RESUMO
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
Giardia lamblia is one of the most common protozoan infectious agents in the world and is responsible for diarrheal disease and chronic postinfectious illness. During the host-parasite interaction, proteases are important molecules related to virulence, invasion, and colonization, not only for Giardia but also for other parasites. We aimed to characterize the cysteine protease activity detected in trophozoite lysates. This proteolytic activity showed the ability to cleave NH-terminal sequences with either a recognition sequence for a viral protease or a recognition sequence for thrombin. This cleavage activity was detected in nonencysting trophozoites and increased with the progression of encystation. This activity was also detected in excretion/secretion products of axenic trophozoites and in trophozoites cocultured with differentiated Caco-2 cells. Based on size exclusion chromatography, we obtained a fraction enriched in low- to medium-molecular-weight proteins that was capable of exerting this cleavage activity and aggregating human platelets. Finally, our results suggest that this proteolytic activity is shared with other protozoan parasites.
Assuntos
Cisteína Proteases/metabolismo , Giardia lamblia/enzimologia , Proteínas de Protozoários/metabolismo , Células CACO-2 , Catepsina B/química , Catepsina B/genética , Catepsina B/metabolismo , Cisteína Proteases/química , Cisteína Proteases/genética , Giardia lamblia/química , Giardia lamblia/genética , Giardíase , Humanos , Proteólise , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Especificidade por Substrato , Trofozoítos/química , Trofozoítos/enzimologia , Trofozoítos/genéticaRESUMO
Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.
Assuntos
Giardia lamblia/química , Vacinas contra Influenza/imunologia , Proteínas de Membrana/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas de Protozoários/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Adjuvantes Imunológicos , Administração Oral , Animais , Apresentação de Antígeno/efeitos dos fármacos , Bioengenharia/métodos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Neuraminidase/genética , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Estabilidade Proteica , Proteínas de Protozoários/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Trofozoítos/química , Vacinação , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
Acanthamoeba castellanii is a free-living organism widely distributed in the environment that may cause disease. This protozoan exists in two forms, an infective trophozoite and a dormant cyst. The trophozoites are able to cause keratitis and granulomatous amoebic encephalitis in humans. Keratitis is an acute, sight threatening infection of cornea with potential to cause permanent blindness without prompt treatment. However, the lack of suspicion and the low awareness about these amoebae besides of the absence of commercially available immunodiagnostic tests may delay an accurate diagnosis. The identification of proteins with potential for use in immunodiagnosis may improve the parasite detection more quickly and specifically. The amoeba adhesion to the host cell is the primary step for infection but there is no full understanding of A. castellanii proteins relevant for host invasion or infection. In this study, an assessment of soluble and surface-enriched protein fractions expressed by A. castellanii trophozoites, based on complementary LC-MS/MS approach using peptides from SDS-PAGE excised bands, was performed. Our proteomic analysis allowed identification of a total of 503 proteins, of which 308 proteins were exclusively identified in the soluble fraction, 119 in surface-enriched fraction and 76 in both. In silico analysis of functional classification revealed several proteins involved in many biological mechanisms in A. castellanii, including pathogen survival and infection of mammalian hosts. The analysis of predicted antigenic peptides allowed the identification of proteins with potential for immunodiagnostic assays.
Assuntos
Acanthamoeba castellanii/química , Proteínas de Membrana/análise , Proteoma/análise , Proteínas de Protozoários/análise , Trofozoítos/química , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Proteômica , Espectrometria de Massas em TandemRESUMO
Giardia intestinalis is a protozoan parasite that colonizes the upper part of the small intestine of its mammalian hosts. The trophozoite, which is the replicative stage, has a complex cytoskeleton that allows it to move and adhere to intestinal cells. It has been proposed that protein phosphatase 2A (PP2A) participates in the regulation of changes to the parasite cytoskeleton during its life cycle. However, how PP2A is involved in this regulation remains unclear since its substrates and regulators have not been characterized. In this work, we report the bioinformatic and experimental analysis of two potential regulatory Bâ³ subunits of PP2A in Giardia, both of which are calcium-binding proteins. In this work, in silico and experimental evidence of the binding of both proteins to calcium is presented; the proteins are shown to interact with the catalytic PP2A subunit in the trophozoite stage, and they exhibit different subcellular localization patterns. Because PP2A is a heterotrimer, homology analysis of the different subunits of PP2A indicates that fewer holoenzyme combinations can be formed in this parasite than in other organisms. Our results suggest that the localization of PP2A may be associated with calcium-dependent signaling through its Bâ³ type regulatory subunits.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Giardia lamblia/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trofozoítos/enzimologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Domínio Catalítico , Giardia lamblia/enzimologia , Giardia lamblia/genética , Proteína Fosfatase 2/genética , Subunidades Proteicas , Proteólise , Proteínas de Protozoários/genética , Trofozoítos/química , Trofozoítos/genética , Trofozoítos/metabolismoRESUMO
BACKGROUND: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B). RESULTS: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites. CONCLUSIONS: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.
Assuntos
Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Giardia lamblia/genética , Giardíase/parasitologia , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Membrana Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/imunologia , Feminino , Giardia lamblia/classificação , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Alinhamento de Sequência , Trofozoítos/química , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismoRESUMO
Trichomonas vaginalis is a parasite that resides in the human urogenital tract and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. Nucleoside triphosphate diphosphohydrolase (NTPDase), which hydrolyzes extracellular di- and triphosphate nucleotides, and ecto-5'-nucleotidase, which hydrolyzes AMP, have been characterized in T. vaginalis. The aim of this study was to characterize the adenosine deaminase (ADA) activity in intact trophozoites of T. vaginalis. A strong inhibition in adenosine deamination was observed in the presence of calcium and magnesium, which was prevented by EDTA. The apparent K(M) value for adenosine was 1.13 ± 0.07mM. The calculated V(max) was 2.61 ± 0.054 nmol NH(3) min(-1) mg(-1) protein. Adenosine deamination was inhibited in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine. Semi-quantitative reverse transcriptase-PCR experiments were performed and both ADA-related genes ada(125) and ada(231) mRNA were expressed, although ada(231) in higher quantity when compared with the ada(125) : α-tubulin ratio. Furthermore, a phylogenetic analysis showed that the T. vaginalis sequences formed a clade with Entamoeba histolytica and Dictyostelium discoideum sequences, and it strongly suggests homologous functions in the T. vaginalis genome. The presence of ADA activity in T. vaginalis may be important to modulate the adenosine/inosine levels during infection and, consequently, to maintain the anti-inflammatory properties through different nucleoside-signalling mechanisms.
Assuntos
Adenosina Desaminase/química , Adenosina Desaminase/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/enzimologia , Trofozoítos/enzimologia , Adenosina Desaminase/genética , Feminino , Humanos , Cinética , Proteínas de Protozoários/genética , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/química , Trichomonas vaginalis/genética , Trichomonas vaginalis/crescimento & desenvolvimento , Trofozoítos/química , Trofozoítos/crescimento & desenvolvimentoRESUMO
Entamoeba histolytica trophozoite cytokinesis is dependent upon cytoskeletal elements such as filamentous actin and myosin. Here we present confocal and transmission electron microscopy studies of this process. A sequence in the formation of the contractile ring was shown with rhodamine-phalloidine staining. Ultrastructural analysis revealed the presence of fibrilar aggregates in the cytoplasm of dividing trophozoites. Among them two filaments of different diameter were identified. These aggregates presented repeating assemblies of thin and thick filaments that in cross section revealed a muscle-like appearance. Our results suggest that these aggregates constitute the contractile ring responsible for the separation of daughter cells.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/análise , Entamoeba histolytica/ultraestrutura , Miosinas/análise , Citoesqueleto de Actina/química , Animais , Citocinese , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Entamoeba histolytica/química , Entamoeba histolytica/citologia , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Trofozoítos/química , Trofozoítos/citologia , Trofozoítos/ultraestruturaRESUMO
Free-living ameba Naegleria fowleri produces an acute and fatal infectious disease called primary amebic meningoencephalitis (PAM), whose pathophysiological mechanism is largely unknown. The aim of this study was to investigate the role of nitric oxide (NO) in PAM. Although NO has a cytotoxic effect on various parasites, it is produced by others as part of the pathology, as is the case with Entamoeba histolytica. To test for the production of NO, we analyzed whether antibodies against mammalian NO synthase isoforms (neuronal, inducible, and endothelial) presented immunoreactivity to N. fowleri proteins. We found that the trophozoites produced NO in vitro. The Western blot results, which showed N. fowleri trophozoites, contained proteins that share epitopes with the three described mammalian NOS, but have relative molecular weights different than those described in the literature, suggesting that N. fowleri may contain undescribed NOS isoforms. Moreover, we found that trophozoites reacted to the NOS2 antibody, in amebic cultures as well as in the mouse brain infected with N. fowleri, suggesting that nitric oxide may participate in the pathogenesis of PAM. Further research aimed at determining whether N. fowleri contains active novel NOS isoforms could lead to the design of new therapies against this parasite.