Formation of oxidised (OX) proteins in Entamoeba histolytica exposed to auranofin and consequences on the parasite virulence.

Metronidazole (MNZ), the first line drug for amoebiasis and auranofin (AF), an emerging antiprotozoan drug, are both inhibiting Entamoeba histolytica thioredoxin reductase. The nature of oxidised proteins (OXs) formed in AF- or MNZ-treated E. histolytica trophozoites is unknown. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the OXs formed in AF- or MNZ-treated E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry (MS). We detected 661 OXs in MNZ-treated trophozoites and 583 OXs in AF-treated trophozoites. More than 50% of these OXs were shared, and their functions include hydrolases, enzyme modulators, transferases, nucleic acid binding proteins, oxidoreductases, cytoskeletal proteins, chaperones, and ligases. Here, we report that the formation of actin filaments (F-actin) is impaired in AF-treated trophozoites. Consequently, their erythrophagocytosis, cytopathic activity, and their motility are impaired. We also observed that less than 15% of OXs present in H2 O2 -treated trophozoites are also present in AF- or MNZ-treated trophozoites. These results strongly suggest that the formation of OXs in AF- or MNZ-treated trophozoites and in H2 O2 -treated trophozoites occurred by two different mechanisms.

Escherichia coli mediated resistance of Entamoeba histolytica to oxidative stress is triggered by oxaloacetate.

Amebiasis, a global intestinal parasitic disease, is due to Entamoeba histolytica. This parasite, which feeds on bacteria in the large intestine of its human host, can trigger a strong inflammatory response upon invasion of the colonic mucosa. Whereas information about the mechanisms which are used by the parasite to cope with oxidative and nitrosative stresses during infection is available, knowledge about the contribution of bacteria to these mechanisms is lacking. In a recent study, we demonstrated that enteropathogenic Escherichia coli O55 protects E. histolytica against oxidative stress. Resin-assisted capture (RAC) of oxidized (OX) proteins coupled to mass spectrometry (OX-RAC) was used to investigate the oxidation status of cysteine residues in proteins present in E. histolytica trophozoites incubated with live or heat-killed E. coli O55 and then exposed to H2O2-mediated oxidative stress. We found that the redox proteome of E. histolytica exposed to heat-killed E. coli O55 is enriched with proteins involved in redox homeostasis, lipid metabolism, small molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound biosynthesis. In contrast, we found that proteins associated with redox homeostasis were the only OX-proteins that were enriched in E. histolytica trophozoites which were incubated with live E. coli O55. These data indicate that E. coli has a profound impact on the redox proteome of E. histolytica. Unexpectedly, some E. coli proteins were also co-identified with E. histolytica proteins by OX-RAC. We demonstrated that one of these proteins, E. coli malate dehydrogenase (EcMDH) and its product, oxaloacetate, are key elements of E. coli-mediated resistance of E. histolytica to oxidative stress and that oxaloacetate helps the parasite survive in the large intestine. We also provide evidence that the protective effect of oxaloacetate against oxidative stress extends to Caenorhabditis elegans.

Target identification and intervention strategies against amebiasis.

Entamoeba histolytica is the etiological agent of amebiasis, which is an endemic parasitic disease in developing countries and is the cause of approximately 70,000 deaths annually. E. histolytica trophozoites usually reside in the colon as a non-pathogenic commensal in most infected individuals (90% of infected individuals are asymptomatic). For unknown reasons, these trophozoites can become virulent and invasive, cause amebic dysentery, and migrate to the liver where they cause hepatocellular damage. Amebiasis is usually treated either by amebicides which are classified as (a) luminal and are active against the luminal forms of the parasite, (b) tissue and are effective against those parasites that have invaded tissues, and (c) mixed and are effective against the luminal forms of the parasite and those forms which invaded the host's tissues. Of the amebicides, the luminal amebicide, metronidazole (MTZ), is the most widely used drug to treat amebiasis. Although well tolerated, concerns about its adverse effects and the possible emergence of MTZ-resistant strains of E. histolytica have led to the development of new therapeutic strategies against amebiasis. These strategies include improving the potency of existing amebicides, discovering new uses for approved drugs (repurposing of existing drugs), drug rediscovery, vaccination, drug targeting of essential E. histolytica components, and the use of probiotics and bioactive natural products. This review examines each of these strategies in the light of the current knowledge on the gut microbiota of patients with amebiasis.

Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation.

A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu.

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes.

Stress granule formation in Entamoeba histolytica: cross-talk between EhMLBP, EhRLE3 reverse transcriptase and polyubiquitinated proteins.

The Entamoeba histolytica-methylated LINE-binding protein (EhMLBP) binds to methylated repetitive DNA and is a positive regulator of a reverse transcriptase of a long interspersed nucleotide element (LINE). This protein protects trophozoites against heat shock by reducing protein aggregation. The presence of EhMLBP and polyubiquitinated proteins in heat shock-induced protein aggregates raised the question whether these proteins interact. This assumption was confirmed by co-immunoprecipitation experiments: ubiquitinated proteins were detected in the perinuclear region of non-stressed E. histolytica trophozoites, whereas ubiquitinated proteins were detected in the perinuclear region and colocalized with EhMLBP in cytoplasmic granules in heat-shocked trophozoites. We also observed that overexpression of the reverse transcriptase of EhRLE3 induced the upregulation of EhMLBP expression and the formation of these EhMLBP-containing granules. Since (i) these EhMLBP-containing granules in the cytoplasm of heat-shocked E. histolytica trophozoites also contain polyubiquitinated proteins and poly(A)(+) mRNA and (ii) their formation is promoted by sodium arsenate, puromycin, and pateamine A and is inhibited by cycloheximide, we propose that these cytoplasmic EhMLBP-containing granules are stress granules. Our data also suggest that the formation of these granules is dependent upon EhMLBP and LINE.

The Entamoeba histolytica Dnmt2 homolog (Ehmeth) confers resistance to nitrosative stress.

Nitric oxide (NO) has antimicrobial properties against many pathogens due to its reactivity as an S-nitrosylating agent. It inhibits many of the key enzymes that are involved in the metabolism and virulence of the parasite Entamoeba histolytica through S-nitrosylation of essential cysteine residues. Very little information is available on the mechanism of resistance to NO by pathogens in general and by this parasite in particular. Here, we report that exposure of the parasites to S-nitrosoglutathione (GSNO), an NO donor molecule, strongly reduces their viability and protein synthesis. However, the deleterious effects of NO were significantly reduced in trophozoites overexpressing Ehmeth, the cytosine-5 methyltransferase of the Dnmt2 family. Since these trophozoites also exhibited high levels of tRNA(Asp) methylation, the high levels suggested that Ehmeth-mediated tRNA(Asp) methylation is part of the resistance mechanism to NO. We previously reported that enolase, another glycolytic enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount of Ehmeth-enolase complex was significantly reduced in GSNO-treated E. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via site-directed mutagenesis that cysteine residues 228 and 229 of Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase and for Ehmeth-mediated resistance to NO. These results indicate that Ehmeth has a central role in the response of the parasite to NO, and they contribute to the growing evidence that NO is a regulator of epigenetic mechanisms.

Identification of dihydropyrimidine dehydrogenase as a virulence factor essential for the survival of Entamoeba histolytica in glucose-poor environments.

Adaptation to nutritional changes is a key feature for successful survival of a pathogen within its host. The protozoan parasite Entamoeba histolytica normally colonizes the human colon and in rare occasions, this parasite spread to distant organs, such as the liver. E. histolytica obtains most of its energy from the fermentation of glucose into ethanol. In this study, we were intrigued to know how this parasite reacts to changes in glucose availability and we addressed this issue by performing a DNA microarray analysis of gene expression. Results show that parasites that were adapted to growth in absence of glucose increased their virulence and altered the transcription of several genes. One of these genes is the dihydropyrimidine dehydrogenase (DPD), which is involved in degradation of pyrimidines. We showed that this gene is crucial for the parasite's growth when the availability of glucose is limited. These data contribute to our understanding of the parasite's ability to survive in glucose-poor environments and reveal a new role for the DPD enzyme.

Unraveling the interplay between unicellular parasites and bacterial biofilms: Implications for disease persistence and antibiotic resistance.

Bacterial biofilms have attracted significant attention due to their involvement in persistent infections, food and water contamination, and infrastructure corrosion. This review delves into the intricate interactions between bacterial biofilms and unicellular parasites, shedding light on their impact on biofilm formation, structure, and function. Unicellular parasites, including protozoa, influence bacterial biofilms through grazing activities, leading to adaptive changes in bacterial communities. Moreover, parasites like Leishmania and Giardia can shape biofilm composition in a grazing independent manner, potentially influencing disease outcomes. Biofilms, acting as reservoirs, enable the survival of protozoan parasites against environmental stressors and antimicrobial agents. Furthermore, these biofilms may influence parasite virulence and stress responses, posing challenges in disease treatment. Interactions between unicellular parasites and fungal-containing biofilms is also discussed, hinting at complex microbial relationships in various ecosystems. Understanding these interactions offers insights into disease mechanisms and antibiotic resistance dissemination, paving the way for innovative therapeutic strategies and ecosystem-level implications.

Impact of Reactive Sulfur Species on Entamoeba histolytica: Modulating Viability, Motility, and Biofilm Degradation Capacity.

Reactive sulfur species (RSS) like hydrogen sulfide (H2S) and cysteine persulfide (Cys-SSH) emerged as key signaling molecules with diverse physiological roles in the body, depending on their concentration and the cellular environment. While it is known that H2S and Cys-SSH are produced by both colonocytes and by the gut microbiota through sulfur metabolism, it remains unknown how these RSS affect amebiasis caused by Entamoeba histolytica, a parasitic protozoan that can be present in the human gastrointestinal tract. This study investigates H2S and Cys-SSH's impact on E. histolytica physiology and explores potential therapeutic implications. Exposing trophozoites to the H2S donor, sodium sulfide (Na2S), or to Cys-SSH led to rapid cytotoxicity. A proteomic analysis of Cys-SSH-challenged trophozoites resulted in the identification of >500 S-sulfurated proteins, which are involved in diverse cellular processes. Functional assessments revealed inhibited protein synthesis, altered cytoskeletal dynamics, and reduced motility in trophozoites treated with Cys-SSH. Notably, cysteine proteases (CPs) were significantly inhibited by S-sulfuration, affecting their bacterial biofilm degradation capacity. Immunofluorescence microscopy confirmed alterations in actin dynamics, corroborating the proteomic findings. Thus, our study reveals how RSS perturbs critical cellular functions in E. histolytica, potentially influencing its pathogenicity and interactions within the gut microbiota. Understanding these molecular mechanisms offers novel insights into amebiasis pathogenesis and unveils potential therapeutic avenues targeting RSS-mediated modifications in parasitic infections.

The Entamoeba histolytica methylated LINE-binding protein EhMLBP provides protection against heat shock.

Adaptation to environmental stress is a key process that allows the unicellular parasite Entamoeba histolytica to survive in its human host. We previously characterized EhMLBP as an essential protein for the growth and the virulence of the parasite. EhMLBP binds to methylated repetitive DNA, and is one of the core proteins of the parasite's epigenetic machinery. Here, we show that EhMLBP and heat shock proteins have common properties. EhMLBP is induced by heat shock and its expression is regulated by a heat shock element binding site that is located in its 5' non-coding region. Following heat shock, the perinuclear localization of EhMLBP in control trophozoites is replaced by an even distribution within the nucleus alongside with an enhanced recruitment of EhMLBP to the reverse transcriptase of a long interspersed nucleotide element (LINE) DNA. Constitutive overexpression of EhMLBP protects trophozoites against heat shock and reduces protein aggregation. This protective function is lost in trophozoites that overexpress a mutated form of EhMLBP which is devoid of its heat shock domain. To the best of our knowledge, this is the first report of a methyl DNA-binding protein that plays a protective role against heat shock.

Insights into the Role of Oxidative Stress and Reactive Oxygen Species in Parasitic Diseases.

Parasitic infections remain a significant public health challenge in many parts of the world, especially in developing countries [...].

Digestive exophagy of biofilms by intestinal amoeba and its impact on stress tolerance and cytotoxicity.

The human protozoan parasite Entamoeba histolytica is responsible for amebiasis, a disease endemic to developing countries. E. histolytica trophozoites colonize the large intestine, primarily feeding on bacteria. However, in the gastrointestinal tract, bacterial cells form aggregates or structured communities called biofilms too large for phagocytosis. Remarkably, trophozoites are still able to invade and degrade established biofilms, utilizing a mechanism that mimics digestive exophagy. Digestive exophagy refers to the secretion of digestive enzymes that promote the digestion of objects too large for direct phagocytosis by phagocytes. E. histolytica cysteine proteinases (CPs) play a crucial role in the degradation process of Bacillus subtilis biofilm. These proteinases target TasA, a major component of the B. subtilis biofilm matrix, also contributing to the adhesion of the parasite to the biofilm. In addition, they are also involved in the degradation of biofilms formed by Gram-negative and Gram-positive enteric pathogens. Furthermore, biofilms also play an important role in protecting trophozoites against oxidative stress. This specific mechanism suggests that the amoeba has adapted to prey on biofilms, potentially serving as an untapped reservoir for novel therapeutic approaches to treat biofilms. Consistently, products derived from the amoeba have been shown to restore antibiotic sensitivity to biofilm cells. In addition, our findings reveal that probiotic biofilms can act as a protective shield for mammalian cells, hindering the progression of the parasite towards them.

A new nuclear function of the Entamoeba histolytica glycolytic enzyme enolase: the metabolic regulation of cytosine-5 methyltransferase 2 (Dnmt2) activity.

Cytosine-5 methyltransferases of the Dnmt2 family function as DNA and tRNA methyltransferases. Insight into the role and biological significance of Dnmt2 is greatly hampered by a lack of knowledge about its protein interactions. In this report, we address the subject of protein interaction by identifying enolase through a yeast two-hybrid screen as a Dnmt2-binding protein. Enolase, which is known to catalyze the conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP), was shown to have both a cytoplasmatic and a nuclear localization in the parasite Entamoeba histolytica. We discovered that enolase acts as a Dnmt2 inhibitor. This unexpected inhibitory activity was antagonized by 2-PG, which suggests that glucose metabolism controls the non-glycolytic function of enolase. Interestingly, glucose starvation drives enolase to accumulate within the nucleus, which in turn leads to the formation of additional enolase-E.histolytica DNMT2 homolog (Ehmeth) complex, and to a significant reduction of the tRNA(Asp) methylation in the parasite. The crucial role of enolase as a Dnmt2 inhibitor was also demonstrated in E.histolytica expressing a nuclear localization signal (NLS)-fused-enolase. These results establish enolase as the first Dnmt2 interacting protein, and highlight an unexpected role of a glycolytic enzyme in the modulation of Dnmt2 activity.

Are Metabolites From the Gut Microbiota Capable of Regulating Epigenetic Mechanisms in the Human Parasite Entamoeba histolytica?

The unicellular parasite Entamoeba histolytica inhabits the human gut. It has to adapt to a complex environment that consists of the host microbiota, nutritional stress, oxidative stress, and nitrosative stress. Adaptation to this complex environment is vital for the survival of this parasite. Studies have shown that the host microbiota shapes virulence and stress adaptation in E. histolytica. Increasing evidence suggests that metabolites from the microbiota mediate communication between the parasite and microbiota. In this review, we discuss the bacterial metabolites that regulate epigenetic processes in E. histolytica and the implications that this knowledge may have for the development of new anti-amebic strategies.

Insights into the Mechanisms of Lactobacillus acidophilus Activity against Entamoeba histolytica by Using Thiol Redox Proteomics.

Amebiasis is an intestinal disease transmitted by the protist parasite, Entamoeba histolytica. Lactobacillus acidophilus is a common inhabitant of healthy human gut and a probiotic that has antimicrobial properties against a number of pathogenic bacteria, fungi, and parasites. The aim of this study was to investigate the amebicide activity of L. acidophilus and its mechanisms. For this purpose, E. histolytica and L. acidophilus were co-incubated and the parasite's viability was determined by eosin dye exclusion. The level of ozidized proteins (OXs) in the parasite was determined by resin-assisted capture RAC (OX-RAC). Incubation with L. acidophilus for two hours reduced the viability of E. histolytica trophozoites by 50%. As a result of the interaction with catalase, an enzyme that degrades hydrogen peroxide (H2O2) to water and oxygen, this amebicide activity is lost, indicating that it is mediated by H2O2 produced by L. acidophilus. Redox proteomics shows that L. acidophilus triggers the oxidation of many essential amebic enzymes such as pyruvate: ferredoxin oxidoreductase, the lectin Gal/GalNAc, and cysteine proteases (CPs). Further, trophozoites of E. histolytica incubated with L. acidophilus show reduced binding to mammalian cells. These results support L. acidophilus as a prophylactic candidate against amebiasis.

Queuine Salvaging in the Human Parasite Entamoeba histolytica.

Queuosine (Q) is a naturally occurring modified nucleoside that occurs in the first position of transfer RNA anticodons such as Asp, Asn, His, and Tyr. As eukaryotes lack pathways to synthesize queuine, the Q nucleobase, they must obtain it from their diet or gut microbiota. Previously, we described the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica and characterized the enzyme EhTGT responsible for queuine incorporation into tRNA. At present, it is unknown how E. histolytica salvages queuine from gut bacteria. We used liquid chromatography-mass spectrometry (LC-MS) and N-acryloyl-3-aminophenylboronic acid (APB) PAGE analysis to demonstrate that E. histolytica trophozoites can salvage queuine from Q or E. coli K12 but not from the modified E. coli QueC strain, which cannot produce queuine. We then examined the role of EhDUF2419, a protein with homology to DNA glycosylase, as a queuine salvage enzyme in E. histolytica. We found that glutathione S-transferase (GST)-EhDUF2419 catalyzed the conversion of Q into queuine. Trophozoites silenced for EhDUF2419 expression are impaired in their ability to form Q-tRNA from Q or from E. coli. We also observed that Q or E. coli K12 partially protects control trophozoites from oxidative stress (OS), but not siEhDUF2419 trophozoites. Overall, our data reveal that EhDUF2419 is central for the direct salvaging of queuine from bacteria and for the resistance of the parasite to OS.

Structure analysis of Entamoeba histolytica enolase.

Entamoeba histolytica enolase (EhENO) reversibly interconverts 2-phosphoglyceric acid (2-PGA) and phosphoenolpyruvic acid (PEP). The crystal structure of the homodimeric EhENO is presented at a resolution of 1.9 Å. In the crystal structure EhENO presents as an asymmetric dimer with one active site in the open conformation and the other active site in the closed conformation. Interestingly, both active sites contain a copurified 2-PGA molecule. While the 2-PGA molecule in the closed active site closely resembles the conformation known from other enolase-2-PGA complexes, the conformation in the open active site is different. Here, 2-PGA is shifted approximately 1.6 Šaway from metal ion I, most likely representing a precatalytic situation.

Entamoeba histolytica-Gut Microbiota Interaction: More Than Meets the Eye.

Amebiasis is a disease caused by the unicellular parasite Entamoeba histolytica. In most cases, the infection is asymptomatic but when symptomatic, the infection can cause dysentery and invasive extraintestinal complications. In the gut, E. histolytica feeds on bacteria. Increasing evidences support the role of the gut microbiota in the development of the disease. In this review we will discuss the consequences of E. histolytica infection on the gut microbiota. We will also discuss new evidences about the role of gut microbiota in regulating the resistance of the parasite to oxidative stress and its virulence.

Entamoeba histolytica Adaption to Auranofin: A Phenotypic and Multi-Omics Characterization.

Auranofin (AF), an antirheumatic agent, targets mammalian thioredoxin reductase (TrxR), an important enzyme controlling redox homeostasis. AF is also highly effective against a diversity of pathogenic bacteria and protozoan parasites. Here, we report on the resistance of the parasite Entamoeba histolytica to 2 µM of AF that was acquired by gradual exposure of the parasite to an increasing amount of the drug. AF-adapted E. histolytica trophozoites (AFAT) have impaired growth and cytopathic activity, and are more sensitive to oxidative stress (OS), nitrosative stress (NS), and metronidazole (MNZ) than wild type (WT) trophozoites. Integrated transcriptomics and redoxomics analyses showed that many upregulated genes in AFAT, including genes encoding for dehydrogenase and cytoskeletal proteins, have their product oxidized in wild type trophozoites exposed to AF (acute AF trophozoites) but not in AFAT. We also showed that the level of reactive oxygen species (ROS) and oxidized proteins (OXs) in AFAT is lower than that in acute AF trophozoites. Overexpression of E. histolytica TrxR (EhTrxR) did not protect the parasite against AF, which suggests that EhTrxR is not central to the mechanism of adaptation to AF.