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.

Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite, Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase (CS, O-acetylserine sulfhydrylase), does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC₅₀ values of 0.31–490 μM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC₅₀ values of 18 and 0.8 μM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC₅₀ values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to CS inhibition.

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. Comparative genomic analysis of pathogens is used to identify genes associated with disease manifestations. In the present study, this approach was used to identify genes, present in a strain of Entamoeba histolytica isolated from dysenteric patients, but absent in strains isolated from asymptomatic infected individuals. Strain KU27 is an “avirulent” strain, or less virulent as compared to the virulence of other strains. KU27 was isolated from an asymptomatic infected individual, and is incapable of producing abscesses in a hamster liver abscess model. A previous genotyping study, using tRNA-linked short tandem repeats as markers, also identified this Japanese strain as a genetically distinct low-virulence strain. We conducted a comparative genomic analysis of strain KU27 against virulent strain of E. histolytica. Our analysis identified an AIG1 gene as a potential determinant of virulence. Further, we demonstrated that the gene product is involved in the formation of protrusions on the plasma membrane and adherence to host cells. To the best of our knowledge, this is the first study to identify a virulence-associated gene by comparative genomics. Here, we demonstrate the causal connection of the identified gene with virulence by the analysis of cell morphology, adhesion competence, and distribution among clinical isolates.

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.

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.