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Macropinocytosis is an evolutionarily conserved endocytic process that plays a vital role in internalizing extracellular fluids and particles in cells. This non-selective endocytic pathway is crucial for various physiological functions such as nutrient uptake, sensing, signaling, antigen presentation, and cell migration. While macropinocytosis has been extensively studied in macrophages and cancer cells, the molecular mechanisms of macropinocytosis in pathogens are less understood. It has been known that Entamoeba histolytica, the causative agent of amebiasis, exploits macropinocytosis for survival and pathogenesis. Since macropinocytosis is initiated by actin polymerization, leading to the formation of membrane ruffles and the subsequent trapping of solutes in macropinosomes, actin cytoskeleton regulation is crucial. Thus, this study focuses on unraveling the role of well-conserved actin cytoskeleton regulators, Rho small GTPase family proteins, in macropinocytosis in E. histolytica. Through gene silencing of highly transcribed Ehrho/Ehrac genes and following flow cytometry analysis, we identified that silencing EhracM enhances dextran macropinocytosis and affects cellular migration persistence. Live imaging and interactome analysis unveiled the cytosolic and vesicular localization of EhRacM, along with its interaction with signaling and membrane traffic-related proteins, shedding light on EhRacM's multiple roles. Our findings provide insights into the specific regulatory mechanisms of macropinocytosis among endocytic pathways in E. histolytica, highlighting the significance of EhRacM in both macropinocytosis and cellular migration.

Autophagy is one of the bulk degradation systems and is conserved throughout eukaryotes. In the enteric protozoan parasite Entamoeba histolytica, the causative agent of human amebiasis, Atg8 is not exclusively involved in autophagy per se but also in other membrane traffic-related pathways such as phagosome biogenesis. We previously reported that repression of atg8 gene expression by antisense small RNA-mediated transcriptional gene silencing (gs) resulted in growth retardation, delayed endocytosis, and reduced acidification of endosomes and phagosomes. In this study, to better understand the role of Atg8 in phagocytosis and trogocytosis, we conducted a comparative proteomic analysis of phagosomes isolated from wild type and atg8-gs strains. We found that 127 and 107 proteins were detected >1.5-fold less or more abundantly, respectively, in phagosomes isolated from the atg8-gs strain, compared to the control strain. Among 127 proteins whose abundance was reduced in phagosomes from atg8-gs, a panel of proteins related to fatty acid metabolism, phagocytosis, and endoplasmic reticulum (ER) homeostasis was identified. Various lysosomal hydrolases and their receptors also tend to be excluded from phagosomes by atg8-gs, reinforcing the notion that Atg8 is involved in phagosomal acidification and digestion. On the contrary, among 107 proteins whose abundance increased in phagosomes from atg8-gs strain, ribosome-related proteins and metabolite interconversion enzymes are enriched. We further investigated the localization of several representative proteins, including adenylyl cyclase-associated protein and plasma membrane calcium pump, both of which were demonstrated to be recruited to phagosomes and trogosomes via an Atg8-dependent mechanism. Taken together, our study has provided the basis of the phagosome proteome to further elucidate molecular events in the Atg8-dependent regulatory network of phagosome/trogosome biogenesis in E. histolytica.

The severity of Entamoeba histolytica infection is determined by host immunology, pathogen virulence, and the intestinal environment. Conventional research for assessing pathogen virulence has been mainly performed using laboratory strains, such as a virulent HM-1: IMSS (HM-1) and an avirulent Rahman, under various artificial environmental conditions because of the difficulties of axenic isolation of the clinical strains. However, it is still unclear whether scientific knowledge based on laboratory strains are universally applicable to the true pathogenesis. Hereby, we performed transcriptomic analysis of clinical strains from patients with different degrees of disease severity, as well as HM-1 under different conditions. Even after several months of axenization, Clinical strains show the distinct profile in gene expression during in vitro passage, moreover, difference between any 2 of these strains was much greater than the changes on the liver challenge. Interestingly, 26 DEGs, which were closely related to the biological functions, were oppositely up- or down regulated between virulent Ax 19 (liver abscess) and avirulent Ax 11 (asymptomatic carrier). Additionally, RNAseq using laboratory strain (HM1) showed more than half of genes were differently expressed between continuously in vitro passaged HM1 (in vitro HM1) and periodically liver passaged HM1 (virulent HM1), which was much greater than the changes on the liver passage of virulent HM1. Also, transcriptomic analysis of a laboratory strain revealed that continuous environmental stress enhances its virulence via a shift in its gene expression profile. Changes in gene expression patterns on liver abscess formation were not consistent between clinical and laboratory strains.

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes including cytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite.

Trogocytosis is a mode of internalization of a part of a live cell by nibbling and is mechanistically distinct from phagocytosis, which implies internalization of a whole cell or a particle. Trogocytosis has been demonstrated in a broad range of cell types in multicellular organisms and is also known to be involved in a plethora of functions. In immune cells, trogocytosis is involved in the “cross-dressing” between antigen presenting cells and T cells, and is thus considered to mediate intercellular communication. On the other hand, trogocytosis has also been reported in a variety of unicellular organisms including the protistan (protozoan) parasite Entamoeba histolytica. E. histolytica ingests human T cell line by trogocytosis and acquires complement resistance and cross-dresses major histocompatibility complex (MHC) class I on the cell surface. Furthermore, trogocytosis and trogocytosis-like phenomena (nibbling of a live cell, not previously described as trogocytosis) have also been reported in other parasitic protists such as Trichomonas, Plasmodium, Toxoplasma, and free-living amoebae. Thus, trogocytosis is conserved in diverse eukaryotic supergroups as a means of intercellular communication. It is depicting the universality of trogocytosis among eukaryotes. In this review, we summarize our current understanding of trogocytosis in unicellular organisms, including the history of its discovery, taxonomical distribution, roles, and molecular mechanisms.

Entamoeba histolytica can kill host cells by trogocytosis, while it ingests dead cells by phagocytosis. Here, Somlata et al. show that EhAGCK1, an AGC family kinase, is specifically involved in trogocytosis, shedding light on the molecular differences between trogocytosis and phagocytosis.

Phosphatidylinositol (PtdIns) metabolism is indispensable in eukaryotes. Phosphoinositides (PIs) are phosphorylated derivatives of PtdIns and consist of seven species generated by reversible phosphorylation of the inositol moieties at the positions 3, 4, and 5. Each of the seven PIs has a unique subcellular and membrane domain distribution. In the enteric protozoan parasite Entamoeba histolytica, it has been previously shown that the PIs phosphatidylinositol 3-phosphate (PtdIns3P), PtdIns(4,5)P2, and PtdIns(3,4,5)P3 are localized to phagosomes/phagocytic cups, plasma membrane, and phagocytic cups, respectively. The localization of these PIs in E. histolytica is similar to that in mammalian cells, suggesting that PIs have orthologous functions in E. histolytica. In contrast, the conservation of the enzymes that metabolize PIs in this organism has not been well-documented. In this review, we summarized the full repertoire of the PI kinases and PI phosphatases found in E. histolytica via a genome-wide survey of the current genomic information. E. histolytica appears to have 10 PI kinases and 23 PI phosphatases. It has a panel of evolutionarily conserved enzymes that generate all the seven PI species. However, class II PI 3-kinases, type II PI 4-kinases, type III PI 5-phosphatases, and PI 4P-specific phosphatases are not present. Additionally, regulatory subunits of class I PI 3-kinases and type III PI 4-kinases have not been identified. Instead, homologs of class I PI 3-kinases and PTEN, a PI 3-phosphatase, exist as multiple isoforms, which likely reflects that elaborate signaling cascades mediated by PtdIns(3,4,5)P3 are present in this organism. There are several enzymes that have the nuclear localization signal: one phosphatidylinositol phosphate (PIP) kinase, two PI 3-phosphatases, and one PI 5-phosphatase; this suggests that PI metabolism also has conserved roles related to nuclear functions in E. histolytica, as it does in model organisms.

Autophagy is a eukaryotic cellular machinery that is able to degrade large intracellular components, including organelles, and plays a pivotal role in cellular homeostasis. Target materials are enclosed by a double membrane vesicle called autophagosome, whose formation is coordinated by autophagy-related proteins (ATGs). Studies of yeast and Metazoa have identified approximately 40 ATGs. Genome projects for unicellular eukaryotes revealed that some ATGs are conserved in all eukaryotic supergroups but others have arisen or were lost during evolution in some specific lineages. In spite of an apparent reduction in the ATG molecular machinery found in parasitic protists, it has become clear that ATGs play an important role in stage differentiation or organelle maintenance, sometimes with an original function that is unrelated to canonical degradative autophagy. In this review, we aim to briefly summarize the current state of knowledge in parasitic protists, in the light of the latest important findings from more canonical model organisms. Determining the roles of ATGs and the diversity of their functions in various lineages is an important challenge for understanding the evolutionary background of autophagy.

Phosphatidylinositol phosphates (PIPs) are involved in many cellular events as important secondary messengers. In Entamoeba histolytica, a human intestinal protozoan parasite, virulence-associated mechanisms such as cell motility, vesicular traffic, trogo- and phagocytosis are regulated by PIPs. It has been well established that PI3P, PI4P, and PI(3,4,5)P3 play specific roles during amoebic trogo- and phagocytosis. In the present study, we demonstrated the nuclear localization of PI4P in E. histolytica trophozoites in steady state with immunofluorescence imaging and immunoelectron microscopy, using anti-PI4P antibodies and PI4P biosensors [substrate of the Icm/ Dot type IV secretion system (SidM)]. We further showed that the nuclear PI4P decreased after a co-culture with human erythrocytes or Chinese hamster ovary (CHO) cells. However, concomitant changes in the localization and the amount of PI(4,5)P2, which is the expected major metabolized (phosphorylated) product of PI4P, were not observed. This phenomenon was specifically caused by whole or ghost erythrocytes and CHO cells, but not artificial beads. The amount of PIP2 and PIP, biochemically estimated by [32P]-phosphate metabolic labeling and thin layer chromatography, was decreased upon erythrocyte adherence. Altogether, our data indicate for the first time in eukaryotes that erythrocyte attachment leads to the metabolism of nuclear PIPs, and metabolites other than PI(4,5)P2 may be involved in the regulation of downstream cellular events such as cytoskeleton rearrangement or transcriptional regulation.

Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and cysteine synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the cysteine-deprived medium compared to the cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the cysteine-deprived medium when compared to the growth in the cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of cysteine than that in the presence of cysteine), although the IC50 value in the cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of cysteine. The cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the cysteine-containing medium, the cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a cysteine-dependent manner with relatively low mammalian cytotoxicity.

[This corrects the article DOI: 10.1371/journal.ppat.1006882.].

The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-induced gene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment.

Encystation is an essential differentiation process for the completion of the life cycle of a group of intestinal protozoa including Entamoeba histolytica, the causative agent of intestinal and extraintestinal amebiasis. However, regulation of gene expression during encystation is poorly understood. To comprehensively understand the process at the molecular level, the transcriptomic profiles of E. invadens, which is a related reptilian species that causes an invasive disease similar to that of E. histolytica, was investigated during encystation. Using a custom-generated Affymetrix platform microarray, we performed time course (0.5, 2, 8, 24, 48, and 120 h) gene expression analysis of encysting E. invadens. ANOVA analysis revealed that a total of 1,528 genes showed ≥3 fold up-regulation at one or more time points, relative to the trophozoite stage. Of these modulated genes, 8% (116 genes) were up-regulated at the early time points (0.5, 2 and 8h), while 63% (962 genes) were up-regulated at the later time points (24, 48, and 120 h). Twenty nine percent (450 genes) are either up-regulated at 2 to 5 time points or constitutively up-regulated in both early and late stages. Among the up-regulated genes are the genes encoding transporters, cytoskeletal proteins, proteins involved in vesicular trafficking (small GTPases), Myb transcription factors, cysteine proteases, components of the proteasome, and enzymes for chitin biosynthesis. This study represents the first kinetic analysis of gene expression during differentiation from the invasive trophozoite to the dormant, infective cyst stage in Entamoeba. Functional analysis on individual genes and their encoded products that are modulated during encystation may lead to the discovery of targets for the development of new chemotherapeutics that interfere with stage conversion of the parasite.

Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. It is estimated approximately 1 % of humans are infected with E histolytica, resulting in an estimate of 100,000 deaths annually. Clinical manifestations of amebic infection range widely from asymptomatic to severe symptoms, including dysentery and extra-intestinal abscesses. Like other infectious diseases, it is assumed that only -20% of infected individuals develop symptoms, and genetic factors of both the parasite and humans as well as the environmental factors, e.g., microbiota, determine outcome of infection. There are multiple essential steps in amebic infection: degradation of and invasion into the mucosal layer, adherence to the intestinal epithelium, invasion into the tissues, and dissemination to other organs. While the mechanisms of invasion and destruction of the host tissues by the amebae during infection have been elucidated at the molecular levels, it remains largely uncharacterized how the parasite survive in the host by evading and attacking host immune system. Recently, the strategies for immune evasion by the parasite have been unraveled, including immunomodulation to suppress IFN-γ production, elimination of immune cells and soluble immune mediators, and metabolic alterations against reactive oxygen and nitrogen species to fend off the attack from immune system. In this review, we summarized the latest knowledge on immune reaction and immune evasion during amebiasis. [ABSTRACT FROM AUTHOR]

Antimicrobial chemotherapy is critical in the fight against infectious diseases caused by Entamoeba histolytica. Among the drugs available for the treatment of amebiasis, metronidazole (MTZ) is considered the drug of choice. Recently, in vitro studies have described MTZ resistance and the potential mechanisms involved. Costs to fitness and adaptive responses associated with resistance, however, have not been investigated. In this study we generated an HM-1 derived strain resistant to 12µM MTZ (MTZR). We examined its phenotypic and transcriptional profile to determine the consequences and mRNA level changes associated with MTZ resistance. Our results indicated increased cell size and granularity, and decreased rates in cell division, adhesion, phagocytosis, cytopathogenicity, and glucose consumption. Transcriptome analysis revealed 142 differentially expressed genes in MTZR. In contrast to other MTZ resistant parasites, MTZR did not down-regulate pyruvate:ferredoxin oxidoreductase, but showed increased expression of genes for a hypothetical protein (HP1) and several iron-sulfur flavoproteins, and downregulation of genes for leucine-rich proteins. Fisher’s exact test showed 24 significantly enriched GO terms in MTZR, and a 3-way comparison of modulated genes in MTZR against those of MTZR cultured without MTZ and HM-1 cultured with MTZ, showed that 88 genes were specific to MTZR. Overall, our findings suggested that MTZ resistance is associated with specific transcriptional changes and decreased parasite virulence. [ABSTRACT FROM AUTHOR]