RESUMO
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.
RESUMO
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.
Assuntos
Amoeba , Entamoeba histolytica , Animais , Humanos , Entamoeba histolytica/metabolismo , Fagocitose , Trato Gastrointestinal , Biofilmes , MamíferosRESUMO
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.
RESUMO
Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in [Formula: see text] Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in [Formula: see text], parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.IMPORTANCEEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.
Assuntos
Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/patogenicidade , Guanina/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Animais , Entamoeba histolytica/genética , Feminino , Guanina/metabolismo , Guanina/farmacologia , Células HeLa , Humanos , Metilação , Camundongos , Camundongos Endogâmicos BALB C , RNA de Transferência/metabolismoRESUMO
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.
Assuntos
Amebíase/tratamento farmacológico , Amebíase/prevenção & controle , Amebicidas/uso terapêutico , Entamoeba histolytica/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Vacinas Protozoárias/administração & dosagem , Amebíase/imunologia , Amebíase/parasitologia , Animais , Produtos Biológicos/uso terapêutico , Colo/efeitos dos fármacos , Colo/parasitologia , Colo/patologia , Reposicionamento de Medicamentos/métodos , Entamoeba histolytica/patogenicidade , Entamoeba histolytica/fisiologia , Microbioma Gastrointestinal/imunologia , Interações Hospedeiro-Parasita/imunologia , Humanos , Fígado/efeitos dos fármacos , Fígado/parasitologia , Fígado/patologia , Metronidazol/uso terapêutico , Interações Microbianas , Probióticos/uso terapêutico , Vacinas Protozoárias/biossíntese , Índice de Gravidade de DoençaRESUMO
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.
Assuntos
Entamoeba histolytica/efeitos dos fármacos , Entamebíase/tratamento farmacológico , Escherichia coli/fisiologia , Ácido Oxaloacético/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Amebíase/tratamento farmacológico , Amebíase/metabolismo , Amebíase/parasitologia , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/parasitologia , Células Cultivadas , Entamebíase/metabolismo , Entamebíase/parasitologia , Células HeLa , Humanos , Intestino Grosso/efeitos dos fármacos , Intestino Grosso/metabolismo , Intestino Grosso/parasitologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBARESUMO
During its life cycle, the unicellular parasite Entamoeba histolytica is challenged by a wide variety of environmental stresses, such as fluctuation in glucose concentration, changes in gut microbiota composition, and the release of oxidative and nitrosative species from neutrophils and macrophages. The best mode of survival for this parasite is to continuously adapt itself to the dynamic environment of the host. Our ability to study the stress-induced responses and adaptive mechanisms of this parasite has been transformed through the development of genomics, proteomics or metabolomics (omics sciences). These studies provide insights into different facets of the parasite's behavior in the host. However, there is a dire need for multi-omics data integration to better understand its pathogenic nature, ultimately paving the way to identify new chemotherapeutic targets against amebiasis. This review provides an integration of the most relevant omics information on the mechanisms that are used by E. histolytica to resist environmental stresses.
Assuntos
Entamebíase/parasitologia , Genômica , Metabolômica , Proteômica , Estresse Fisiológico , Adaptação Biológica , Animais , Cisteína/metabolismo , Entamoeba histolytica/fisiologia , Microbioma Gastrointestinal , Genômica/métodos , Glucose/metabolismo , Interações Hospedeiro-Parasita , Humanos , Ferro/metabolismo , Metabolômica/métodos , Estresse Oxidativo , Proteômica/métodosRESUMO
We have recently reported that Entamoeba histolytica trophozoites can adapt to toxic levels of the nitric oxide (NO) donor, S-nitrosoglutathione (GSNO). Even if the consequences of this adaptation on the modulation of gene expression in NO-adapted trophozoites (NAT) have been previously explored, insight on S-nitrosylated (SNO) proteins in NAT is missing. Our study aims to fill this knowledge gap by performing a screening of SNO proteins in NAT. Employing SNO resin-assisted capture (RAC), we identified 242 putative SNO proteins with key functions in calcium binding, enzyme modulation, redox homeostasis, and actin cytoskeleton. Of the SNO proteins in NAT, proteins that are associated with actin family cytoskeleton protein are significantly enriched. Here we report that the formation of actin filaments (F-actin) is impaired in NAT. Consequently, the ability of NAT to ingest erythrocytes and their motility and their cytopathic activity are impaired. These phenotypes can be imitated by treating control parasite with cytochalasin D (CytD), a drug that binds to F-actin polymer and prevent polymerization of actin monomers. Removal of GSNO from the culture medium of NAT restored the sensitivity of the parasite to nitrosative stress (NS) and its ability to form F-actin formation and its virulence. These results establish the central role of NO in shaping the virulence of the parasite through its effect on F-actin formation and highlight the impressive ability of this parasite to adapt to NS.
Assuntos
Actinas/metabolismo , Entamoeba histolytica/química , Entamoeba histolytica/metabolismo , Estresse Nitrosativo , Proteínas de Protozoários/metabolismo , S-Nitrosotióis/química , Virulência , Citoesqueleto de Actina/metabolismo , Actinas/ultraestrutura , Movimento Celular/fisiologia , Cisteína/análogos & derivados , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/patogenicidade , Eritrócitos/parasitologia , Expressão Gênica , Microscopia Confocal , Óxido Nítrico/farmacologia , Proteólise , Proteínas de Protozoários/genética , Trofozoítos/metabolismo , Virulência/efeitos dos fármacosRESUMO
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.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Ácidos Nucleicos/metabolismo , Animais , Sítios de Ligação , Catálise , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/classificação , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Metilação , Ácidos Nucleicos/química , Ácidos Nucleicos/genética , Filogenia , Ligação Proteica , Retroelementos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Entamoeba histolytica is a gastrointestinal protozoan parasite that causes amebiasis, a disease which has a worldwide distribution with substantial morbidity and mortality. Nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that E. histolytica encounters during its life cycle. Although the effects of nitric oxide (NO) on the regulation of gene expression in this parasite have been previously investigated, our knowledge on S-nitrosylated proteins in E.histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale detection of S-nitrosylated (SNO) proteins in E.histolytica trophozoites that were treated with the NO donor, S-nitrosocysteine by resin-assisted capture (RAC). We found that proteins involved in glycolysis, gluconeogenesis, translation, protein transport, and adherence to target cells such as the heavy subunit of Gal/GalNac lectin are among the S-nitrosylated proteins that were enriched by SNO-RAC. We also found that the S-nitrosylated cysteine residues in the carbohydrate recognition domain (CRD) of Gal/GalNAc lectin impairs its function and contributes to the inhibition of E.histolytica adherence to host cells. Collectively, these results advance our understanding of the mechanism of reduced E.histolytica adherence to mammalian cells by NO and emphasize the importance of NO as a regulator of key physiological functions in E.histolytica.
Assuntos
Cisteína/análogos & derivados , Entamoeba histolytica/química , Lectinas/química , Óxido Nítrico/química , Nitrogênio/química , S-Nitrosotióis/química , Carboidratos/química , Adesão Celular , Cromatografia de Afinidade , Cisteína/química , Entamebíase/imunologia , Entamebíase/parasitologia , Glicólise , Células HeLa , Humanos , Transporte Proteico , Proteoma , Proteômica , Proteínas de Protozoários/genéticaRESUMO
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.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Resistência a Medicamentos/genética , Entamoeba histolytica/genética , Doadores de Óxido Nítrico/farmacologia , Proteínas de Protozoários/genética , S-Nitrosoglutationa/farmacologia , Cisteína/química , Cisteína/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/metabolismo , Epigênese Genética , Metilação , Mutagênese Sítio-Dirigida , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , RNA de Transferência/metabolismo , S-Nitrosoglutationa/metabolismo , Transdução de Sinais , Estresse Fisiológico , Trofozoítos/efeitos dos fármacos , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismoRESUMO
In eukaryotes, DNA methylation is an important epigenetic modification that is generally involved in gene regulation. Methyltransferases (MTases) of the DNMT2 family have been shown to have a dual substrate specificity acting on DNA as well as on three specific tRNAs (tRNA(Asp), tRNA(Val), tRNA(Gly)). Entamoeba histolytica is a major human pathogen, and expresses a single DNA MTase (EhMeth) that belongs to the DNMT2 family and shows high homology to the human enzyme as well as to the bacterial DNA MTase M.HhaI. The molecular basis for the recognition of the substrate tRNAs and discrimination of non-cognate tRNAs is unknown. Here we present the crystal structure of the cytosine-5-methyltransferase EhMeth at a resolution of 2.15 Å, in complex with its reaction product S-adenosyl-L-homocysteine, revealing all parts of a DNMT2 MTase, including the active site loop. Mobility shift assays show that in vitro the full length tRNA is required for stable complex formation with EhMeth.
Assuntos
DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Entamoeba histolytica/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X/métodos , Metilação de DNA , Humanos , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA de Transferência/química , S-Adenosil-Homocisteína/químicaRESUMO
The unicellular parasite, Entamoeba histolytica, is exposed to numerous adverse conditions, such as nutrient deprivation, during its life cycle stages in the human host. In the present study, we examined whether the parasite virulence could be influenced by glucose starvation (GS). The migratory behaviour of the parasite and its capability to kill mammalian cells and to lyse erythrocytes is strongly enhanced following GS. In order to gain insights into the mechanism underlying the GS boosting effects on virulence, we analyzed differences in protein expression levels in control and glucose-starved trophozoites, by quantitative proteomic analysis. We observed that upstream regulatory element 3-binding protein (URE3-BP), a transcription factor that modulates E.histolytica virulence, and the lysine-rich protein 1 (KRiP1) which is induced during liver abscess development, are upregulated by GS. We also analyzed E. histolytica membrane fractions and noticed that the Gal/GalNAc lectin light subunit LgL1 is up-regulated by GS. Surprisingly, amoebapore A (Ap-A) and cysteine proteinase A5 (CP-A5), two important E. histolytica virulence factors, were strongly down-regulated by GS. While the boosting effect of GS on E. histolytica virulence was conserved in strains silenced for Ap-A and CP-A5, it was lost in LgL1 and in KRiP1 down-regulated strains. These data emphasize the unexpected role of GS in the modulation of E.histolytica virulence and the involvement of KRiP1 and Lgl1 in this phenomenon.
Assuntos
Entamoeba histolytica/metabolismo , Entamoeba histolytica/patogenicidade , Glucose/metabolismo , Estresse Oxidativo/fisiologia , Proteínas de Protozoários/metabolismo , Fatores de Virulência/metabolismo , Animais , Cricetinae , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Entamebíase/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Abscesso Hepático Amebiano/patologia , Masculino , Mesocricetus , Proteínas de Protozoários/genética , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismo , Virulência , Fatores de Virulência/genéticaRESUMO
Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, Dictyostelium discoideum and Drosophila. Our recent work has shown that Ehmeth methylates tRNA(Asp), and this finding indicates that this enzyme has a dual DNA/tRNA(Asp) methyltransferase activity. This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Entamoeba histolytica/enzimologia , RNA Fúngico/metabolismo , RNA de Transferência/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , Entamoeba histolytica/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Metilação , RNA Fúngico/genética , RNA de Transferência/genéticaRESUMO
Histone deacetylation is associated with a repressed chromatin state, and histone acetylase and deacetylase activities have been previously described in Entamoeba histolytica. To investigate their roles in the control of Entamoeba gene expression, the parasite was grown in 50 nM trichostatin A (TSA), an inhibitor of histone deacetylase. TSA enhanced the cytopathic and hemolytic activity of the parasite and its resistance to oxidative stress. We first focused our attention on peroxiredoxin, a protein previously associated with E. histolytica virulence and resistance to oxidative stress. We found that the expression of peroxiredoxin was increased after TSA treatment, but were unable to confirm that this was a direct consequence of histone modification at the promoter. By microarray analysis, we found that some other mRNAs encoding some other virulence factors, such as the galactose-inhibitable lectin small subunits, were also increased. The pattern of gene expression was surprisingly different from that previously described after treatment with 150 nM TSA.
Assuntos
Entamoeba histolytica/patogenicidade , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Peroxirredoxinas/biossíntese , Fatores de Virulência/biossíntese , Animais , Antipruriginosos/farmacologia , Sobrevivência Celular , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/genética , Células Epiteliais/parasitologia , Perfilação da Expressão Gênica , Células HeLa , Hemólise , Inibidores de Histona Desacetilases , Humanos , Lectinas/biossíntese , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Oxidantes/farmacologia , Estresse Oxidativo , Proteínas de Protozoários/biossíntese , Transcrição Gênica , VirulênciaRESUMO
BACKGROUND & AIMS: The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. METHODS: Cysteine protease-deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [(35)S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. RESULTS: Trophozoites with reduced cysteine protease activity were ineffective at degrading [(35)S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease-deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. CONCLUSIONS: These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis.
Assuntos
Colo/metabolismo , Cisteína Endopeptidases/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Entamoeba histolytica/enzimologia , Mucinas/metabolismo , RNA Antissenso/farmacologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Cisteína Endopeptidases/genética , Humanos , TransfecçãoRESUMO
Invasive microorganisms efface enteric microvilli to establish intimate contact with the apical surface of enterocytes. To understand the molecular basis of this effacement in amebic colitis, we seeded Entamoeba histolytica trophozoites on top of differentiated human Caco-2 cell layers. Western blots of detergent lysates from such cocultures showed proteolysis of the actin-bundling protein villin within 1 min of direct contact of living trophozoites with enterocytes. Mixtures of separately prepared lysates excluded detergent colysis as the cause of villin proteolysis. Caspases were not responsible as evidenced by the lack of degradation of specific substrates and the failure of a specific caspase inhibitor to prevent villin proteolysis. A crucial role for amebic cysteine proteinases was shown by prevention of villin proteolysis and associated microvillar alterations through the treatment of trophozoites before coculture with synthetic inhibitors that completely blocked amebic cysteine proteinase activity on zymograms. Moreover, trophozoites of amebic strains pSA8 and SAW760 with strongly reduced cysteine proteinase activity showed a reduced proteolysis of villin in coculture with enteric cells. Salmonella typhimurium and enteropathogenic Escherichia coli disturb microvilli without villin proteolysis, indicating that the latter is not a consequence of the disturbance of microvilli. In conclusion, villin proteolysis is an early event in the molecular cross-talk between enterocytes and amebic trophozoites, causing a disturbance of microvilli.
Assuntos
Proteínas de Transporte/metabolismo , Cisteína Endopeptidases/metabolismo , Entamoeba histolytica/enzimologia , Mucosa Intestinal/parasitologia , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/parasitologia , Animais , Adesão Celular , Técnicas de Cocultura , Neoplasias Colorretais , Entamoeba histolytica/fisiologia , Humanos , Células Tumorais Cultivadas , Ureia/metabolismoRESUMO
Nitric oxide is involved in the neutrophil and macrophage killing of the protozoan parasite Entamoeba histolytica. In the present study, we found that cysteine proteinases, significant contributors to amebic virulence and alcohol dehydrogenase 2, an enzyme absolutely required for the survival of the parasite, are both significantly inhibited by S-nitroso-glutathione, a physiological nitric oxide donor, within the concentration range 0.5-2.0 m M.