Characterization of Entamoeba fatty acid elongases; validation as targets and provision of promising leads for new drugs against amebiasis.

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26-30 carbons) and/or medium (8-12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba.

Entamoeba histolytica: EhADH, an Alix Protein, Participates in Several Virulence Events through Its Different Domains.

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1-400 aa), Linker (246-446 aa) and Adh (444-687 aa) to evaluate their role in virulence. The TrophozBro11-400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226-446 also increased the virulence properties, but with lower effect than the TrophozBro11-400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444-687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.

Entamoeba moshkovskii as a potential model organism for Gal/GalNAc lectin intermediate subunit exhibition and functional identification.

In humans, Entamoeba histolytica is the main pathogen causing various amoebiases, while E. moshkovskii falls between being a pathogen and non-pathogen. The two species have similar behavior patterns but differ significantly in pathogenicity, with previous studies and clinical data indicating that E. moshkovskii has a low level of pathogenicity. Meaningfully, the biological characteristics of E. moshkovskii make it a potential model organism and a protein display platform for studying the functions of important Entamoeba proteins. Here, an Amoeba-pcDNA3.1 vector capable of overexpressing E. histolytica-sourced Igl-C protein was constructed and successfully transfected into E. moshkovskii. High levels of expression of the Igl-C, EGFP, and NeoR genes were identified in Igl-C-transfected trophozoites using qRT-PCR, and they were subsequently confirmed using immunoblotting. Transfection of Igl-C protein improved the adherence and phagocytosis of E. moshkovskii, demonstrating that E. histolytica Igl mediated amoebic adhesion. Moreover, as a manifestation of protein virulence, the ability of post-transfected trophozoites to induce inflammation in host macrophages was also enhanced. In conclusion, this study utilizing the characteristics of E. moshkovskii confirmed its potential to serve as a model organism. E. moshkovskii could replace E. histolytica as the target of gene editing, allowing more efficient study of amoebic pathogenicity.

Characterization of a unique attachment organelle: Single-cell force spectroscopy of Giardia duodenalis trophozoites.

The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. In its trophozoite form, G. duodenalis can adhere to the human intestinal epithelium and a variety of other, artificial surfaces. Its attachment is facilitated by a unique microtubule-based attachment organelle, the so-called ventral disc. The mechanical function of the ventral disc, however, is still debated. Earlier studies postulated that a dynamic negative pressure under the ventral disc, generated by persistently beating flagella, mediates the attachment. Later studies suggested a suction model based on structural changes of the ventral discs, substrate clutching or grasping, or unspecific contact forces. In this study, we aim to contribute to the understanding of G. duodenalis attachment by investigating detachment characteristics and determining adhesion forces of single trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) by fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS). Briefly, viable adherent trophozoites were approached with a FluidFM micropipette, immobilized to the micropipette aperture by negative pressure, and detached from the surface by micropipette retraction while retract force curves were recorded. These force curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Respective adhesion forces reached 7.7 ± 4.2 nN at 1 µm s-1 pulling speed. Importantly, this unique force pattern was different from that of other eukaryotic cells such as Candida albicans or oral keratinocytes, considered for comparison in this study. The latter both displayed a force pattern with force peaks of different values or force plateaus (for keratinocytes) indicative of breakage of molecular bonds of cell-anchored classes of adhesion molecules or membrane components. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces, when the pulling speeds were raised up to 10 µm s-1 and adhesion forces increased to 28.7 ± 10.5 nN. Taken together, comparative SCSF revealed novel and unique retract force curve characteristics for attached G. duodenalis, suggesting a ligand-independent suction mechanism, that differ from those of other well described eukaryotes.

Identification of End-Binding 1 Protein as Novel α-4 Giardin-Binding Partners in Giardia lamblia Trophozoites.

BACKGROUND: Giardia lamblia (syn. G. intestinalis, G. duodenalis) is a primitive opportunistic protozoon, and one of the earliest differentiated eukaryotes. Despite its primitive nature, G. lamblia has a sophisticated cytoskeleton system, which is closely related to its proliferation and pathogenicity. Meanwhile, α giardin is a G. lamblia-specific cytoskeleton protein, which belongs to the annexin superfamily. Interestingly, G. lamblia has 21 annexin-like α giardins, i.e., more than higher eukaryotes. The functional differences among α giardin members are not fully understood. METHODS: We took α-4 giardin, a member of α giardin family, as a research object. A morpholino-mediated knockdown experiment was performed to identify the effect of α-4 giardin on G. lamblia trophozoites biological traits. A yeast two-hybrid cDNA library of G. lamblia strain C2 trophozoites was screened for interaction partners of α-4 giardin. Co-immunoprecipitation and fluorescent colocalization confirmed the relationship between G. lamblia EB1 (gEB1) and α-4 giardin. RESULTS: α-4 Giardin could inhibit the proliferation and adhesion of G. lamblia trophozoites. In addition, it interacted with G. lamblia EB1 (gEB1). CONCLUSIONS: α-4 Giardin was involved in proliferation and adhesion in G. lamblia trophozoites, and EB1, a crucial roles in mitosis, was an interacting partner of α-4 giardin.

Plasmodium falciparum utilizes pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.

Trophozoite fitness dictates the intestinal epithelial cell response to Giardia intestinalis infection.

Giardia intestinalis is a non-invasive, protozoan parasite infecting the upper small intestine of most mammals. Symptomatic infections cause the diarrhoeal disease giardiasis in humans and animals, but at least half of the infections are asymptomatic. However, the molecular underpinnings of these different outcomes of the infection are still poorly defined. Here, we studied the early transcriptional response to G. intestinalis trophozoites, the disease-causing life-cycle stage, in human enteroid-derived, 2-dimensional intestinal epithelial cell (IEC) monolayers. Trophozoites preconditioned in media that maximise parasite fitness triggered only neglectable inflammatory transcription in the IECs during the first hours of co-incubation. By sharp contrast, "non-fit" or lysed trophozoites induced a vigorous IEC transcriptional response, including high up-regulation of many inflammatory cytokines and chemokines. Furthermore, "fit" trophozoites could even suppress the stimulatory effect of lysed trophozoites in mixed infections, suggesting active G. intestinalis suppression of the IEC response. By dual-species RNA-sequencing, we defined the IEC and G. intestinalis gene expression programs associated with these differential outcomes of the infection. Taken together, our results inform on how G. intestinalis infection can lead to such highly variable effects on the host, and pinpoints trophozoite fitness as a key determinant of the IEC response to this common parasite.

Giardia lamblia G6PD::6PGL Fused Protein Inhibitors Decrease Trophozoite Viability: A New Alternative against Giardiasis.

Treatments to combat giardiasis have been reported to have several drawbacks, partly due to the drug resistance and toxicity of current antiparasitic agents. These constraints have prompted many researchers to investigate new drugs that act against protozoan parasites. Enzyme inhibition is an important means of regulating pathogen metabolism and has recently been identified as a significant alternative target in the search for new treatments. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase (G6PD::6PGL) is a bifunctional enzyme involved in the pentose phosphate pathway (PPP) in Giardia lamblia (G. lamblia). The G. lamblia enzyme is unusual since, unlike the human enzyme, it is a fused enzyme. Here, we show, through inhibition assays, that an in-house chemical library of 120 compounds and four target compounds, named CNZ-7, CNZ-8, CMC-1, and FLP-2, are potent inhibitors of the G. lamblia G6PD::6PGL fused enzyme. With a constant (k2) of 2.3, 3.2, and 2.8 M−1 s−1, respectively, they provoke alterations in the secondary and tertiary protein structure and global stability. As a novel approach, target compounds show antigiardial activity, with IC50 values of 8.7, 15.2, 15.3, and 24.1 µM in trophozoites from G. lamblia. Moreover, these compounds show selectivity against G. lamblia, since, through counter-screening in Caco-2 and HT29 human cells, they were found to have low toxicity. This finding positions these compounds as a potential and attractive starting point for new antigiardial drugs.

Giardia duodenalis enolase is secreted as monomer during trophozoite-epithelial cell interactions, activates plasminogen and induces necroptotic damage.

Enolase, a multifunctional protein expressed by multiple pathogens activates plasminogen to promote proteolysis on components of the extracellular matrix, an important event in early host-pathogen interactions. A secreted form of enolase that is released upon the interaction of trophozoites with epithelial cells has been detected in the secretome of G. duodenalis. However, the role of enolase in the host-pathogen interactions remains largely unknown. In this work, the effects of G. duodenalis enolase (Gd-eno) on the epithelial cell model (IEC-6) were analyzed. Firstly, the coding sequence of Giardia enolase was cloned and the recombinant protein used to raise antibodies that were then used to define the localization and role of enolase in epithelial cell-trophozoite interactions. Gd-eno was detected in small cytoplasmic vesicles as well as at the surface and is enriched in the region of the ventral disk of Giardia trophozoites. Moreover, the blocking of the soluble monomeric form of the enzyme, which is secreted upon interaction with IEC-6 cells by the anti-rGd-eno antibodies, significantly inhibited trophozoite attachment to intestinal IEC-6 cell monolayers. Further, rGd-eno was able to bind human plasminogen (HsPlg) and enhanced plasmin activity in vitro when the trophozoites were incubated with the intrinsic plasminogen activators of epithelial cells. In IEC-6 cells, rGd-eno treatment induced a profuse cell damage characterized by copious vacuolization, intercellular separation and detachment from the substrate; this effect was inhibited by either anti-Gd-eno Abs or the plasmin inhibitor ϵ- aminocaproic acid. Lastly, we established that in epithelial cells rGd-eno treatment induced a necroptotic-like process mediated by tumor necrosis factor α (TNF-α) and the apoptosis inducing factor (AIF), but independent of caspase-3. All together, these results suggest that Giardia enolase is a secreted moonlighting protein that stimulates a necroptotic-like process in IEC-6 epithelial cells via plasminogen activation along to TNFα and AIF activities and must be considered as a virulence factor.

Alpha-terthienyl increases filamentous actin of Entamoeba histolytica.

This study aimed to know if alpha terthienyl (α-T) affects E. histolytica viability and to analyze its effect on the actin cytoskeleton. Trophozoites of E. histolytica HM1-IMSS were treated with α-T, then, cell viability and morphology were evaluated using tetrazolium salts and scanning electron microscopy, respectively; while actin filaments (F-actin) were stained with rhodamine-phalloidin, observed by confocal microscopy and quantified by fluorometry. Data showed that α-T inhibited cell viability of trophozoites (IC50, 19.43 µg / mL), affected the cell morphology, and increased the F-actin in a dose-dependent manner. Production of reactive oxygen species and RhoA-GTP levels remained normal in α-T-treated amebas. Two inhibitors that affect the organization of the trophozoites cytoskeleton, one that interacts directly with actin, Cytochalasin D (CD), and one that affects the Rho signaling pathway by inhibiting the downstream effector Rock, Y27632, were tested. Y27632 did not affect the increase of polymerized actin observed with α-T, this compound partially ameliorates the potent disrupting effects of CD on actin filaments. Docking results suggest that α-T could be an antagonist of CD for the same interaction zone in actin, however, more studies are needed to define the action mechanism of this compound.

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole.

Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite.

Role of cathepsin B of Naegleria fowleri during primary amebic meningoencephalitis.

Naegleria fowleri causes primary amoebic meningoencephalitis in humans and experimental animals. It has been suggested that cysteine proteases of parasites play key roles in metabolism, nutrient uptake, host tissue invasion, and immune evasion. The aim of this work was to evaluate the presence, expression, and role of cathepsin B from N. fowleri in vitro and during PAM. Rabbit-specific polyclonal antibodies against cathepsin B were obtained from rabbit immunization with a synthetic peptide obtained by bioinformatic design. In addition, a probe was designed from mRNA for N. fowleri cathepsin B. Both protein and messenger were detected in fixed trophozoites, trophozoites interacted with polymorphonuclear and histological sections of infected mice. The main cathepsin B distribution was observed in cytoplasm or membrane mainly pseudopods and food-cups while messenger was in nucleus and cytoplasm. Surprisingly, both the messenger and enzyme were observed in extracellular medium. To determine cathepsin B release, we used trophozoites supernatant recovered from nasal passages or brain of infected mice. We observed the highest release in supernatant from recovered brain amoebae, and when we analyzed molecular weight of secreted proteins by immunoblot, we found 30 and 37 kDa bands which were highly immunogenic. Finally, role of cathepsin B during N. fowleri infection was determined; we preincubated trophozoites with E-64, pHMB or antibodies with which we obtained 60%, 100%, and 60% of survival, respectively, in infected mice. These results suggest that cathepsin B plays a role during pathogenesis caused by N. fowleri mainly in adhesion and contributes to nervous tissue damage.

Proteomic and Functional Analysis of the Effects of Quinoxaline Derivatives on Entamoeba histolytica.

Quinoxalines are heterocyclic compounds that contain a benzene ring and a pyrazine ring. The oxidation of both nitrogen of the pyrazine ring results in quinoxaline derivatives (QdNO), which exhibit a variety of biological properties, including antiparasitic activity. However, its activity against Entamoeba histolytica, the protozoan that causes human amebiasis, is poorly understood. Recently, our group reported that various QdNOs produce morphological changes in E. histolytica trophozoites, increase reactive oxygen species, and inhibit thioredoxin reductase activity. Notably, T-001 and T-017 derivatives were among the QdNOs with the best activity. In order to contribute to the characterization of the antiamebic effect of QdNOs, in this work we analyzed the proteomic profile of E. histolytica trophozoites treated with the QdNOs T-001 and T-017, and the results were correlated with functional assays. A total number of 163 deregulated proteins were found in trophozoites treated with T-001, and 131 in those treated with T-017. A set of 21 overexpressed and 24 under-expressed proteins was identified, which were mainly related to cytoskeleton and intracellular traffic, nucleic acid transcription, translation and binding, and redox homeostasis. Furthermore, T-001 and T-017 modified the virulence of trophozoites, since they altered their erythrophagocytosis, migration, adhesion and cytolytic capacity. Our results show that in addition to alter reactive oxygen species, and thioredoxin reductase activity, T-001 and T-017 affect essential functions related to the actin cytoskeleton, which eventually affects E. histolytica virulence and survival.

CDC50 Orthologues in Plasmodium falciparum Have Distinct Roles in Merozoite Egress and Trophozoite Maturation.

In model organisms, type IV ATPases (P4-ATPases) require cell division control protein 50 (CDC50) chaperones for their phospholipid flipping activity. In the malaria parasite Plasmodium falciparum, guanylyl cyclase alpha (GCα) is an integral membrane protein that is essential for release (egress) of merozoites from their host erythrocytes. GCα is unusual in that it contains both a C-terminal cyclase domain and an N-terminal P4-ATPase domain of unknown function. We sought to investigate whether any of the three CDC50 orthologues (termed A, B, and C) encoded by P. falciparum are required for GCα function. Using gene tagging and conditional gene disruption, we demonstrate that CDC50B and CDC50C but not CDC50A are expressed in the clinically important asexual blood stages and that CDC50B is a binding partner of GCα whereas CDC50C is the binding partner of another putative P4-ATPase, phospholipid-transporting ATPase 2 (ATP2). Our findings indicate that CDC50B has no essential role for intraerythrocytic parasite maturation but modulates the rate of parasite egress by interacting with GCα for optimal cGMP synthesis. In contrast, CDC50C is essential for blood stage trophozoite maturation. Additionally, we find that the CDC50C-ATP2 complex may influence parasite endocytosis of host cell hemoglobin and consequently hemozoin formation. IMPORTANCE Malaria morbidity arises due to successive rounds of replication of Plasmodium parasites within red blood cells. Mature daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that a unique bifunctional guanylyl cyclase, GCα, initiates egress by synthesis of cGMP. GCα has an N-terminal P4-ATPase domain of unknown function. In model organisms, P4-ATPases function through interaction with a CDC50 partner protein. Here, we investigate the role of CDC50 orthologues in P. falciparum and show that GCα binds CDC50B, an interaction that regulates egress efficiency. We also find that CDC50C is essential and binds a putative P4-ATPase, ATP2, in a complex that influences endocytosis of host hemaglobin. Our results highlight the heterogenous and critical role of CDC50 proteins in P. falciparum.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.

Kinesin-13, a Motor Protein, is Regulated by Polo-like Kinase in Giardia lamblia.

Kinesin-13 (Kin-13), a depolymerizer of microtubule (MT), has been known to affect the length of Giardia. Giardia Kin-13 (GlKin-13) was localized to axoneme, flagellar tips, and centrosomes, where phosphorylated forms of Giardia polo-like kinase (GlPLK) were distributed. We observed the interaction between GlKin-13 and GlPLK via co-immunoprecipitation using transgenic Giardia cells expressing Myc-tagged GlKin-13, hemagglutinin-tagged GlPLK, and in vitro-synthesized GlKin-13 and GlPLK proteins. In vitro-synthesized GlPLK was demonstrated to auto-phosphorylate and phosphorylate GlKin-13 upon incubation with [γ-32P]ATP. Morpholino-mediated depletion of both GlKin-13 and GlPLK caused an extension of flagella and a decreased volume of median bodies in Giardia trophozoites. Our results suggest that GlPLK plays a pertinent role in formation of flagella and median bodies by modulating MT depolymerizing activity of GlKin-13.

Dual RNA Sequencing Reveals Key Events When Different Giardia Life Cycle Stages Interact With Human Intestinal Epithelial Cells In Vitro.

Giardia intestinalis is a protozoan parasite causing diarrheal disease, giardiasis, after extracellular infection of humans and other mammals' intestinal epithelial cells (IECs) of the upper small intestine. The parasite has two main life cycle stages: replicative trophozoites and transmissive cysts. Differentiating parasites (encysting cells) and trophozoites have recently been shown to be present in the same regions of the upper small intestine, whereas most mature cysts are found further down in the intestinal system. To learn more about host-parasite interactions during Giardia infections, we used an in vitro model of the parasite's interaction with host IECs (differentiated Caco-2 cells) and Giardia WB trophozoites, early encysting cells (7 h), and cysts. Dual RNA sequencing (Dual RNAseq) was used to identify differentially expressed genes (DEGs) in both Giardia and the IECs, which might relate to establishing infection and disease induction. In the human cells, the largest gene expression changes were found in immune and MAPK signaling, transcriptional regulation, apoptosis, cholesterol metabolism and oxidative stress. The different life cycle stages of Giardia induced a core of similar DEGs but at different levels and there are many life cycle stage-specific DEGs. The metabolic protein PCK1, the transcription factors HES7, HEY1 and JUN, the peptide hormone CCK and the mucins MUC2 and MUC5A are up-regulated in the IECs by trophozoites but not cysts. Cysts specifically induce the chemokines CCL4L2, CCL5 and CXCL5, the signaling protein TRKA and the anti-bacterial protein WFDC12. The parasite, in turn, up-regulated a large number of hypothetical genes, high cysteine membrane proteins (HCMPs) and oxidative stress response genes. Early encysting cells have unique DEGs compared to trophozoites (e.g. several uniquely up-regulated HCMPs) and interaction of these cells with IECs affected the encystation process. Our data show that different life cycle stages of Giardia induce different gene expression responses in the host cells and that the IECs in turn differentially affect the gene expression in trophozoites and early encysting cells. This life cycle stage-specific host-parasite cross-talk is an important aspect to consider during further studies of Giardia's molecular pathogenesis.

PTEN differentially regulates endocytosis, migration, and proliferation in the enteric protozoan parasite Entamoeba histolytica.

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.

PI Kinase-EhGEF2-EhRho5 axis contributes to LPA stimulated macropinocytosis in Entamoeba histolytica.

Entamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis.

Docosahexaenoic acid is potent against the growth of mature stages of Plasmodium falciparum; inhibition of hematin polymerization a possible target.

The present study investigates the potential effect of externally added unsaturated fatty acids on P. falciparum growth. Our results indicate that polyunsaturated fatty acids (PUFAs) inhibit the growth of Plasmodium in proportional to their degree of unsaturation. At higher concentration the PUFA Docosahexaenoic acid (DHA) induces pyknotic nuclei in infected erythrocytes. When Plasmodium stages were treated transiently with DHA, the ring stage culture recovered from the drug effect and parasitemia was increased post DHA removal with delayed growth of 12 h, compared to untreated control. Schizont stage treated culture displayed a 36 h delay in growth to infect fresh erythrocytes signifying its recovery is less than the ring stage. However the trophozoite stage failed to recover and showed a decrease in parasitemia, similar to that of continuous treated culture. PUFAs inhibited ß- hematin polymerization by binding to free heme derived from hemoglobin degradation. Digestive vacuole neutral lipid bodies, which are pivotal for ß- hematin polymerization, decreased and subsequently abrogated with increasing concentration of DHA in trophozoite stage treated culture. Our study concludes that DHA interacts with heme monomers and inhibits the ß- hematin polymerization and growth of mature stages i.e., trophozoite and schizont stages of plasmodium.