RESUMO
Enolase, a multifunctional protein expressed by multiple pathogens activates plasminogen to promote proteolysis on components of the extracellular matrix, an important event in early host-pathogen interactions. A secreted form of enolase that is released upon the interaction of trophozoites with epithelial cells has been detected in the secretome of G. duodenalis. However, the role of enolase in the host-pathogen interactions remains largely unknown. In this work, the effects of G. duodenalis enolase (Gd-eno) on the epithelial cell model (IEC-6) were analyzed. Firstly, the coding sequence of Giardia enolase was cloned and the recombinant protein used to raise antibodies that were then used to define the localization and role of enolase in epithelial cell-trophozoite interactions. Gd-eno was detected in small cytoplasmic vesicles as well as at the surface and is enriched in the region of the ventral disk of Giardia trophozoites. Moreover, the blocking of the soluble monomeric form of the enzyme, which is secreted upon interaction with IEC-6 cells by the anti-rGd-eno antibodies, significantly inhibited trophozoite attachment to intestinal IEC-6 cell monolayers. Further, rGd-eno was able to bind human plasminogen (HsPlg) and enhanced plasmin activity in vitro when the trophozoites were incubated with the intrinsic plasminogen activators of epithelial cells. In IEC-6 cells, rGd-eno treatment induced a profuse cell damage characterized by copious vacuolization, intercellular separation and detachment from the substrate; this effect was inhibited by either anti-Gd-eno Abs or the plasmin inhibitor ϵ- aminocaproic acid. Lastly, we established that in epithelial cells rGd-eno treatment induced a necroptotic-like process mediated by tumor necrosis factor α (TNF-α) and the apoptosis inducing factor (AIF), but independent of caspase-3. All together, these results suggest that Giardia enolase is a secreted moonlighting protein that stimulates a necroptotic-like process in IEC-6 epithelial cells via plasminogen activation along to TNFα and AIF activities and must be considered as a virulence factor.
Assuntos
Giardia lamblia , Giardíase , Animais , Comunicação Celular , Giardia/metabolismo , Giardia lamblia/metabolismo , Humanos , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Trofozoítos/metabolismoRESUMO
Transcription is the first step of gene expression regulation and is a fundamental mechanism for establishing the viability and development of a cell. The TATA box-binding protein (TBP) interaction with a TATA box in a promoter is one of the best studied mechanisms in transcription initiation. TBP is a transcription factor that is highly conserved from archaea to humans and is essential for the transcription initiated by each of the three RNA polymerases. In addition, the discovery of TBP-related factor 1 (TRF1) and other factors related to TBP shed light on the variability among transcription initiation complexes, thus demonstrating that the compositions of these complexes are, in fact, more complicated than originally believed. Despite these facts, the majority of studies on transcription have been performed on animal, plant and fungal cells, which serve as canonical models, and information regarding protist cells is relatively scarce. The aim of this work is to review the diversity of the TBPs that have been documented in protists and describe some of the specific features that differentiate them from their counterparts in higher eukaryotes.
Assuntos
Eucariotos/genética , TATA Box , Proteína de Ligação a TATA-Box , Transcrição Gênica , Eucariotos/metabolismo , Genes de Protozoários , Variação Genética , Giardia/genética , Giardia/metabolismo , Leishmania/genética , Leishmania/metabolismo , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Trypanosoma/genética , Trypanosoma/metabolismoRESUMO
BACKGROUND: Giardia duodenalis is conventionally diagnosed in fecal samples using parasitological methods. However, sensitivity is poor when only a single sample is analyzed, due to intermittent excretion of cysts in feces. Alternatively, the serum antibodies to G. duodenalis can be used for parasite diagnosis and epidemiological studies to determine previous exposure. We compared the rate of G. duodenalis infection between serum anti-Giardia IgG and IgA antibodies and fecal examination in Brazilian children. METHODS: Fecal and serum samples were tested from 287 children at a clinical laboratory and from 187 children at daycare centers. Fecal samples were processed using conventional parasitological methods and coproantigen detection for Giardia diagnosis. Serum samples were tested using an in-house ELISA for detection of anti-Giardia IgG and IgA. RESULTS: G. duodenalis was found in 8.2% (N=39) of the 474 children analyzed. The sensitivity and specificity of ELISA were 80.0% and 90.0% for IgG and 80.0% and 83.3% for IgA, respectively. The total positivity rate of anti-Giardia IgG and IgA in the sera was 13.9% (N=66) and 23.6% (N=112). The agreement between the positivity of specific antibodies and the detection of G. duodenalis in feces was moderate for ELISA-IgG, kappa index (95% CI)=0.543 (0.422-0.664), and mild for ELISA-IgA, kappa index (95% CI)=0.283 (0.162-0.404). Among the children infected with other enteroparasites, 11.6% (N=10) and 24.4% (N=21) showed reactivity to anti-Giardia IgG and to IgA, respectively. This cross-reactivity was more frequent in samples from children infected with Endolimax nana and Entamoeba coli. CONCLUSIONS: The higher frequency of specific antibody reactivity compared with G. duodenalis diagnosis in feces could reflect continuous exposure of children to G. duodenalis infection, resulting in long-lasting immunological memory and/or cross-reactivity with other intestinal amoebas.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Giardia/imunologia , Giardíase/diagnóstico , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Criança , Pré-Escolar , Estudos Transversais , Endolimax/imunologia , Fezes/parasitologia , Feminino , Giardia/isolamento & purificação , Giardia/metabolismo , Giardíase/parasitologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Lactente , Recém-Nascido , Masculino , Sensibilidade e EspecificidadeRESUMO
Lactoferrin (LF) is an 80 KDa iron-binding glycoprotein that plays a significant role in the innate immune system and is considered to be an important microbicide molecule. It has been suggested to be effective in the treatment of giardiasis, an intestinal disease caused by the protozoan parasite G. lamblia. However, the molecular mechanisms by which LF exerts its effect on this parasite are unknown. Most of the microbicidal activity of human or bovine LF (hLF or bLF) has been associated with the N-terminal region of the mature LF - lactoferricin (LFcin). LFcin is produced by pepsin cleavage of the native protein in vitro and likely in vivo. In this work, we analyse the participation of the endocytic machinery of G. lamblia in the internalization of bLF and bLFcin and their effects on cell homeostasis. Our results show that, when bLF or bLFcin are internalized by receptor-mediated endocytosis, cell growth stops, and morphological changes are produced in the trophozoites, which ultimately will produce immature cysts. Our findings contribute to disclose the fine mechanism by which bLF and bLFcin may function as an antigiardial molecule and why they have therapeutic potential to eradicate giardiasis.
Assuntos
Cistos/patologia , Giardia/efeitos dos fármacos , Giardia/metabolismo , Lactoferrina/farmacocinética , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cistos/metabolismo , Cistos/parasitologia , Cistos/prevenção & controle , Relação Dose-Resposta a Droga , Endocitose/fisiologia , Giardia/crescimento & desenvolvimento , Giardíase/parasitologia , Giardíase/patologia , Humanos , Lactoferrina/farmacologia , Ligação Proteica , Receptores de LDL/metabolismoRESUMO
Giardiasis is a worldwide parasitic disease that affects mainly children and immunosuppressed people. Side effects and the emergence of resistance over current used drugs make imperative looking for new antiparasitics through discovering of new biological targets and designing of novel drugs. Recently, it has determined that gastric proton-pump inhibitors (PPI) have anti-giardiasic activity. The glycolytic enzyme, triosephosphate isomerase (GlTIM), is one of its potential targets. Therefore, we employed the scaffold of PPI to design new compounds aimed to increase their antigiardial capacity by inactivating GlTIM. Here we demonstrated that two novel PPI-derivatives (BHO2 and BHO3), have better anti-giardiasic activity than omeprazole in concentrations around 120-130 µM, without cytotoxic effect on mammal cell cultures. The derivatives inactivated GlTIM through the chemical modification of Cys222 promoting local structural changes in the enzyme. Furthermore, derivatives forms adducts linked to Cys residues through a C-S bond. We demonstrated that PPI can be used as scaffolds to design better antiparasitic molecules; we also are proposing a molecular mechanism of reaction for these novel derivatives.
Assuntos
Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Giardia/metabolismo , Inibidores da Bomba de Prótons/química , Triose-Fosfato Isomerase/metabolismo , Antiprotozoários/química , Sítios de Ligação , Giardia/efeitos dos fármacos , Giardíase/tratamento farmacológico , Humanos , Estrutura Molecular , Omeprazol/farmacologia , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/metabolismo , Triose-Fosfato Isomerase/químicaRESUMO
The retromer is a pentameric protein complex that mediates the retrograde transport of acid hydrolase receptors between endosomes and the trans-Golgi network and is conserved across all eukaryotes. Unlike other eukaryotes, the endomembrane system of Giardia trophozoite is simple and is composed only of the endoplasmic reticulum and peripheral vesicles (PVs), which may represent an ancient organellar system converging compartments such as early and late endosomes and lysosomes. Sorting and trafficking of membrane proteins and soluble hydrolases from the endoplasmic reticulum to the PVs have been described as specific and conserved but whether the giardial retromer participates in receptor recycling remains elusive. Homologs of the retromer Vacuolar Protein Sorting (Vps35p, Vps26p, and Vps29p) have been identified in this parasite. Cloning the GlVPS35 subunit and antisera production enabled the localization of this protein in the PVs as well as in the cytosol. Tagged expression of the subunits was used to demonstrate their association with membranes, and immunofluorescence confocal laser scanning revealed high degrees of colabeling between the retromer subunits and also with the endoplasmic reticulum and PV compartment markers. Protein-protein interaction data revealed interaction between the subunits of GlVPS35 and the cytosolic domain of the hydrolase receptor GlVps. Altogether our data provide original information on the molecular interactions that mediate assembly of the cargo-selective retromer subcomplex and its involvement in the recycling of the acid hydrolase receptor in this parasite.
Assuntos
Giardia/metabolismo , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Membrana Celular/metabolismo , Centrifugação , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Protozoários/química , Frações Subcelulares/metabolismoRESUMO
The ubiquity and importance of Giardia and Cryptosporidium as pathogens are reflected in the increasing number of publications concerning these organisms, but they are not the only reason why researchers are increasingly turning their attention to studying Giardia and Cryptosporidium. As new tools and databases become available, it is now possible to investigate fundamental issues related to their biology and relationship with their hosts. In this article, we highlight recent advances in research and outline questions arising that need to be addressed as a way of focusing the attention of the research and health communities and encouraging further dialogue and collaboration.
Assuntos
Cryptosporidium , Giardia , Animais , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Criptosporidiose/fisiopatologia , Cryptosporidium/genética , Cryptosporidium/metabolismo , Cryptosporidium/patogenicidade , Cryptosporidium/fisiologia , Perfilação da Expressão Gênica , Genoma de Protozoário , Giardia/genética , Giardia/metabolismo , Giardia/patogenicidade , Giardia/fisiologia , Giardíase/epidemiologia , Giardíase/parasitologia , Giardíase/fisiopatologia , Interações Hospedeiro-Parasita , Humanos , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.
Assuntos
Endocitose , Eucariotos , Animais , Entamoeba/metabolismo , Entamoeba/fisiologia , Entamoeba/ultraestrutura , Eucariotos/metabolismo , Eucariotos/fisiologia , Eucariotos/ultraestrutura , Giardia/metabolismo , Giardia/fisiologia , Giardia/ultraestrutura , Histocitoquímica , Leishmania/metabolismo , Leishmania/fisiologia , Leishmania/ultraestrutura , Microscopia Eletrônica , Plasmodium/metabolismo , Plasmodium/fisiologia , Plasmodium/ultraestrutura , Trichomonas/metabolismo , Trichomonas/fisiologia , Trichomonas/ultraestrutura , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/fisiologia , Trypanosoma brucei brucei/ultraestrutura , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/ultraestruturaRESUMO
Giardia duodenalis has been described as 'anucleolated'. In this work we analysed the subcellular distribution of several nucleolar markers in Giardia nuclei using silver and immunostaining techniques for electron and confocal laser microscopy as well as expression of epitope-tagged proteins in transgenic trophozoites. We identified anteronuclear fibrogranular structures corresponding to nucleolar organising regions with recruited ribonucleoprotein complexes, rRNA and epitope-tagged fibrillarin and rRNA-pseudouridine synthase (CBF5). Recombinant fibrillarin and CBF5 were targeted to this subcompartment. This study demonstrates the presence of nucleoli in G. duodenalis and provides a model to analyse minimal requirements for nucleolar assembly and maintenance in eukaryotic cells.
Assuntos
Nucléolo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Giardia/ultraestrutura , Região Organizadora do Nucléolo/ultraestrutura , Animais , Nucléolo Celular/metabolismo , Evolução Molecular , Giardia/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica , Região Organizadora do Nucléolo/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
The present investigation was undertaken to identify and characterize trophozoite proteases of five axenic strains of Giardia duodenalis isolated in Brazil and the reference strain Portland 1 isolated in the United States. Trophozoite cell lysates of each strain were analysed for the pattern of proteins and for proteolytic activity. Samples were tested in SDS-polyacrylamide gel electrophoresis for the protein profiles, and the detection of proteases in cell lysates was performed using substrate gel electrophoresis [gelatin, collagen, bovine serum albumin (BSA) and haemoglobin] and azocasein assays. Indeed, synthetic inhibitors were included in the assays to characterize the protease classes. Differences on the hydrolysis patterns of protein substrates were observed in relation to the substrate composition as much as the Giardia trophozoite strain. The substrate-containing gels revealed hydrolysis bands with molecular masses ranging from >97 to 20-15 kDa, and most zones were common to the five strains. However, some pronounced differences could be detected in the BTU-11 pattern. Azocasein was also degraded; however, depending on the lysate assayed, the degree of substrate degradation was variable. It was observed that inhibitory effects are substrate-dependent since the activity was predominantly due to cysteine proteases against gelatin, collagen, BSA and azocasein substrates and due to serine against haemoglobin. The presence of aspartic protease and aminopeptidase activity in the lysates was also indicated.
Assuntos
Giardia/enzimologia , Giardia/crescimento & desenvolvimento , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas/metabolismo , Trofozoítos/enzimologia , Animais , Brasil , Colágeno/metabolismo , Gelatina/metabolismo , Giardia/efeitos dos fármacos , Giardia/metabolismo , Hemoglobinas/metabolismo , Humanos , Peptídeo Hidrolases/efeitos dos fármacos , Soroalbumina Bovina/metabolismo , Trofozoítos/metabolismoRESUMO
BACKGROUND: It is well documented that Giardia duodenalis undergoes surface antigenic variation both in vivo and in vitro. Proteins involved have been characterized and referred to as VSP (variable surface protein). METHODS: Two cloned cDNA inserts of 0.45 and 1.95 kb were obtained from G. duodenalis expression library and sequenced. Comparison sequence analyses were made against Genbank. PCR analysis was performed on G. duodenalis isolates to identify isolates bearing genes encoding such a peptide. Specific antiserum was prepared against 450-bp encoded peptide and tested by Western blot, immunofluorescence, and inhibition of adhesion of G. duodenalis to target cells. RESULTS: We cloned and characterized a G. duodenalis 450-bp DNA fragment; its DNA sequence analysis revealed that this fragment displayed 99% identity with vsp9B10A gene. Predicted amino acid sequence for this fragment also had significant (99%) identity to VSP9B10A. A second 1.95-kb insert, which encompassed the 450-bp cDNA fragment, was also isolated; its DNA and amino acid sequence displayed 99.5% identity with vsp9B10A gene and 99.2% with the corresponding inferred protein, respectively. This inferred protein contained 24 Cys-X-X-Cys motifs and long ORF of 642 aminoacids. PCR analysis showed that DNA sequence encoding a fragment of this gene was present in P1, CIEA:0487:2-C-8 clone and in INP:180800-B2 G. duodenalis human isolates, while it was absent in sheep isolate of G. duodenalis INP:150593-J10. CONCLUSIONS: Immunofluorescence analysis using antibodies raised against the peptide encoded by 450-bp fragment showed that expression of this epitope varies on trophozoite surface of the C-8 Mexican clone and is involved in parasite adhesion to target epithelial cells.
Assuntos
Giardia/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Sequência de Bases , Western Blotting , Adesão Celular , Linhagem Celular , Pré-Escolar , Clonagem Molecular , DNA/química , DNA Complementar/metabolismo , Cães , Epitopos/química , Biblioteca Gênica , Variação Genética , Giardíase/imunologia , Humanos , Cinética , México , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Dados de Sequência Molecular , Peptídeos/química , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas Recombinantes de Fusão/química , Ovinos , Fatores de TempoRESUMO
Analysis of the attachment basis of Giardia lamblia 1/Portland strain trophozoites to confluent MDCK (Madin Darby Canine Kidney) cell monolayers and type I collagen films, demonstrated, by the use of light and electron microscopy, that the ventral disk as well as the ventrolateral border are the structures involved in the substrate adhesion. Furthermore, we noticed that trophozoite attachment is more effective to collagen than to the apical surface of the epithelium. We described, for the first time, a rounded shape structure evaginated from the naked area that seems to be the ventral disk contact site between the trophozoite and the substrate. This structure has also been observed when the trophozoite is attached to coverslip glass or among trophozoites in close contact.
Assuntos
Giardia/patogenicidade , Animais , Adesão Celular , Células Cultivadas , Colágeno/metabolismo , Cães , Células Epiteliais , Epitélio/metabolismo , Giardia/metabolismo , Giardia/ultraestrutura , Vidro , RimRESUMO
El análisis de las bases estructurales de la adhesión de trofozoítos de Giardialamblia cepa 1/Potland a monocapas confluentes de células MDCK (Madin Darby Canine Kidney) y a capas de colágena del tipo I proporciona evidencias a nivel de microcospía fotónica y electrónica de que el disco ventral así como el borde ventrolateral son las estructuras de adhesión al sustrato. Además se observó que la adhesión del trofozoítos es más efectiva sobre la colágena que sobre al superficie apical del epitelio. Se describe por primera vez la presencia de una estructura circular en forma de bolsa evaginada del área desnuda que parece ser el sitio del disco ventral de contacto íntimo del trofozoíto con el sustrato. Dicha estructura ha sido también observada cuando se da la adhesión del trofozoíto a la superficie de vidrio del cubreobjeto o aún cuando se establece el contacto estrecho entre trofozoítos
Assuntos
Animais , Cães , Giardia/patogenicidade , Adesão Celular , Células Cultivadas , Colágeno/metabolismo , Epitélio/citologia , Epitélio/metabolismo , Giardia/metabolismo , Giardia/ultraestrutura , Vidro , RimRESUMO
El análisis de las bases estructurales de la adhesión de trofozoítos de Giardialamblia cepa 1/Potland a monocapas confluentes de células MDCK (Madin Darby Canine Kidney) y a capas de colágena del tipo I proporciona evidencias a nivel de microcospía fotónica y electrónica de que el disco ventral así como el borde ventrolateral son las estructuras de adhesión al sustrato. Además se observó que la adhesión del trofozoítos es más efectiva sobre la colágena que sobre al superficie apical del epitelio. Se describe por primera vez la presencia de una estructura circular en forma de bolsa evaginada del área desnuda que parece ser el sitio del disco ventral de contacto íntimo del trofozoíto con el sustrato. Dicha estructura ha sido también observada cuando se da la adhesión del trofozoíto a la superficie de vidrio del cubreobjeto o aún cuando se establece el contacto estrecho entre trofozoítos (AU)
Assuntos
Estudo Comparativo , Animais , Cães , Giardia/patogenicidade , Giardia/metabolismo , Giardia/ultraestrutura , Epitélio/citologia , Epitélio/metabolismo , Colágeno/metabolismo , Células Cultivadas , Rim , Vidro , Adesão CelularRESUMO
The surface charge of Giardia lamblia trophozoites from axenic cultures of strains recently isolated in Mexico from human cases of symptomatic and asymptomatic giardiasis was studied by means of cellular microelectrophoresis and ultrastructural cytochemistry. It is concluded that ionogenic surface groups confer a negative surface charge on trophozoites of G. lamblia and that no significant differences exist between the surface charge of trophozoites of symptomatic and asymptomatic origin.
Assuntos
Giardia/metabolismo , Giardíase/parasitologia , Animais , Eletroforese , Giardia/ultraestrutura , Histocitoquímica , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Propriedades de SuperfícieRESUMO
Con el propósito de estudiar si la G. lamblia se acompaña en el intestino de una gran población de bacterias productoras de indol, se investigó la concentración de sulfato de indoxil en la orina, antes y después de erradicar este parásito. Los resultados plantean, indirectamente, que la giardiasis se asocia a una proliferación de enterobacterias que sobrepasa en magnitud la que ordinariamente existe en el intestino