Trogocytosis in Unicellular Eukaryotes.

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.

The atypical protein arginine methyltrasferase of Entamoeba histolytica (EhPRMTA) is involved in cell proliferation, heat shock response and in vitro virulence.

Protein arginine methylation regulates several cellular events, including epigenetics, splicing, translation, and stress response, among others. This posttranslational modification is catalyzed by protein arginine methyltransferases (PRMTs), which according to their products are classified from type I to type IV. The type I produces monomethyl arginine and asymmetric dimethyl arginine; in mammalian there are six families of this PRMT type (PRMT1, 2, 3, 4, 6, and 8). The protozoa parasite Entamoeba histolytica has four PRMTs related to type I; three of them are similar to PRMT1, but the other one does not show significant homology to be grouped in any known PRMT family, thus we called it as atypical PRMT (EhPRMTA). Here, we showed that EhPRMTA does not contain several of the canonical amino acid residues of type I PRMTs, confirming that it is an atypical PRMT. A specific antibody against EhPRMTA localized this protein in cytoplasm. The recombinant EhPRMTA displayed catalytic activity on commercial histones and the native enzyme modified its expression level during heat shock and erythrophagocytosis. Besides, the knockdown of EhPRMTA produced an increment in cell growth, and phagocytosis, but decreases cell migration and the survival of trophozoites submitted to heat shock, suggesting that this protein is involved in regulate negatively or positively these events, respectively. Thus, results suggest that this methyltransferase regulates some cellular functions related to virulence and cell surviving.

Characterization of Extracellular Vesicles from Entamoeba histolytica Identifies Roles in Intercellular Communication That Regulates Parasite Growth and Development.

Extracellular vesicles (EVs) secreted by eukaryotic and prokaryotic cells to transport lipids, proteins, and nucleic acids to the external environment have important roles in cell-cell communication through cargo transfer. We identified and characterized EVs from Entamoeba histolytica, a protozoan parasite and a human pathogen. Conditioned medium from amebic parasites contained particles consistent with the expected size and morphology of EVs. Mass spectrometry was used to characterize the EV proteome and showed that it was enriched in common exosome marker proteins, including proteins associated with vesicle formation, cell signaling, and metabolism, as well as cytoskeletal proteins. Additionally, the EVs were found to selectively package small RNAs (sRNA), which were protected within the vesicles against RNase treatment. Sequencing analysis of the sRNA contained in EVs revealed that the majority were 27 nucleotides (nt) in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba RNA interference (RNAi) pathway proteins, including Argonaute, were also present in amebic EVs. Interestingly, we found that the amebic EVs impacted intercellular communication between parasites and altered encystation efficiency. EVs isolated from encysting parasites promoted encystation in other parasites, whereas EVs from metabolically active trophozoites impeded encystation. Overall, the data reveal that Entamoeba secrete EVs that are similar in size and shape to previously characterized exosomes from other organisms and that these EVs contain a defined protein and small RNA cargo and have roles in intercellular communication among parasites and influence growth kinetics.

Crystal structure of O-Acetylserine sulfhydralase (OASS) isoform 3 from Entamoeba histolytica: Pharmacophore-based virtual screening and validation of novel inhibitors.

The l-cysteine is crucial for growth, survival, defense against oxidative stress, and pathogenesis of Entamoeba histolytica. The de novo biosynthesis of l-cysteine in E. histolytica, has a two-step pathway, where O-acetylserine sulfhydrylase (OASS) catalyses the last step by converting OAS to l-cysteine. This pathway is absent in humans and hence represents a promising target for novel therapeutics. E. histolytica expresses three isoforms of OASS and knockdown studies showed the importance of these enzymes for the survival of the pathogen. Here, we report the crystal structure of OASS isoform 3 from E. histolytica to 1.54 Å resolution. The active site geometries and kinetics of EhOASS3 and EhOASS1 structures were found to be very similar. Small-molecule libraries were screened against EhOASS3 and compounds were shortlisted based on the docking scores. F3226-1387 showed best inhibition with IC50 of 38 µM against EhOASS3 and was able to inhibit the growth of the organism to 72%.

'Entamoeba histolytica' identified by stool microscopy from children with acute diarrhoea in Peru is not E. histolytica.

Entamoeba histolytica is a rare but feared pathogen owing to its related morbidity and mortality. Physicians in an ambulatory clinic in Cusco noted frequent reports of E. histolytica diagnosed by microscopy. Other non-pathogenic species of Entamoeba have an identical microscopic appearance. To determine whether the organisms were actually E. histolytica, faecal specimens from children aged six months to three years with diarrhoea were tested by a species-specific ELISA for E. histolytica antigen. Although 19/73 patients (26.0%) were presumptively diagnosed with amoebiasis based on microscopy, none were confirmed by ELISA. Most cases diagnosed as E. histolytic by microscopy in Peru are not infected by the pathogenic species and are probably colonised by non-pathogenic amoeba such as Entamoeba dispar.

Morphodynamics of the Actin-Rich Cytoskeleton in Entamoeba histolytica.

Entamoeba histolytica is the anaerobic protozoan parasite responsible for human amoebiasis, the third most deadly parasitic disease worldwide. This highly motile eukaryotic cell invades human tissues and constitutes an excellent experimental model of cell motility and cell shape deformation. The absence of extranuclear microtubules in Entamoeba histolytica means that the actin-rich cytoskeleton takes on a crucial role in not only amoebic motility but also other processes sustaining pathogenesis, such as the phagocytosis of human cells and the parasite's resistance of host immune responses. Actin is highly conserved among eukaryotes, although diverse isoforms exist in almost all organisms studied to date. However, E. histolytica has a single actin protein, the structure of which differs significantly from those of its human homologs. Here, we studied the expression, structure and dynamics of actin in E. histolytica. We used molecular and cellular approaches to evaluate actin gene expression during intestinal invasion by E. histolytica trophozoites. Based on a three-dimensional structural bioinformatics analysis, we characterized protein domains differences between amoebic actin and human actin. Fine-tuned molecular dynamics simulations enabled us to examine protein motion and refine the three-dimensional structures of both actins, including elements potentially accounting for differences changes in the affinity properties of amoebic actin and deoxyribonuclease I. The dynamic, multifunctional nature of the amoebic cytoskeleton prompted us to examine the pleiotropic forms of actin structures within live E. histolytica cells; we observed the cortical cytoskeleton, stress fibers, "dot-like" structures, adhesion plates, and macropinosomes. In line with these data, a proteomics study of actin-binding proteins highlighted the Arp2/3 protein complex as a crucial element for the development of macropinosomes and adhesion plaques.

The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation in Entamoeba histolytica.

One of the hallmarks of amoebic colitis is the detection of Entamoeba histolytica (Eh) trophozoites with ingested erythrocytes. Therefore, erythrophagocytosis is traditionally considered as one of the most important criteria to identify the pathogenic behavior of the amoebic trophozoites. Phagocytosis is an essential process for the proliferation and virulence of this parasite. Phagocytic cargo, upon internalization, follows a defined trafficking route to amoebic lysosomal degradation machinery. Here, we demonstrated the role of EhRab35 in the early and late phases of erythrophagocytosis by the amoeba. EhRab35 showed large vacuolar as well as punctate vesicular localization. The spatiotemporal dynamics of vacuolar EhRab35 and its exchange with soluble cytosolic pool were monitored by fluorescence recovery after photobleaching experiments. Using extensive microscopy and biochemical methods, we demonstrated that upon incubation with RBCs EhRab35 is recruited to the site of phagocytic cups as well as to the nascent phagosomes that harbor Gal/GalNAc lectin and actin. Overexpression of a dominant negative mutant of EhRab35 reduced phagocytic cup formation and thereby reduced RBC internalization, suggesting a potential role of the Rab GTPase in the cup formation. Furthermore, we also performed a phagosomal maturation assay and observed that the activated form of EhRab35 significantly increased the rate of RBC degradation. Interestingly, this mutant also significantly enhanced the number of acidic compartments in the trophozoites. Taken together, our results suggest that EhRab35 is involved in the initial stage of phagocytosis as well as in the phagolysosomal biogenesis in E. histolytica and thus contributes to the pathogenicity of the parasite.

Endoplasmic reticulum-resident Rab8A GTPase is involved in phagocytosis in the protozoan parasite Entamoeba histolytica.

Phagocytosis is indispensable for the pathogenesis of the intestinal protozoan parasite Entamoeba histolytica. Here, we showed that in E. histolytica Rab8A, which is generally involved in trafficking from the trans-Golgi network to the plasma membrane in other organisms but was previously identified in phagosomes of the amoeba in the proteomic analysis, primarily resides in the endoplasmic reticulum (ER) and participates in phagocytosis. We demonstrated that down-regulation of EhRab8A by small antisense RNA-mediated transcriptional gene silencing remarkably reduced adherence and phagocytosis of erythrocytes, bacteria and carboxylated latex beads. Surface biotinylation followed by SDS-PAGE analysis revealed that the surface expression of several proteins presumably involved in target recognition was reduced in the EhRab8A gene-silenced strain. Further, overexpression of wild-type EhRab8A augmented phagocytosis, whereas expression of the dominant-negative form of EhRab8A resulted in reduced phagocytosis. These results indicated that EhRab8A regulates transport of surface receptor(s) for the prey from the ER to the plasma membrane. To our knowledge, this is the first report that the ER-resident Rab GTPase is involved in phagocytosis through the regulation of trafficking of a surface receptor, supporting a premise of direct involvement of the ER in phagocytosis.

Calpain-like: A Ca(2+) dependent cystein protease in Entamoeba histolytica cell death.

Entamoeba histolytica programmed cell death (PCD) induced by G418 is characterized by the release of important amounts of intracellular calcium from reservoirs. Nevertheless, no typical caspases have been detected in the parasite, the PCD phenotype is inhibited by the cysteine protease inhibitor E-64. These results strongly suggest that Ca(2+)-dependent proteases could be involved in PCD. In this study, we evaluate the expression and activity of a specific dependent Ca(2+) protease, the calpain-like protease, by real-time quantitative PCR (RTq-PCR), Western blot assays and a enzymatic method during the induction of PCD by G418. Alternatively, using cell viability and TUNEL assays, we also demonstrated that the Z-Leu-Leu-Leu-al calpain inhibitor reduced the rate of cell death. The results demonstrated 4.9-fold overexpression of calpain-like gene 1.5 h after G418 PCD induction, while calpain-like protein increased almost two-fold with respect to basal calpain-like expression after 3 h of induction, and calpain activity was found to be approximately three-fold higher 6 h after treatment compared with untreated trophozoites. Taken together, these results suggest that this Ca(2+)-dependent protease could be involved in the executory phase of PCD.

Erythrophagocytosis in Entamoeba histolytica and Entamoeba dispar: a comparative study.

Entamoeba histolytica is the causative agent of human intestinal and liver amebiasis. The extraordinary phagocytic activity of E. histolytica trophozoites has been accepted as one of the virulence mechanisms responsible for their invasive capacity. The recognition of the noninvasive Entamoeba dispar as a different species has raised the question as to whether the lack of pathogenic potential of this ameba correlates with a limited phagocytic capacity. We have therefore compared the process of erythrophagocytosis in both species by means of light and video microscopy, hemoglobin measurement, and the estimation of reactive oxygen species (ROS). In the present study, we confirmed that E. dispar has lower erythrophagocytic capacity. We also observed by video microscopy a new event of erythrocyte opsonization-like in both species, being more characteristic in E. histolytica. Moreover, E. dispar showed a lower capacity to produce ROS compared with the invasive species and also showed a large population of amoebae that did not engulf any erythrocyte over time. Our results demonstrate that E. histolytica has a higher phagocytic capacity than E. dispar, including a higher rate of production of ROS in the course of ingesting red blood cells.

Caecal amebic colitis mimicking obstructing right sided colonic carcinoma with liver metastases: a rare case.

Intestinal Entamoeba Histolytica infection can lead to colitis, abscess formation, colonic perforation and rarely amoeboma. We report a case of colonic amoebiasis, in which the presenting symptoms and radiological findings closely resembled an obstructing right-sided colonic carcinoma, with liver metastases.

The flavonoid (-)-epicatechin affects cytoskeleton proteins and functions in Entamoeba histolytica.

Human amoebiasis is an intestinal disease with a global distribution. Due to reports of parasite resistance or susceptibility reduction to metronidazole treatment, there is a renewed interest for the search of new molecules with antiamoebic activity. The flavonoid (-)-epicatechin that was isolated from the Mexican medicinal plant Geranium mexicanum HBK has an in vitro activity against E. histolytica trophozoites, however its molecular effects have been poorly documented. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that E. histolytica cytoskeleton proteins exhibit differential abundance in response to (-)-epicatechin treatment. Moreover, functional assays revealed modification on pathogenic mechanisms associated with cytoskeleton functionality, namely, adhesion, migration, phagocytosis and cytolysis. Consequently, these data suggested that (-)-epicatechin could affect virulence properties of this human pathogen. BIOLOGICAL SIGNIFICANCE: This work contributes with some advances in the action mechanisms involved in the antiamoebic effect of the flavonoid (-)-epicatechin. We found that this flavonoid has an unusual effect on trophozoites growth that is dependent of its concentration. Additionally, we reported that (-)-epicatechin affects mainly amebic cytoskeleton proteins, which results in alteration on important virulence mechanisms, like adhesion, migration, phagocytosis and cytolysis. This study provides new knowledge about a potential alternative therapy directed to the treatment of 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.

Bioassay-guided fractionation of extracts from Codiaeum variegatum against Entamoeba histolytica discovers compounds that modify expression of ceramide biosynthesis related genes.

Leaves of Codiaeum variegatum ("garden croton") are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica.

Localization of phosphatidylinositol 4,5-bisphosphate to lipid rafts and uroids in the human protozoan parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal protozoan parasite and is the causative agent of amoebiasis. During invasive infection, highly motile amoebae destroy the colonic epithelium, enter the blood circulation, and disseminate to other organs such as liver, causing liver abscess. Motility is a key factor in E. histolytica pathogenesis, and this process relies on a dynamic actomyosin cytoskeleton. In other systems, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is known to regulate a wide variety of cellular functions, including signal transduction, actin remodeling, and cell motility. Little is known about the role of PI(4,5)P2 in E. histolytica pathogenicity. In this study, we demonstrate that PI(4,5)P2 is localized to cholesterol-rich microdomains, lipid rafts, and the actin-rich fractions of the E. histolytica membrane. Microscopy revealed that the trailing edge of polarized trophozoites, uroids, are highly enriched in lipid rafts and their constituent lipid, PI(4,5)P2. Polarization and enrichment of uroids and rafts with PI(4,5)P2 were enhanced upon treatment of E. histolytica cells with cholesterol. Exposure to cholesterol also increased intracellular calcium, which is a downstream effector of PI(4,5)P2, with a concomitant increase in motility. Together, our data suggest that in E. histolytica, PI(4,5)P2 may signal from lipid rafts and cholesterol may play a role in triggering PI(4,5)P2-mediated signaling to enhance the motility of this pathogen.

mRNA decay proteins are targeted to poly(A)+ RNA and dsRNA-containing cytoplasmic foci that resemble P-bodies in Entamoeba histolytica.

In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies). In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them "P-body-like structures". These foci contain additional mRNA degradation factors, including the EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5'-to-3' mRNA decay. The number of EhCAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i) EhCAF1 colocalized with poly(A)(+) RNA and (ii) during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P-body-like structures in E. histolytica. Our findings should open up opportunities for deciphering the mechanisms of mRNA degradation and RNA-based gene silencing in this deep-branching eukaryote.

Autophosphorylation of Ser428 of EhC2PK plays a critical role in regulating erythrophagocytosis in the parasite Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica can invade both intestinal and extra intestinal tissues resulting in amoebiasis. During the process of invasion E. histolytica ingests red blood and host cells using phagocytic processes. Though phagocytosis is considered to be a key virulence determinant, the mechanism is not very well understood in E. histolytica. We have recently demonstrated that a novel C2 domain-containing protein kinase, EhC2PK is involved in the initiation of erythrophagocytosis. Because cells overexpressing the kinase-dead mutant of EhC2PK displayed a reduction in erythrophagocytosis, it appears that kinase activity is necessary for initiation. Biochemical analysis showed that EhC2PK is an unusual Mn(2+)-dependent serine kinase. It has a trans-autophosphorylated site at Ser(428) as revealed by mass spectrometric and biochemical analysis. The autophosphorylation defective mutants (S428A, KDΔC) showed a reduction in auto and substrate phosphorylation. Time kinetics of in vitro kinase activity suggested two phases, an initial short slow phase followed by a rapid phase for wild type protein, whereas mutations in the autophosphorylation sites that cause defect (S428A) or conferred phosphomimetic property (S428E) displayed no distinct phases, suggesting that autophosphorylation may be controlling kinase activity through an autocatalytic mechanism. A reduction and delay in erythrophagocytosis was observed in E. histolytica cells overexpressing S428A and KDΔC proteins. These results indicate that enrichment of EhC2PK at the site of phagocytosis enhances the rate of trans-autophosphorylation, thereby increasing kinase activity and regulating the initiation of erythrophagocytosis in E. histolytica.

Lipids induce expression of serum-responsive transmembrane kinase EhTMKB1-9 in an early branching eukaryote Entamoeba histolytica.

Mechanisms underlying the initiation of proliferative response are known only for a few organisms, and are not understood for the medically important organisms including Entamoeba histolytica. The trans membrane kinase EhTMKB1-9 of E. histolytica is one of the early indicators of proliferation and its' expression is regulated by serum, one of the components necessary for cellular proliferation in vitro. In this study we show that bovine serum albumin (BSA) can induce EhTMKB1-9 expression in place of serum, and that both follow the same mechanism. Both serum and BSA use the same promoter element and the activation process is initiated through a PI3 kinase-mediated pathway. We further show that BSA activates EhTMKB1-9 due to the lipids associated with it and that unsaturated fatty acids are responsible for activation. These results suggest that lipid molecules are ligand(s) for initiation of a signaling system that stimulates EhTMKB1-9 expression.

Control of Entamoeba histolytica adherence involves metallosurface protease 1, an M8 family surface metalloprotease with homology to leishmanolysin.

Invasive amebiasis due to Entamoeba histolytica infection is an important cause of morbidity in developing countries. The E. histolytica genome contains two homologues to the metalloprotease leishmanolysin gene, Entamoeba histolytica MSP-1 (EhMSP-1) and EhMSP-2, while the commensal ameba Entamoeba dispar has lost EhMSP-1. In this study, we sought to characterize E. histolytica metallosurface protease 1 (EhMSP-1). Using immunoprecipitation and a model substrate, we found that EhMSP-1 was a functional metalloprotease. Confocal microscopy and flow cytometry revealed that EhMSP-1 localized to the cell surface and revealed the existence of distinct, nonclonal trophozoite populations with high and low EhMSP-1 surface abundance that became synchronized following serum starvation. Phenotypic assays were performed after silencing EhMSP-1. Adherence of EhMSP-1-deficient trophozoites to tissue culture cell monolayers was more than five times greater than that of control amebas, but surface staining of several antigens, including the galactose adherence lectin, was unchanged. EhMSP-1 silencing similarly increased adherence to both viable and apoptotic Jurkat lymphocytes. Tissue culture cell monolayer destruction was reduced by EhMSP-1 silencing, although it was blocked almost completely by inhibiting cysteine proteases. Consistent with a primary defect in regulation of amebic adherence, EhMSP-1 silencing also resulted in reduced mobility on tissue culture cell monolayers and in increased phagocytosis. In conclusion, EhMSP-1 was shown to be a surface metalloprotease involved in regulation of amebic adherence, with additional effects on cell motility, cell monolayer destruction, and phagocytosis.