Fibronectin induces a transition from amoeboid to a fan morphology and modifies migration in Entamoeba histolytica.

Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.

An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1.

The parasite Entamoeba histolytica is the etiological agent of amoebiasis, a major cause of morbidity and mortality due to parasitic diseases in developing countries. Phagocytosis is an essential mode of obtaining nutrition and has been associated with the virulence behaviour of E. histolytica. Signalling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remains to be elucidated in this parasite. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica and have described some of the molecules that play key roles in the process. Here we showed the involvement of non-Dbl Rho Guanine Nucleotide Exchange Factor, EhGEF in regulation of amoebic phagocytosis by regulating activation of EhRho1. EhGEF was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. Our observation from imaging, pull down experiments and down regulating expression of different molecules suggest that EhGEF interacts with EhRho1 and it is required during initiation of phagocytosis and phagosome formation. Also, biophysical, and computational analysis reveals that EhGEF mediates GTP exchange on EhRho1 via an unconventional pathway. In conclusion, we describe a non-Dbl EhGEF of EhRho1 which is involved in endocytic processes of E. histolytica.

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

Lipid transfer proteins (LTPs) are the key contributor of organelle-specific lipid distribution and cellular lipid homeostasis. Here, we report a novel implication of LTPs in phagocytosis, trogocytosis, pinocytosis, biosynthetic secretion, recycling of pinosomes, and motility of the parasitic protist E. histolytica, the etiological agent of human amoebiasis. We show that two StAR-related lipid transfer (START) domain-containing LTPs (named as EhLTP1 and 3) are involved in these biological pathways in an LTP-specific manner. Our findings provide novel implications of LTPs, which are relevant to the elucidation of pathophysiology of the diseases caused by parasitic protists.

Coordinated activity of amoebic formin and profilin are essential for phagocytosis.

For the protist parasite Entamoeba histolytica, endocytic processes, such as phagocytosis, are essential for its survival in the human gut. The actin cytoskeleton is involved in the formation of pseudopods and phagosomal vesicles by incorporating a number of actin-binding and modulating proteins along with actin in a temporal manner. The actin dynamics, which comprises polymerization, branching, and depolymerization is very tightly regulated and takes place directionally at the sites of initiation of phagocytosis. Formin and profilin are two actin-binding proteins that are known to regulate actin cytoskeleton dynamics and thereby, endocytic processes. In this article, we report the participation of formin and profilin in E. histolytica phagocytosis and propose that these two proteins interact with each other and their sequential recruitment at the site is required for the successful completion of phagocytosis. The evidence is based on detailed microscopic, live imaging, interaction studies, and expression downregulation. The cells downregulated for expression of formin show absence of profilin at the site of phagocytosis, whereas downregulation of profilin does not affect formin localization.

The non-LTR retrotransposons of Entamoeba histolytica: genomic organization and biology.

Genome sequence analysis of Entamoeba species revealed various classes of transposable elements. While E. histolytica and E. dispar are rich in non-long terminal repeat (LTR) retrotransposons, E. invadens contains predominantly DNA transposons. Non-LTR retrotransposons of E. histolytica constitute three families of long interspersed nuclear elements (LINEs), and their short, nonautonomous partners, SINEs. They occupy ~ 11% of the genome. The EhLINE1/EhSINE1 family is the most abundant and best studied. EhLINE1 is 4.8 kb, with two ORFs that encode functions needed for retrotransposition. ORF1 codes for the nucleic acid-binding protein, and ORF2 has domains for reverse transcriptase (RT) and endonuclease (EN). Most copies of EhLINEs lack complete ORFs. ORF1p is expressed constitutively, but ORF2p is not detected. Retrotransposition could be demonstrated upon ectopic over expression of ORF2p, showing that retrotransposition machinery is functional. The newly retrotransposed sequences showed a high degree of recombination. In transcriptomic analysis, RNA-Seq reads were mapped to individual EhLINE1 copies. Although full-length copies were transcribed, no full-length 4.8 kb transcripts were seen. Rather, sense transcripts mapped to ORF1, RT and EN domains. Intriguingly, there was strong antisense transcription almost exclusively from the RT domain. These unique features of EhLINE1 could serve to attenuate retrotransposition in E. histolytica.

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

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.

Two isotypes of phosphatidylinositol 3-phosphate-binding sorting nexins play distinct roles in trogocytosis in Entamoeba histolytica.

Phosphatidylinositol phosphates (PIPs) function as important second messengers in many cellular events. In the human intestinal protist Entamoeba histolytica, where phagocytosis/trogocytosis plays an indispensable role in proliferation and pathophysiology during infection, various PIPs are involved in multiple steps of phago/trogocytosis. PI3-phosphate (PI3P) plays a pivotal role in the biogenesis of phagosome/trogosomes via recruitment of PI3P effectors. Because no known PI3P downstream effectors are conserved in E. histolytica, we exploited a unique method to identify the proteins PI3P dependently recruited to phagosomes. We rationalised that overexpression of PI3P-binding GFP-HrsFYVE competes for PI3P on phagosomal membranes and results in dissociation of PI3P effectors from phagosomes. EhVps26 and EhVps35, but not sorting nexins (SNXs), of the retromer complex were detected from phagosomes only without GFP-HrsFYVE overexpression. Two potential SNXs, EhSNX1 and EhSNX2, identified in the genome, possess only phox homology domain and specifically bound to PI3P, but retromer components, EhVps26 and EhVps35, did not bind to PI3P. Live and immunofluorescence imaging showed that EhSNX1 was recruited to the trogocytic cup and tunnel-like structures, and subsequently, EhSNX2 was recruited to trogosomes. Furthermore, EhSNX1, but not EhSNX2, specifically bound to Arp2/3 and EhVps26, which were localised to the tunnel-like structures and the trogosomes, respectively. EhSNX2 gene silencing increased trogocytosis, suggesting that EhSNX2 plays an inhibitory role in trogocytosis.

Extracellular vesicles released by anaerobic protozoan parasites: Current situation.

Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism for transferring information between cells and organisms across all three kingdoms of life. Parasitic unicellular eukaryotes use EVs as vehicles for intercellular communication and host manipulation. Pathogenic protozoans are able to modulate the immune system of the host and establish infection by transferring a wide range of molecules contained in different types of EVs. In addition to effects on the host, EVs are able to transfer virulence factors, drug-resistance genes and differentiation factors between parasites. In this review we cover the current knowledge on EVs from anaerobic or microaerophilic extracellular protozoan parasites, including Trichomonas vaginalis, Tritrichomonas foetus, Giardia intestinalis and Entamoeba histolytica, with a focus on their potential role in the process of infection. The role of EVs in host: parasite communication adds a new level of complexity to our understanding of parasite biology, and may be a key to understand the complexity behind their mechanism of pathogenesis.

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites.

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.

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.

Morphological and Motility Features of the Stable Bleb-Driven Monopodial Form of Entamoeba and Its Importance in Encystation.

Entamoeba histolytica and its reptilian counterpart and encystation model Entamoeba invadens formed a polarized monopodial morphology when treated with pentoxifylline. This morphology was propelled by retrograde flow of the cell surface resulting from a cyclic sol-gel conversion of cytoplasm and a stable bleb at the leading edge. Pentoxifylline treatment switched the unpolarized, adherent trophozoites to the nonadherent, stable bleb-driven form and altered the motility pattern from slow and random to fast, directionally persistent, and highly chemotactic. Interestingly, exogenously added adenosine produced multiple protrusions and random motility, an opposite phenotype to that of pentoxifylline. Thus, pentoxifylline, an adenosine antagonist, may be inducing the monopodial morphology by preventing lateral protrusions and restricting the leading edge to one site. The polarized form of E. invadens was aggregation competent, and time-lapse microscopy of encystation revealed its appearance during early hours, mediating the cell aggregation by directional cell migration. The addition of purine nucleotides to in vitro encystation culture prevented the formation of polarized morphology and inhibited the cell aggregation and, thus, the encystation, which further showed the importance of the polarized form in the Entamoeba life cycle. Cell polarity and motility are essential in the pathogenesis of Entamoeba parasites, and the stable bleb-driven polarized morphology of Entamoeba may also be important in invasive amoebiasis.

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.

Target identification and intervention strategies against amebiasis.

Entamoeba histolytica is the etiological agent of amebiasis, which is an endemic parasitic disease in developing countries and is the cause of approximately 70,000 deaths annually. E. histolytica trophozoites usually reside in the colon as a non-pathogenic commensal in most infected individuals (90% of infected individuals are asymptomatic). For unknown reasons, these trophozoites can become virulent and invasive, cause amebic dysentery, and migrate to the liver where they cause hepatocellular damage. Amebiasis is usually treated either by amebicides which are classified as (a) luminal and are active against the luminal forms of the parasite, (b) tissue and are effective against those parasites that have invaded tissues, and (c) mixed and are effective against the luminal forms of the parasite and those forms which invaded the host's tissues. Of the amebicides, the luminal amebicide, metronidazole (MTZ), is the most widely used drug to treat amebiasis. Although well tolerated, concerns about its adverse effects and the possible emergence of MTZ-resistant strains of E. histolytica have led to the development of new therapeutic strategies against amebiasis. These strategies include improving the potency of existing amebicides, discovering new uses for approved drugs (repurposing of existing drugs), drug rediscovery, vaccination, drug targeting of essential E. histolytica components, and the use of probiotics and bioactive natural products. This review examines each of these strategies in the light of the current knowledge on the gut microbiota of patients with amebiasis.

Structure of Ca2+-binding protein-6 from Entamoeba histolytica and its involvement in trophozoite proliferation regulation.

Cell cycle of Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (abbreviated hereafter as EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that EhCaBP6 modulates microtubule dynamics by increasing the rate of tubulin polymerization. Our results, including structural inferences, suggest that EhCaBP6 is an unusual CaBP involved in regulating cell proliferation in E. histolytica similar to nuclear Calmodulin.

EhRho1 regulates phagocytosis by modulating actin dynamics through EhFormin1 and EhProfilin1 in Entamoeba histolytica.

The protist parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries and a major cause of morbidity and mortality. Invasive infection in amoebiasis mostly affects intestinal epithelial cell lining but can also involve other organs, such as liver, lungs, or brain. Phagocytosis is an essential mode of nutrition in amoeba and has often been associated with virulence behaviour of E. histolytica. E. histolytica possesses a highly dynamic and actin-rich cytoskeleton that is thought to be involved in many processes, such as motility, pseudopod formation, and pathogenesis. Rho GTPases are known to be key regulators of the actin cytoskeleton and consequently influence the shape and movement of cells. Our study is mainly focused to understand the role of EhRho1 in the phagocytosis process of E. histolytica. EhRho1 got enriched in the phagocytic cups along with EhActin and remains attached with phagosomal membrane. However, there was no direct binding of EhRho1 with G- or F-actin, though binding was observed with the actin nucleating proteins EhFormin1 and EhProfilin1. Overexpression of dominant negative mutant or lowering the expression by antisense RNA of EhRho1 in trophozoites caused delocalisation of EhFormin1 and EhProfilin1 from phagocytic cups, which results in impairment of phagocytic process and decrease in F-actin content. The overall results show that EhRho1 regulates phagocytosis by modulating actin dynamics through recruitment of EhFormin1 and EhProfilin1 at the phagocytosis nucleation site in E. histolytica.

Identification of the gene encoding the TATA box-binding protein-associated factor 1 (TAF1) and its putative role in the heat shock response in the protozoan parasite Entamoeba histolytica.

Transcription factor IID (TFIID) is a cornerstone in the transcription initiation in eukaryotes. It is composed of TBP and approximately 14 different subunits named TBP-associated factors (TAFs). TFIID has a key role in transcription of many genes involved in cell proliferation, cell growth, cell cycle, cell cycle checkpoint, and various other processes as well. Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, represents a major global health concern. Our research group has previously reported the genes coding the TATA box-binding protein (EhTBP) and TBP-related factor 1 (EhTRF1), which displayed different mRNA levels in trophozoites under different stress conditions. In this work, we identified the TBP-associated factor 1 (Ehtaf1) gene in the E. histolytica genome, which possess a well-conserved DUF domain and a Bromo domain located in the middle and C-terminus of the protein, respectively. The EhTAF1-DUF domain tertiary structure is similar to the corresponding HsTAF1 DUF domain. RT-qPCR experiments with RNA isolated from trophozoites harvested at different time points of the growth curve and under different stress conditions revealed that the Ehtaf1 gene was found slightly upregulated in the death phase of growth curve, but under heat shock stress, it was found upregulated 10 times, suggesting that Ehtaf1 might have an important role in the heat shock stress response. We also found that EhTAF1 is expressed in the nucleus and cytoplasm at 37 °C, but under heat shock stress, it is overexpressed in both the nucleus and cytoplasm, and partially colocalized with EhHSP70 in cytoplasm.

EFFECT OF LACTOBACILLUS POSTBIOTICS ON ENTAMOEBA HISTOLYTICA TROPHOZOITES.

BACKGROUND: Amebiasis is an infectious disease caused by Entamoeba histolytica. It represents one of the three worldwide leading causes of death by parasites and a public health problem due to its frequency, morbidity, mortality, and easy dispersion. OBJECTIVE: The study was aimed to evaluate the in vitro effect of Lactobacillus spp. postbiotics on E. histolytica trophozoites (HM1-IMSS strain) and to determine morphometric changes in trophozoite membrane by atomic force microscopy (AFM). METHODS: Bioassays on trophozoites were conducted with lyophilized postbiotics at 0.1, 0.3, and 0.5 mg/mL concentrations, and trophozoite samples were obtained for AFM analysis. RESULTS: Results indicated postbiotic inhibitory activity; the highest percentage inhibition was 89.63% at 0.5 mg/mL. Trophozoites nanomechanical analysis showed 28.32% increase in ruggedness and 56% decrease in size with treatments compared to the control. CONCLUSION: Our study showed that the synergy of Lactobacillus postbiotics inhibited E. histolytica HM1-IMSS in vitro growth under axenic conditions, inducing morphometric alterations in trophozoites' cell membrane. These results would allow designing strategies or treatments aimed at E. histolytica control in the future.

Amoeboid movement in protozoan pathogens.

Entamoeba histolytica, the causative agent of amoebiasis, is a protozoan parasite characterised by its amoeboid motility, which is essential to its survival and invasion of the human host. Elucidating the molecular mechanisms leading to invasion of human tissues by E. histolytica requires a quantitative understanding of how its cytoskeleton deforms and tailors its mode of migration to the local microenvironment. Here we review the wide range of methods available to extract biophysical information from amoeboid cells, from interventional techniques to computational modelling approaches, and discuss how recent developments in bioimaging and bioimage informatics can complement our understanding of cellular morphodynamics at the intracellular level.

The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5ß1 Integrin at the Macrophage-Amebae Intercellular Junction.

Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5ß1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5ß1 integrin and NLRP3 into the intercellular junction, where α5ß1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5ß1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5ß1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5ß1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5ß1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection.