RESUMO
Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1-400 aa), Linker (246-446 aa) and Adh (444-687 aa) to evaluate their role in virulence. The TrophozBro11-400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226-446 also increased the virulence properties, but with lower effect than the TrophozBro11-400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444-687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.
Assuntos
Entamoeba histolytica , Proteínas de Protozoários , Entamoeba histolytica/patogenicidade , Entamoeba histolytica/metabolismo , Animais , Camundongos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Humanos , Virulência , Fagocitose , Domínios Proteicos , Entamebíase/parasitologia , Entamebíase/metabolismo , Cricetinae , Trofozoítos/metabolismoRESUMO
Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.
Assuntos
Entamoeba histolytica/fisiologia , Entamebíase/metabolismo , Entamebíase/parasitologia , Interações Hospedeiro-Parasita , Fagocitose , Proteínas de Protozoários/metabolismo , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismo , Sítios de Ligação , Citofagocitose , Entamoeba histolytica/classificação , Entamebíase/imunologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Genoma de Protozoário , Humanos , Modelos Moleculares , Fagossomos , Filogenia , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , SumoilaçãoRESUMO
Lipids are essential players in parasites pathogenesis. In particular, the highly phagocytic trophozoites of Entamoeba histolytica, the causative agent of amoebiasis, exhibit a dynamic membrane fusion and fission, in which lipids strongly participate; particularly during the overstated motility of the parasite to reach and attack the epithelia and ingest target cells. Synthesis and metabolism of lipids in this protozoan present remarkable difference with those performed by other eukaryotes. Here, we reviewed the current knowledge on lipids in E. histolytica. Trophozoites synthesize phosphatidylcholine and phosphatidylethanolamine by the Kennedy pathway; and sphingolipids, phosphatidylserine, and phosphatidylinositol, by processes similar to those used by other eukaryotes. However, trophozoites lack enzymes for cholesterol and fatty acids synthesis, which are scavenged from the host or culture medium by specific mechanisms. Cholesterol, a fundamental molecule for the expression of virulence, is transported from the medium into the trophozoites by EhNPC1 and EhNPC2 proteins. Inside cells, lipids are distributed by different pathways, including by the participation of the endosomal sorting complex required for transport (ESCRT), involved in vesicle fusion and fission. Cholesterol interacts with the phospholipid lysobisphosphatidic acid (LBPA) and EhADH, an ALIX family protein, also involved in phagocytosis. In this review, we summarize the known information on phospholipids synthesis and cholesterol transport as well as their metabolic pathways in E. histolytica; highlighting the mechanisms used by trophozoites to dispose lipids involved in the virulence processes.
Assuntos
Entamoeba histolytica/metabolismo , Entamoeba histolytica/patogenicidade , Entamebíase/parasitologia , Metabolismo dos Lipídeos , Trofozoítos/metabolismo , Fatores de Virulência/metabolismo , Animais , Colesterol/biossíntese , Colesterol/metabolismo , Entamoeba histolytica/química , Entamebíase/metabolismo , Ácidos Graxos/biossíntese , Humanos , Lipídeos/análise , Fagocitose , Fosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Trofozoítos/química , VirulênciaRESUMO
Epidemiological studies on species-specific Entamoeba infections are scarce due to the morphological similarity of pathogenic Entamoeba histolytica and nonpathogenic E. dispar and E. moshkovskii. The diagnosis of E. histolytica is frequently based on coproantigen (E. histolytica-Gal/GalNAc lectin specific) detection by immunoassays. However, specific E. histolytica-lectin is not expressed in cysts, which are eliminated by asymptomatic individuals leading to false-negative results and an underestimation of amebiasis prevalence. Molecular techniques based on the amplification of parasite DNA have been shown to be a highly sensitive and specific method that allows the detection of different Entamoeba species. This study aimed to assess the frequency of the species from E. histolytica/dispar/moshkovskii complex by molecular and immunological techniques in individuals attended at a public health system in Salvador-Bahia, Brazil. A cross-sectional study involving 55,218 individuals was carried out. The diagnosis was based on microscopy revealing E. histolytica/dispar/moshkovskii complex. The species differentiation was performed by E. histolytica-specific antigen, serological evaluation and by molecular technique. The overall prevalence of E. histolytica/dispar/moshkovskii complex determined by microscopy was approximately 0.49% (273/55,218). E. histolytica-specific antigen detection and molecular characterization returned 100% negativity for E. histolytica. However, serological evaluation returned an 8.9% positivity (8/90). In the stool samples analysed by PCR, it was not possible to identify E. histolytica and E. moshkovskii, although circulating IgG anti-E. histolytica has been detected.
Assuntos
Antígenos de Protozoários , DNA de Protozoário , Entamoeba histolytica , Entamebíase , Adolescente , Adulto , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Brasil , Criança , Estudos Transversais , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Entamoeba histolytica/classificação , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Entamebíase/genética , Entamebíase/metabolismo , Entamebíase/parasitologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , PrevalênciaRESUMO
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.
Assuntos
Membrana Celular/fisiologia , Entamoeba histolytica/enzimologia , Entamebíase/parasitologia , Esfingomielina Fosfodiesterase/metabolismo , Trofozoítos/metabolismo , Cálcio/metabolismo , Entamebíase/metabolismo , Humanos , Esfingomielina Fosfodiesterase/genética , Trofozoítos/crescimento & desenvolvimentoRESUMO
Neutrophil extracellular traps (NETs) are DNA fibers decorated with histones and antimicrobial proteins from cytoplasmic granules released into the extracellular space in a process denominated NETosis. The molecular pathways involved in NETosis have not been completely understood. Classical NETosis mechanisms involve the neutrophil elastase (NE) translocation to nucleus due to the generation of reactive oxygen species (ROS) by NADPH oxidase (NOX2) or the peptidyl arginine deiminase 4 (PAD4) activation in response to an increase in extracellular calcium influx; both mechanisms result in DNA decondensation. Previously, we reported that trophozoites and lipopeptidophosphoglycan from Entamoeba histolytica trigger NET release in human neutrophils. Here, we demonstrated in a quantitative manner that NETs were rapidly form upon treatment with amoebic trophozoites and involved both nuclear and mitochondrial DNA (mtDNA). NETs formation depended on amoeba viability as heat-inactivated or paraformaldehyde-fixed amoebas were not able to induce NETs. Interestingly, ROS were not detected in neutrophils during their interaction with amoebas, which could explain why NOX2 inhibition using apocynin did not affect this NETosis. Surprisingly, whereas calcium chelation reduced NET release induced by amoebas, PAD4 inhibition by GSK484 failed to block DNA extrusion but, as expected, abolished NETosis induced by the calcium ionophore A23187. Additionally, NE translocation to the nucleus and serine-protease activity were necessary for NET release caused by amoeba. These data support the idea that E. histolytica trophozoites trigger NETosis by a rapid non-classical mechanism and that different mechanisms of NETs release exist depending on the stimuli used.
Assuntos
Entamoeba histolytica/metabolismo , Entamebíase/metabolismo , Armadilhas Extracelulares/metabolismo , NADPH Oxidases/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trofozoítos/metabolismo , Acetofenonas/antagonistas & inibidores , Apoptose , Cálcio/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Entamebíase/parasitologia , Armadilhas Extracelulares/parasitologia , Humanos , Elastase de Leucócito/metabolismo , Viabilidade Microbiana , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADPH Oxidases/efeitos dos fármacos , Necrose , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Oxirredução/efeitos dos fármacos , Peptidoglicano/metabolismo , Fosfolipídeos/metabolismo , Proteína-Arginina Desiminase do Tipo 4 , Inibidores de Serina Proteinase/metabolismo , Trofozoítos/genéticaRESUMO
In Entamoeba histolytica, iron modulates virulence and gene expression via unknown regulatory mechanisms. The existence of a posttranscriptional iron regulatory system parallel with the iron-responsive element (IRE)/iron regulatory protein (IRP) system in the protozoan Trichomonas vaginalis has recently been reported. Due to their evolutionary closeness and the importance of iron for growth and virulence in these protozoa, we hypothesized the existence of an IRE/IRP-like mechanism in E. histolytica. To determine the presence of IRE-like elements in some mRNAs from this parasite, we performed in silico analyses of the 5'- and 3'-UTRs of mRNAs encoding virulence factors and cytoskeleton, ribosomal and metabolism proteins. The Zuker mfold software predicted IRE-like secondary structures in 52 of the 135 mRNAs analysed. However, only nine structures shared sequence similarity with the apical loop sequence (CAGUGN) of the previously reported human IRE-ferritin, whereas the GUU/UUG protozoan-specific motif was detected in 23 stem-loop structures. A new motif, AUU/AUUU, was also observed in 23 structures, suggesting the possible existence of an amoeba-specific motif. Additionally, cross-linking and RNA electrophoretic mobility shift assays showed specific RNA-protein interactions, using as a model two amoebic IRE-like elements from iron-regulated mRNAs and HeLa, T. vaginalis and E. histolytica cytoplasmic proteins. Our data suggest the presence of a posttranscriptional iron regulatory IRE/IRP-like mechanism in E. histolytica.
Assuntos
Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Regulação da Expressão Gênica , Ferro/metabolismo , Elementos de Resposta , Células Cultivadas , Entamebíase/metabolismo , Entamebíase/parasitologia , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Entamoeba histolytica is the causative agent of amebiasis in humans and is responsible for 100,000 deaths annually, making it the third leading cause of death due to a protozoan parasite. Pathogenesis appears to result from the potent cytotoxic activity of the parasite, which kills host cells within minutes. Although the mechanism is unknown, it is well established to be contact-dependent. The life cycle of the parasite alternates with two forms: the resistant cyst and the invasive trophozoite. The adhesive interactions between the parasite and surface glycoconjugates of host cells, as well as those lining the epithelia, are determinants for invasion of human tissues, for its cytotoxic activity, and finally for the outcome of the disease. In this review we present an overview of the information available on the amebic lectins and adhesins that are responsible of those adhesive interactions and we also refer to their effect on the host immune response. Finally, we present some concluding remarks and perspectives in the field.
Assuntos
Moléculas de Adesão Celular/metabolismo , Entamoeba histolytica/metabolismo , Entamebíase/metabolismo , Lectinas/metabolismo , Proteínas de Protozoários/metabolismo , Trofozoítos/metabolismo , Animais , Entamoeba histolytica/patogenicidade , Entamebíase/patologia , HumanosRESUMO
Entamoeba histolytica is a protozoan parasite that presents a risk to the health of millions of people worldwide. Due to the existence of different clinical forms caused by the parasite and also different virulence levels presented by one strain, one would expect differences in the profile of gene transcripts between virulent and nonvirulent cultures. In this study we used the differential display to select gene segments related to invasiveness of amoeba. One Brazilian strain of E. histolytica in two conditions, able or not to cause lesions in experimental animals, was used. RNA from this strain, was used to study the differential expression of genes. 29 specific gene fragments differentially expressed in the virulent strain were selected. By real-time PCR, six of these genes had confirmed their differential expression in the virulent culture. These genes may have important roles in triggering invasive amoebiasis and may be related to adaptation of trophozoites to difficulties encountered during colonization of the intestinal epithelium and liver tissue. Future studies with these genes may elucidate its actual role in tissue invasion by E. histolytica generating new pathways for diagnosis and treatment of amoebiasis.
Assuntos
Entamoeba histolytica/metabolismo , Entamebíase/metabolismo , Regulação da Expressão Gênica , RNA de Protozoário/biossíntese , Animais , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidade , Entamebíase/genética , Entamebíase/terapia , Humanos , Camundongos , RNA de Protozoário/genética , RatosRESUMO
Iron is essential for nearly all organisms; in mammals, it is part of proteins such as haemoglobin, and it is captured by transferrin and lactoferrin. Transferrin is present in serum, and lactoferrin is secreted by the mucosa and by neutrophils at infection sites, as a host iron-withholding response, sequestering iron away from invading microorganisms. Additionally, all cells contain ferritin, which sequesters iron when its intracellular levels are increased, detoxifying and preventing damage. Liver ferritin contains 50% of iron corporal reserves. During evolution, pathogens have evolved diverse strategies to obtain iron from their hosts in order to survive. The protozoan Entamoeba histolytica invades the intestinal mucosa, causing dysentery, and the trophozoites often travel to the liver producing hepatic abscesses; thus, intestine and liver proteins could be important iron supplies for E. histolytica. We found that E. histolytica trophozoites can grow in both ferrous and ferric iron, and that they can use haemoglobin, holo-transferrin, holo-lactoferrin, and ferritin as in vitro iron sources. These proteins supported the amoeba growth throughout consecutive passages, similarly to ferric citrate. By confocal microscopy and immunoblotting, iron-binding proteins were observed specifically bound to the amoeba surface, and they were endocytosed, trafficked through the endosomal/lysosomal route, and degraded by neutral and acidic cysteine-proteases. Transferrin and ferritin were mainly internalized through clathrin-coated vesicles, and holo-lactoferrin was mainly internalized by caveola-like structures. In contrast, apo-lactoferrin bound to membrane lipids and cholesterol, inducing cell death. The results suggest that in vivo trophozoites secrete products that can destroy enterocytes, erythrocytes, and hepatocytes, releasing transferrin, haemoglobin, ferritin, and other iron-containing proteins, which, together with lactoferrin derived from neutrophils and acinar cells, could be used as abundant iron supplies by amoebas.
Assuntos
Endocitose , Entamoeba histolytica/fisiologia , Entamebíase/metabolismo , Entamebíase/parasitologia , Proteínas de Ligação ao Ferro/metabolismo , Trofozoítos/fisiologia , Animais , Bactérias/metabolismo , Bactérias/patogenicidade , Entamoeba histolytica/citologia , Entamoeba histolytica/patogenicidade , Entamebíase/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Ferro/metabolismo , Microscopia Confocal , Trofozoítos/citologia , VirulênciaRESUMO
The pattern of protein from membrane and crude homogenate of Entamoeba histolytica strain 462 axenically cultivated (462ac) and submitted to hamster liver passage (462hp) was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrains 462ac and 462hp were compared by zymodeme analysis, erythrophagocytosis, cytopathic effect upon mammalian cells and the capability to induce abscess in hamster liver. The results showed no differences for erythrophagocytosis, cytopathic effect or zymodene for substrains 462ac and 462hp. A type II pathogenic zymodene was observed. Substrain 462ac did not induce liver abscess, but 462hp induced abscesses in 70% of the inoculated animals. The pattern of proteins from plasma membrane and crude homogenate were different. One protein of approximately 45kDa and another of 23 kDa showed at no detectable levels in the membrane of 462ac. A third component of approximately 90 kDa showed more intensively expressed in the 462ac.