and our knowledge of the bloodstream form flagellum is limited. We have previously shown that trypanin functions as part of a flagellar dynein regulatory system that transmits regulatory signals from the central pair apparatus and radial spokes to axonemal dyneins. Here we investigate the requirement for this dynein regulatory system in bloodstream form trypanosomes. We demonstrate that trypanin is localized to the flagellum of bloodstream form trypanosomes, in a pattern identical to that seen in procyclic cells. Surprisingly, trypanin RNA interference is lethal in the bloodstream form. These knockdown mutants fail to initiate cytokinesis, but undergo multiple rounds of organelle replication, accumulating multiple flagella, nuclei, kinetoplasts, mitochondria, and flagellum attachment zone structures. These findings suggest that normal flagellar beat is essential in bloodstream form trypanosomes and underscore the emerging concept that there is a dichotomy between trypanosome lifecycle stages with respect to factors that contribute to cell division and cell morphogenesis. This is the first time that a defined dynein regulatory complex has been shown to be essential in any organism and implicates the dynein regulatory complex and other enzymatic regulators of flagellar motility as candidate drug targets for the treatment of African sleeping sickness.

The flagellum of Trypanosoma brucei: new tricks from an old dog.

African trypanosomes, i.e. Trypanosoma brucei and related sub-species, are devastating human and animal pathogens that cause significant human mortality and limit sustained economic development in sub-Saharan Africa. T. brucei is a highly motile protozoan parasite and coordinated motility is central to both disease pathogenesis in the mammalian host and parasite development in the tsetse fly vector. Therefore, understanding unique aspects of the T. brucei flagellum may uncover novel targets for therapeutic intervention in African sleeping sickness. Moreover, studies of conserved features of the T. brucei flagellum are directly relevant to understanding fundamental aspects of flagellum and cilium function in other eukaryotes, making T. brucei an important model system. The T. brucei flagellum contains a canonical 9+2 axoneme, together with additional features that are unique to kinetoplastids and a few closely-related organisms. Until recently, much of our knowledge of the structure and function of the trypanosome flagellum was based on analogy and inference from other organisms. There has been an explosion in functional studies in T. brucei in recent years, revealing conserved as well as novel and unexpected structural and functional features of the flagellum. Most notably, the flagellum has been found to be an essential organelle, with critical roles in parasite motility, morphogenesis, cell division and immune evasion. This review highlights recent discoveries on the T. brucei flagellum.

Inhibition of Amebic Lysosomal Acidification Blocks Amebic Trogocytosis and Cell Killing.

Entamoeba histolytica ingests fragments of live host cells in a nibbling-like process termed amebic trogocytosis. Amebic trogocytosis is required for cell killing and contributes to tissue invasion, which is a hallmark of invasive amebic colitis. Work done prior to the discovery of amebic trogocytosis showed that acid vesicles are required for amebic cytotoxicity. In the present study, we show that acidified lysosomes are required for amebic trogocytosis and cell killing. Interference with lysosome acidification using ammonium chloride, a weak base, or concanamycin A, a vacuolar H+ ATPase inhibitor, decreased amebic trogocytosis and amebic cytotoxicity. Our data suggest that the inhibitors do not impair the ingestion of an initial fragment but rather block continued trogocytosis and the ingestion of multiple fragments. The acidification inhibitors also decreased phagocytosis, but not fluid-phase endocytosis. These data suggest that amebic lysosomes play a crucial role in amebic trogocytosis, phagocytosis, and cell killing.IMPORTANCEE. histolytica is a protozoan parasite that is prevalent in low-income countries, where it causes potentially fatal diarrhea, dysentery, and liver abscesses. Tissue destruction is a hallmark of invasive E. histolytica infection. The parasite is highly cytotoxic to a wide range of human cells, and parasite cytotoxic activity is likely to drive tissue destruction. E. histolytica is able to kill human cells through amebic trogocytosis. This process also contributes to tissue invasion. Trogocytosis has been observed in other organisms; however, little is known about the mechanism in any system. We show that interference with lysosomal acidification impairs amebic trogocytosis, phagocytosis, and cell killing, indicating that amebic lysosomes are critically important for these processes.

Taking a bite: Amoebic trogocytosis in Entamoeba histolytica and beyond.

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.

Chew on this: amoebic trogocytosis and host cell killing by Entamoeba histolytica.

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.

Three-dimensional structure of the Trypanosome flagellum suggests that the paraflagellar rod functions as a biomechanical spring.

Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture. Trypanosoma brucei is a uniflagellated protozoan parasite that causes African sleeping sickness. The T. brucei flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the T. brucei flagellum. We define the T. brucei axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57 nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of T. brucei cell motility.

Tissue destruction and invasion by Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amebiasis, a disease that is a major source of morbidity and mortality in the developing world. The potent cytotoxic activity of the parasite appears to underlie disease pathogenesis, although the mechanism is unknown. Recently, progress has been made in determining that the parasite activates apoptosis in target cells and some putative effectors have been identified. Recent studies have also begun to unravel the host genetic determinants that influence infection outcome. Thus, we are beginning to get a clearer picture of how this parasite manages to infect, invade and ultimately inflict devastating tissue destruction.