Identification and Characterization of Entamoeba histolytica Choline Kinase.

PURPOSE: Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future. METHODOLOGY: E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes. RESULTS: EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1 µmol/min/mg. Its Km for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting. CONCLUSIONS: With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

Activation of human RNA lariat debranching enzyme Dbr1 by binding protein TTDN1 occurs though an intrinsically disordered C-terminal domain.

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease.

Probing the Peculiarity of EhRabX10, a pseudoRab GTPase, from the Enteric Parasite Entamoeba histolytica through In Silico Modeling and Docking Studies.

Entamoeba histolytica (Eh) is a pathogenic eukaryote that often resides silently in humans under asymptomatic stages. Upon indeterminate stimulus, it develops into fulminant amoebiasis that causes severe hepatic abscesses with 50% mortality. This neglected tropical pathogen relies massively on membrane modulation to flourish and cause disease; these modulations range from the phagocytic mode for food acquisition to a complex trogocytosis mechanism for tissue invasion. Rab GTPases form the largest branch of the Ras-like small GTPases, with a diverse set of roles across the eukaryotic kingdom. Rab GTPases are vital for the orchestration of membrane transport and the secretory pathway responsible for transporting the pathogenic effectors, such as cysteine proteases (EhCPs) which help in tissue invasion. Rab GTPases thus play a crucial role in executing the cytolytic effect of E. histolytica. First, they interact with Gal/Nac lectins required for adhering to the host cells, and then, they assist in the secretion of EhCPs. Additionally, amoebic Rab GTPases are vital for encystation because substantial vesicular trafficking is required to create dormant amoebic cysts. These cysts are the infective agent and help to spread the disease. The absence of a "bonafide" vesicular transport machinery in Eh and the existence of a diverse repertoire of amoebic Rab GTPases (EhRab) hint at their contribution in supporting this atypical machinery. Here, we provide insights into a pseudoRab GTPase, EhRabX10, by performing physicochemical analysis, predictive 3D structure modeling, protein-protein interaction studies, and in silico molecular docking. Our group is the first one to classify EhRabX10 as a pseudoRab GTPase with four nonconserved G-motifs. It possesses the basic fold of the P-loop containing nucleotide hydrolases. Through this in silico study, we provide an introduction to the characterization of the atypical EhRabX10 and set the stage for future explorations into the mechanisms of nucleotide recognition, binding, and hydrolysis employed by the pseudoEhRab GTPase family.

A Novel TLR4-Binding Domain of Peroxiredoxin From Entamoeba histolytica Triggers NLRP3 Inflammasome Activation in Macrophages.

Macrophages promote early host responses to infection by releasing pro-inflammatory cytokines, and they are crucial to combat amoebiasis, a disease affecting millions of people worldwide. Macrophages elicit pro-inflammatory responses following direct cell/cell interaction of Entamoeba histolytica, inducing NLRP3 inflammasome activation with high-output IL-1ß/IL-18 secretion. Here, we found that trophozoites could upregulate peroxiredoxins (Prx) expression and abundantly secrete Prxs when encountering host cells. The C-terminal of Prx was identified as the key functional domain in promoting NLRP3 inflammasome activation, and a recombinant C-terminal domain could act directly on macrophage. The Prxs derived from E. histolytica triggered toll-like receptor 4-dependent activation of NLRP3 inflammasome in a cell/cell contact-independent manner. Through genetic, immunoblotting or pharmacological inhibition methods, NLRP3 inflammasome activation was induced through caspase-1-dependent canonical pathway. Our data suggest that E. histolytica Prxs had stable and durable cell/cell contact-independent effects on macrophages following abundantly secretion during invasion, and the C-terminal of Prx was responsible for activating NLRP3 inflammasome in macrophages. This new alternative pathway may represent a potential novel therapeutic approach for amoebiasis, a global threat to millions.

Cysteine proteases: Battling pathogenic parasitic protozoans with omnipresent enzymes.

Millions of people worldwide lie at the risk of parasitic protozoic infections that kill over a million people each year. The rising inefficacy of conventional therapeutics to combat these diseases, mainly due to the development of drug resistance to a handful of available licensed options contributes substantially to the rising burden of these ailments. Cysteine proteases are omnipresent enzymes that are critically implicated in the pathogenesis of protozoic infections. Despite their significance and druggability, cysteine proteases as therapeutic targets have not yet been translated into the clinic. The review presents the significance of cysteine proteases of members of the genera Plasmodium, Entamoeba, and Leishmania, known to cause Malaria, Amoebiasis, and Leishmaniasis, respectively, the protozoic diseases with the highest morbidity and mortality. Further, projecting them as targets for molecular tools like the CRISPR-Cas technology for favorable manipulation, exploration of obscure genomes, and achieving a better insight into protozoic functioning. Overcoming the hurdles that prevent us from gaining a better insight into the functioning of these enzymes in protozoic systems is a necessity. Managing the burden of parasitic protozoic infections pivotally depends upon the betterment of molecular tools and therapeutic concepts that will pave the path to an array of diagnostic and therapeutic applications.

Structure-Function Relationship Study of a Secretory Amoebic Phosphatase: A Computational-Experimental Approach.

Phosphatases are hydrolytic enzymes that cleave the phosphoester bond of numerous substrates containing phosphorylated residues. The typical classification divides them into acid or alkaline depending on the pH at which they have optimal activity. The histidine phosphatase (HP) superfamily is a large group of functionally diverse enzymes characterized by having an active-site His residue that becomes phosphorylated during catalysis. HP enzymes are relevant biomolecules due to their current and potential application in medicine and biotechnology. Entamoeba histolytica, the causative agent of human amoebiasis, contains a gene (EHI_146950) that encodes a putative secretory acid phosphatase (EhHAPp49), exhibiting sequence similarity to histidine acid phosphatase (HAP)/phytase enzymes, i.e., branch-2 of HP superfamily. To assess whether it has the potential as a biocatalyst in removing phosphate groups from natural substrates, we studied the EhHAPp49 structural and functional features using a computational-experimental approach. Although the combined outcome of computational analyses confirmed its structural similarity with HP branch-2 proteins, the experimental results showed that the recombinant enzyme (rEhHAPp49) has negligible HAP/phytase activity. Nonetheless, results from supplementary activity evaluations revealed that rEhHAPp49 exhibits Mg2+-dependent alkaline pyrophosphatase activity. To our knowledge, this study represents the first computational-experimental characterization of EhHAPp49, which offers further insights into the structure-function relationship and the basis for future research.

Structural analysis of EhPSP in complex with 3-phosphoglyceric acid from Entamoeba histolytica reveals a basis for its lack of phosphoglycerate mutase activity.

Entamoeba histolytica phosphoserine phosphatase (EhPSP), a regulatory enzyme in the serine biosynthetic pathway, is also a structural homolog of cofactor-dependent phosphoglycerate mutase (dPGM). However, despite sharing many of its catalytic residues with dPGM, EhPSP displays no significant mutase activity. In the current work, we determined a crystal structure of EhPSP in complex with 3-PGA to 2.5 Å resolution and observed striking differences between the orientation of 3-PGA bound to EhPSP and that to its other homologous structures. We also performed computational modeling and simulations of the intermediate 2,3-bisphosphoglyceric acid into the active site of EhPSP to better understand its mechanistic details. Based on these results and those of a similar study with the dPGMs from E. coli and B. pseudomallei, the affinity of EhPSP for 2,3-BPG was concluded to be lower than those of the other proteins. Moreover, a different set of 2,3-BPG interacting residues was observed in EhPSP compared to dPGMs, with all of the crucial interacting residues of dPGMs either missing or substituted with weakly interacting residues. This study has expanded our understanding, at the structural level, of the inability of EhPSP to catalyze the mutase reaction and has strengthened earlier conclusions indicating it to be a true phosphatase.

Low molecular weight protein tyrosine phosphatase (LMW-PTP2) protein can potentially modulate virulence of the parasite Entamoeba histolytica.

The Entamoeba histolytica parasite is the causative agent of amebiasis, infecting approximately 1% of the world population and causing 100,000 deaths per year. It binds to Fibronectin (FN), activating signaling pathways regulated by kinases and phosphatases. EhLMW-PTPs genes from E. histolytica encode for Low Molecular Weight Tyrosine Phosphatases expressed in trophozoites and amoebic cysts. The role of these phosphatases in the virulence of the parasite has not yet been well characterized. Our results showed a differential expression of the EhLMW-PTPs, at the mRNA and protein levels, in an asynchronous trophozoites culture. Furthermore, we observed that trophozoites transfected that overexpressed EhLMW-PTP2 phagocytized fewer erythrocytes, possibly due to decreased phagocytic cups, and showed deficiencies in adherence to FN and less cytopathic effect. These analyzes suggest that the parasite's EhLMW-PTPs have an essential role in the mechanisms of proliferation, adhesion, and phagocytosis, regulating its pathogenicity.

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.

ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.

Characterization of low molecular weight protein tyrosine phosphatases of Entamoeba histolytica.

Entamoeba histolytica is an intestinal protozoan parasite of humans and is endemic in developing countries. E. histolytica has two low molecular weight protein tyrosine phosphatase (LMW-PTP) genes, EhLMW-PTP1 and EhLMW-PTP2, which are expressed in cultured trophozoites, clinical isolates, and cysts. The amino acid sequences of proteins EhLMW-PTP1 and EhLMW-PTP2 showed only one amino acid difference between them at position A85V, respectively. Both genes are expressed in cultured trophozoites, mainly EhLMW-PTP2, and in trophozoites recovered from amoebic liver abscess, the expression of EhLMW-PTP1 is downregulated. We cloned the two genes and purified the corresponding recombinant (rEhLMW-PTPs) proteins. Antibodies anti-rEhLMW-PTP2 showed that during red blood cells uptake by E. histolytica, the EhLMW-PTPs were found in the phagocytic cups based on analysis of fluorescence signals. On the other hand, rEhLMW-PTPs showed an optimum phosphatase activity at pH 6.0 with p-nitrophenyl phosphate as the substrate. They dephosphorylate phosphotyrosine and 3-O-methylfluorescein phosphate, but not phosphoserine or phosphothreonine, and the enzymatic activity is inhibited by orthovanadate. rEhLMW-PTP1 and rEhLMW-PTP2 exhibited optimum temperatures of activities at 60 °C and 58 °C, respectively, with high thermal stability at 50 °C. Also, the rEhLMW-PTPs showed high specific activities and specific km value with pNPP or OMFP as the substrates at the physiological temperature (37 °C).

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%.

Role of kinases in virulence and pathogenesis of protozoan parasite E. Histolytica.

Protein kinases are known to regulate several cellular processes like metabolism, motility and endocytosis through phosphorylation of specific target proteins which forms a communication system relaying extracellular signals to intracellular milieu for an adaptive response. One of the protozoan parasite Entamoeba histolytica, which causes amoebiasis and is one of the prominent reason for causing diarrhoea in infants of developing countries, where it remains the third leading cause of deaths in infants(1). The genome of this parasite codes for 331 putative protein kinases which accounts for 3.7% of the proteome. The kinome of the parasite is composed of several conserved and as well as kinase with unusual domain architecture. About one-third of kinome codes for transmembrane kinases (TMK) which is proposed to help the parasite to sense and adapt to the gut environment which is constantly changing. Many kinases are known to be involved in virulence but, the kinome of this important parasite is unexplored. In this review, we present an overview of E. histolytica kinases and their role in amoebic biology understood till now.

Biological activity of esters of quinoxaline-7-carboxylate 1,4-di-N-oxide against E. histolytica and their analysis as potential thioredoxin reductase inhibitors.

Amoebiasis is caused by the protozoan Entamoeba histolytica that affects millions of people throughout the world. The standard treatment is metronidazole, however, this drug causes several side effects, and is also mutagenic and carcinogenic. Therefore, the search for therapeutic alternatives is necessary. Quinoxaline 1,4-di-N-oxides (QdNOs) derivatives have been shown to exhibit activity against different protozoan. In the present study, the effects of esters of quinoxaline-7-carboxylate 1,4-di-N-oxide (7-carboxylate QdNOs) derivatives on E. histolytica proliferation, morphology, ultrastructure, and oxidative stress were evaluated, also their potential as E. histolytica thioredoxin reductase (EhTrxR) inhibitors was analyzed. In vitro tests showed that 12 compounds from n-propyl and isopropyl series, were more active (IC50 = 0.331 to 3.56 µM) than metronidazole (IC50 = 4.5 µM). The compounds with better biological activity have a bulky, trifluoromethyl and isopropyl group at R1-, R2-, and R3-position, respectively. The main alterations found in trophozoites treated with some of these compounds included changes in chromatin, cell granularity, redistribution of vacuoles with cellular debris, and an increase in reactive oxygen species. Interestingly, docking studies suggested that 7-carboxylate QdNOs derivatives could interact with amino acid residues of the NADPH-binding domain and/or the redox-active site of EhTrxR. Enzymatic assays demonstrated that selected 7-carboxylate QdNOs inhibits EhTrxR disulfide reductase activity, and diaphorase activity shows that these compounds could act as electron acceptor substrates for the enzyme. Taken together, these data indicate that among the mechanisms involved in the antiamoebic effect of the 7-carboxylate QdNOs derivatives studied, is the induction of oxidative stress and the inhibition of EhTrxR activity.

Biochemical and biophysical characterization of the smallest pyruvate kinase from Entamoeba histolytica.

Entamoeba histolytica infection is highly prevalent in developing countries across the globe. The ATP synthesis in this pathogen is solely dependent on the glycolysis pathway where pyruvate kinase (Pyk) catalyzes the final reaction. Here, we have cloned, overexpressed and purified the pyruvate kinase (EhPyk) from E. histolytica. EhPyk is the shortest currently known Pyk till date as it contains only two of the three characterized domains when compared to the other homologues and our phylogenetic analysis places it on a distinct branch from the known type I/II Pyks. Our purification results suggested that it exists as a homodimer in solution. The kinetic characterization showed that EhPyk has maximum activity at pH 7.5 where it exhibited Michaelis-Menten's kinetics for phosphoenolpyruvate with a Km of 0.23 mM, and it lost its activity at both the acidic pH 4.0 and basic pH 10.0. We also determined the key secondary structural elements of EhPyk at different pH values. MD simulation of EhPyk structure at different pH values suggested that it is most stable at pH 7.0, while least stable at pH 10.0 followed by pH 4.0. Together, our computational simulations correlate well with the experimental studies. In summary, this study expands the current understanding of the EhPyk identified earlier in the amoebic genome and provides the first characterization of this bacterially expressed protein.

Vorinostat, a possible alternative to metronidazole for the treatment of amebiasis caused by Entamoeba histolytica.

AbbreviationsSAHAsuberoylanilide hydroxamic acidEhHDACHistone Deacetylase from Entamoeba histolyticaRgRadius of gyrationRMSDroot-mean-square deviationRMSFroot-mean-square fluctuationMDSmolecular dynamics simulationVMDVisual Molecular DynamicsNAMDNanoscale Molecular DynamicsPBCperiodic boundary conditionsPMEParticle Mesh Ewald3Dthree-dimensionalCαalpha carbonFDAFood and Drug AdministrationnsnanosecondsGPU CUDAGraphics Processing Unit Compute Unified Device ArchitectureCommunicated by Ramaswamy H. Sarma.

Stress-responsive Entamoeba topoisomerase II: a potential antiamoebic target.

Topoisomerases, the ubiquitous enzymes involved in all DNA processes across the biological world, are targets for various anticancer and antimicrobial agents. In Entamoeba histolytica, the causative agent of amebiasis, we found one of seven unexplored putative topoisomerases to be highly upregulated during heat shock and oxidative stress, and also during the late hours of encystation. Further analysis revealed the upregulated enzyme to be a eukaryotic type IIA topoisomerase (TopoII) with demonstrable activity in vitro. This enzyme is localized to newly forming nuclei during encystation. Gene silencing of the TopoII reduces viability and encystation efficiency. Notable susceptibility of Entamoeba TopoII to prokaryotic topoisomerase inhibitors opens up the possibility for exploring this enzyme as a new antiamoebic target.

Activation of α-, ß-, γ- δ-, ζ- and η- class of carbonic anhydrases with amines and amino acids: a review.

Eight genetically distinct carbonic anhydrase (EC 4.2.1.1) enzyme families (α-, ß-, γ- δ-, ζ-, η-, θ- and ι-CAs) were described to date. On the other hand, 16 mammalian α-CA isoforms are known to be involved in many diseases such as glaucoma, edema, epilepsy, obesity, hypoxic tumors, neuropathic pain, arthritis, neurodegeneration, etc. Although CA inhibitors were investigated for the management of a variety of such disorders, the activators just started to be investigated in detail for their in vivo effects. This review summarizes the activation profiles of α-, ß, γ-, δ-, ζ- and η- CAs from various organisms (animals, fungi, protozoan, bacteria and archaea) with the most investigated classes of activators, the amines and the amino acids.

Characterization of Entamoeba histolytica adenosine 5'-phosphosulfate (APS) kinase; validation as a target and provision of leads for the development of new drugs against amoebiasis.

BACKGROUND: Amoebiasis, caused by Entamoeba histolytica infection, is a global public health problem. However, available drugs to treat amoebiasis are currently limited, and no effective vaccine exists. Therefore, development of new preventive measures against amoebiasis is urgently needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, to develop new drugs against amoebiasis, we focused on E. histolytica adenosine 5'-phosphosulfate kinase (EhAPSK), an essential enzyme in Entamoeba sulfolipid metabolism. Fatty alcohol disulfates and cholesteryl sulfate, sulfolipids synthesized in Entamoeba, play important roles in trophozoite proliferation and cyst formation. These processes are closely associated with clinical manifestation and severe pathogenesis of amoebiasis and with disease transmission, respectively. We validated a combination approach of in silico molecular docking analysis and an in vitro enzyme activity assay for large scale screening. Docking simulation ranked the binding free energy between a homology modeling structure of EhAPSK and 400 compounds. The 400 compounds were also screened by a 96-well plate-based in vitro APSK activity assay. Among fifteen compounds identified as EhAPSK inhibitors by the in vitro system, six were ranked by the in silico analysis as having high affinity toward EhAPSK. Furthermore, 2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide, 3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide, and auranofin, which were identified as EhAPSK inhibitors by both in silico and in vitro analyses, halted not only Entamoeba trophozoite proliferation but also cyst formation. These three compounds also dose-dependently impaired the synthesis of sulfolipids in E. histolytica. CONCLUSIONS/SIGNIFICANCE: Hence, the combined approach of in silico and in vitro-based EhAPSK analyses identified compounds that can be evaluated for their effects on Entamoeba. This can provide leads for the development of new anti-amoebic and amoebiasis transmission-blocking drugs. This strategy can also be applied to identify specific APSK inhibitors, which will benefit research into sulfur metabolism and the ubiquitous pathway terminally synthesizing essential sulfur-containing biomolecules.

Plasma membrane damage repair is mediated by an acid sphingomyelinase in Entamoeba histolytica.

Entamoeba histolytica is a pathogen that during its infective process confronts the host defenses, which damages the amoebic plasma membrane (PM), resulting in the loss of viability. However, it is unknown whether amoebic trophozoites are able to repair their PM when it is damaged. Acid sphingomyelinases (aSMases) have been reported in mammalian cells to promote endocytosis and removal of PM lesions. In this work, six predicted amoebic genes encoding for aSMases were found to be transcribed in the HM1:IMSS strain, finding that the EhaSM6 gene is the most transcribed in basal growth conditions and rendered a functional protein. The secreted aSMase activity detected was stimulated by Mg+2 and inhibited by Co+2. Trophozoites that overexpress the EhaSM6 gene (HM1-SM6HA) exhibit an increase of 2-fold in the secreted aSMase activity. This transfectant trophozoites exposed to pore-forming molecules (SLO, Magainin, ß-Defensin 2 and human complement) exhibited an increase from 6 to 25-fold in the secreted aSMase activity which correlated with higher amoebic viability in a Ca+2 dependent process. However, other agents that affect the PM such as hydrogen peroxide also induced an increase of secreted aSMase, but to a lesser extent. The aSMase6 enzyme is N- and C-terminal processed. Confocal and transmission electron microscopy showed that trophozoites treated with SLO presented a migration of lysosomes containing the aSMase towards the PM, inducing the formation of membrane patches and endosomes in the control strain. These cellular structures were increased in the overexpressing strain, indicating the involvement of the aSMase6 in the PM injury repair. The pore-forming molecules induced an increase in the expression of EhaSM1, 2, 5 and 6 genes, meanwhile, hydrogen peroxide induced an increase in all of them. In all the conditions evaluated, the EhaSM6 gene exhibited the highest levels of induction. Overall, these novel findings show that the aSMase6 enzyme from E. histolytica promotes the repair of the PM damaged with pore-forming molecules to prevent losing cell integrity. This novel system could act when encountered with the lytic defense systems of the host.