RESUMO
PURPOSE: Fascioliasis is caused by Fasciola hepatica of almost worldwide distribution and F. gigantica in wide regions of Asia and Africa. Their adult stage develops in the biliary canals and gallbladder. Infection follows an initial, 3-4 month long invasive, migratory or acute phase, and a several year-long biliary, chronic or obstructive phase. METHODS: The unexpected finding of a fasciolid inside the gallbladder during a cholecystectomy for obstructive lithiasis suspicion in a patient is reported from an area of Iran where human infection had been never reported before and studies on fascioliasis in livestock are absent. RESULTS: The fluke obtained was phenotypically classified as F. hepatica by morphometry and genotypically as F. gigantica by mtDNA cox1 fragment sequencing, although with F. hepatica scattered mutations in species-differing nucleotide positions. The clinical, radiological, and biological signs observed at the acute and chronic phases often lead to some misdiagnosis. Serological methods may be useful in cases of negative coprology. Diagnostic techniques with insufficient resolution leading to unnecessary invasive interventions are analyzed. The way to avoid unnecessary surgery is described, including analyses to be made, diagnostic tools to be used, and aspects to be considered. CONCLUSION: Reaching a correct diagnosis in the confusing presentations avoids procedure delays and unnecessary surgery. A correct drug treatment may be sufficient. Except in extreme pathological presentations, lesions decrease in number and size and finally disappear or calcify after a successful treatment. Finally, the need to increase awareness of physicians about fascioliasis is highlighted, mainly in non-human endemic areas.
Assuntos
Fasciola hepatica , Fasciola , Fasciolíase , Animais , Adulto , Humanos , Fasciolíase/diagnóstico , Fasciolíase/epidemiologia , Fasciola/genética , Fasciola hepatica/genética , Ásia , ColecistectomiaRESUMO
Liver flukes, Fasciola spp., are veterinary and medically important parasites infecting numerous species of economically important animals in addition to humans on a global scale. The components of transforming growth factor beta (TGF-ß) signalling are widely distributed throughout the animal kingdom and are considerably conserved. Through shared common signal transduction mechanisms, crosstalk of TGF-ß signalling between a host and the parasite during infection is possible. Herein, we have identified and undertaken the molecular characterisation of a putative TGF-ß homologue from the tropical liver fluke F. gigantica (FgTLM). A FgTLM cDNA was 3557 bp in length, it encoded for 620 amino acid polypeptide which consisted of 494 amino acids of prodomain and 126 amino acids comprising the mature protein. FgTLM displayed characteristic structures of mammalian TGF-ß ligands that were unique to the inhibin-ß chain, monomer of activin. A phylogenetic analysis revealed the high degree of conservation with TGF-ß molecules from trematode species. Interestingly, the sequence of amino acid in the active domain of FgTLM was completely identical to FhTLM from F. hepatica. FgTLM expressed throughout the lifecycle of F. gigantica but was highly expressed in developmental active stages. The dynamics of expression of FgTLM during the developmental stages of F. gigantica was comparable to the pattern of TGF-ß expression in F. hepatica. Our findings demonstrated that FgTLM exhibits a high level of similarity to FhTLM in the context of both amino acid sequence and the life stage expression patterns. These similarities underline the possibility that the FgTLM molecule might have the same properties and functions as FhTLM in biological processes of the immature parasites and host immune evasion. Consequently, the specific biological functions of FgTLM on either parasite or relevant hosts need to be defined experimentally.
Assuntos
Fasciola hepatica , Fasciola , Fasciolíase , Animais , Humanos , Fasciola/genética , Fasciola hepatica/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Filogenia , Fasciolíase/parasitologia , Mamíferos , Aminoácidos/genética , Aminoácidos/metabolismoRESUMO
BACKGROUND: The liver fluke Fasciola gigantica secretes excretory-secretory proteins during infection to mediate its interaction with the host. In this study, we investigated the immunomodulatory effects of a recombinant tegumental calcium-binding EF-hand protein 4 of F. gigantica (rFg-CaBP4) on goat monocytes. METHODS: The rFg-CaBP4 protein was induced and purified by affinity chromatography. The immunogenic reaction of rFg-CaBP4 against specific antibodies was detected through western blot analysis. The binding of rFg-CaBP4 on surface of goat monocytes was visualized by immunofluorescence assay. The localization of CaBP4 within adult fluke structure was detected by immunohistochemical analysis. The cytokine transcription levels in response to rFg-CaBP4 were examined using ABI 7500 real-time PCR system. The expression of the major histocompatibility complex (MHC) class-II molecule (MHC-II) in response to rFg-CaBP4 protein was analyzed using Flow cytometry. RESULTS: The isopropyl-ß-D-thiogalactopyranoside-induced rFg-CaBP4 protein reacted with rat sera containing anti-rFg-CaBP4 polyclonal antibodies in a western blot analysis. The adhesion of rFg-CaBP4 to monocytes was visualized by immunofluorescence and laser scanning confocal microscopy. Immunohistochemical analysis localized native CaBP4 to the oral sucker, pharynx, genital pore, acetabulum and tegument of adult F. gigantica. Co-incubation of rFg-CaBP4 with concanavalin A-stimulated monocytes increased the transcription levels of interleukin (IL)-2, IL-4, interferon gamma and transforming growth factor-ß. However, a reduction in the expression of IL-10 and no change in the expression of tumor necrosis factor-α were detected. Additionally, rFg-CaBP4-treated monocytes exhibited a marked increase in the expression of the major histocompatibility complex (MHC) class-II molecule (MHC-II) and a decrease in MHC-I expression, in a dose-dependent manner. CONCLUSIONS: These findings provide additional evidence that calcium-binding EF-hand proteins play roles in host-parasite interaction. Further characterization of the immunomodulatory role of rFg-CaBP4 should expand our understanding of the strategies used by F. gigantica to evade the host immune responses.
Assuntos
Proteínas de Ligação ao Cálcio/imunologia , Fasciola/química , Fasciola/imunologia , Imunomodulação , Monócitos/imunologia , Animais , Proteínas de Ligação ao Cálcio/farmacologia , Citocinas/genética , Citocinas/imunologia , Fasciola/genética , Fasciolíase/parasitologia , Cabras/imunologia , Monócitos/efeitos dos fármacos , Proteínas Recombinantes/farmacologiaRESUMO
The excretory-secretory products released by the liver fluke Fasciola gigantica (FgESPs) play important roles in regulating the host immune response during the infection. Identification of hepatic miRNAs altered by FgESPs may improve our understanding of the pathogenesis of F. gigantica infection. In this study, we investigated the alterations in the hepatic microRNAs (miRNAs) in mice treated with FgESPs using high-throughput small RNA (sRNA) sequencing and bioinformatics analysis. The expression of seven miRNAs was confirmed by quantitative stem-loop reverse transcription quantitative PCR (qRT-PCR). A total of 1,313 miRNAs were identified in the liver of mice, and the differentially expressed (DE) miRNAs varied across the time lapsed post exposure to FgESPs. We identified 67, 154 and 53 dysregulated miRNAs at 1, 4 and 12 weeks post-exposure, respectively. 5 miRNAs (miR-126a-3p, miR-150-5p, miR-155-5p, miR-181a-5p and miR-362-3p) were commonly dysregulated at the three time points. We also found that most of the DE miRNAs were induced by FgESPs in the mouse liver after 4 weeks of exposure. These were subjected to Gene Ontology (GO) enrichment analysis, which showed that the predicted targets of the hepatic DE miRNAs of mice 4 weeks of FgESPs injection were enriched in GO terms, including cell membrane, ion binding, cellular communication, organelle and DNA damage. KEGG analysis indicated that the predicted targets of the most downregulated miRNAs were involved in 15 neural activity-related pathways, 6 digestion-related pathways, 20 immune response-related pathways and 17 cancer-related pathways. These data provide new insights into how FgESPs can dysregulate hepatic miRNAs, which play important roles in modulating several aspects of F. gigantica pathogenesis.
Assuntos
Biologia Computacional , Fasciola/genética , Fasciolíase/parasitologia , MicroRNAs/genética , Animais , Regulação para Baixo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Imunidade , Fígado/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Glutathione S-transferases (GSTs) are multifunctional enzymes that play major roles in a wide range of biological processes, including cellular detoxification, biosynthesis, metabolism, and transport. The dynamic structural scaffold and diverse functional roles of GSTs make them important for enzyme engineering and for exploring novel biotechnological applications. The present study reported a significant gain-of-function activity in GST caused by a point mutation at the conserved F136 residue. The fluorescence quenching and kinetic data suggested that both binding affinity and catalytic efficiency of the mutant enzyme to the substrates 1-chloro-2,4-dinitrobenzene (CDNB), as well as the glutathione (GSH), is increased. Molecular docking showed that the mutation improves the binding interactions of the GSH with several binding-site residues. The simulation of molecular dynamics revealed that the mutant enzyme gained increased structural rigidity than the wild-type enzyme. The mutation also altered the residue interaction network (RIN) of the GSH-binding residues. These phenomena suggested that mutations led to conformational alterations and dominant differential motions in the enzyme that lead to increased rigidity and modifications in RIN. Collectively, engineering GST with a single point mutation at conserved F136 can significantly increase its xenobiotic activity by increasing the catalytic efficiency that may be exploited for biotechnological applications.
Assuntos
Glutationa Transferase/genética , Inativação Metabólica/genética , Mutação Puntual/genética , Xenobióticos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Catálise , Fasciola/genética , Glutationa/genética , Cinética , Simulação de Acoplamento Molecular/métodos , Alinhamento de SequênciaRESUMO
AIM: Serodiagnosis of Fasciola gigantica natural infection in buffaloes with recombinant cathepsin L1-D and native cathepsin-L protease antigens. METHODS: The recombinant cat L1-D antigen was expressed in prokaryotic expression system and native cathepsin-L proteases were purified by alcoholic fractionation from adult F. gigantica flukes. Buffaloes (n = 325) were screened for anti-Fasciola antibodies with the above antigens in immunoglobulin-G-enzyme linked immunosorbent assay (IgG-ELISA). RESULTS: The recombinant cat L1-D antigen showed positive reactivity with 101/122 necropsy positive animals but 21/122 necropsy confirmed positive animals were negative in this ELISA (sensitivity 82.8%). However, 30/203 (14.8%) necropsy negative animals for Fasciola were seropositive with specificity of 85.2%. With native cat-L protease, 104/122 necropsy confirmed positive animals were ELISA positive but 18/122 necropsy positive animals were seronegative, thereby depicting the sensitivity of 85.2%. But ELISA with this antigen showed 27/203 (13.3%) necropsy negative animals as positive (specificity 86.7%). CONCLUSIONS: Comparative evaluation of both the antigens showed that they are suitable for serodiagnosis of F. gigantica infection in buffalo herds.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Búfalos/parasitologia , Catepsinas/imunologia , Cisteína Endopeptidases/imunologia , Fasciola/imunologia , Fasciolíase/veterinária , Proteínas de Helminto/imunologia , Animais , Western Blotting/veterinária , Catepsinas/genética , Catepsinas/metabolismo , Bovinos , Clonagem Molecular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática/veterinária , Fasciola/genética , Fasciola/isolamento & purificação , Fasciolíase/diagnóstico , Fasciolíase/parasitologia , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Fígado/parasitologia , Reação em Cadeia da Polimerase/veterinária , RNA de Helmintos/genética , RNA de Helmintos/isolamento & purificação , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Sensibilidade e EspecificidadeRESUMO
An inverse correlation between helminth infection and the autoimmune disease appears to be contributed by the anti-inflammatory factors produced by these organisms. Suppressing osteoclast function without affecting the systemic immunological response is an emerging therapeutic strategy for rheumatoid arthritis (RA). We observed that a synthetic peptide corresponding to 34 amino acids of C-terminal sequence of Fasciola helminth defense molecule-1 (C-FhHDM-1) inhibited RANKL-induced osteoclast formation and lysosomal acidification with an attendant upregulation of sequestome1/p62, a negative regulator of NF-κB expression. C-FhHDM-1 also suppressed RANKL production from osteoblasts. Macrophages are the major inflammatory cells in the joints of RA and C-FhHDM-1 suppressed ICAM-1 (an inflammatory surrogate) expression in these cells. In a murine model of collagen II-induced arthritis (CIA), C-FhHDM-1 improved clinical score, protected against cartilage destruction, and maintained bone mass and bone architecture of joints compared with the CIA group. C-FhHDM-1 suppressed the CIA-induced expression of TNF, IL-17, and IFN-γ in joints but not their serum levels. The peptide also had no effect on the CIA-induced suppression of T regulatory response. We conclude that C-FhHDM-1 has a joint-specific protective effect in experimental arthritis without mitigating systemic inflammation, and thus could become an adjuvant anti-arthritis therapy to prevent RA-induced osteopenia.
Assuntos
Artrite Experimental/metabolismo , Osteoclastos/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Diferenciação Celular , Fasciola/genética , Fasciolíase/imunologia , Proteínas de Helminto/genética , Imunidade , Molécula 1 de Adesão Intercelular/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Peptídeos/química , Ligante RANK/metabolismoRESUMO
Liver flukes of animals are parasitic flatworms of major socioeconomic importance in many countries. Particularly, Fasciola gigantica is a leading cause of production losses to the livestock (mainly sheep and cattle) and meat industries due to clinical disease, reduced weight gain and milk production, and deaths. Immune responses induced by helminth have been extensively studied, but there is limited information on this aspect by F. gigantica, especially on macrophages induced with this parasite. Studies have shown that host immune responses induced by parasitic infection is greatly correlated with the macrophage polarization axis. In the present study, we used the murine model of F. gigantica to explore the interaction of host and F. gigantica. We found F. gigantica NEJs promoted pathology and fibrosis of mice liver, and the enlargement of mice spleen. We also showed that macrophages were recruited to mice peritoneal cavity at 5 days post infection. By evaluating the expression of genetic markers of M2 macrophages such as Arg-1, Ym1 and RELMÉ, and genetic marker of M1 macrophages iNOS, we showed that M2 macrophages were induced by F. gigantica. M2 macrophages are central to the immune response during helminth infection, and our findings in this study provided insight into the immune interaction between F. gigantica and host.
Assuntos
Fasciola hepatica/fisiologia , Fasciola/fisiologia , Fasciolíase/parasitologia , Cirrose Hepática/parasitologia , Macrófagos/parasitologia , Animais , Fasciola/genética , Fasciola/crescimento & desenvolvimento , Fasciola hepatica/crescimento & desenvolvimento , Fasciolíase/imunologia , Fasciolíase/patologia , Feminino , Humanos , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Macrófagos/imunologia , Masculino , Camundongos , FenótipoRESUMO
Fasciolosis caused by trematode parasites of the genus Fasciola is a global disease of livestock, particularly cattle, sheep, water buffalo and goats. It is also a major human zoonosis with reports suggesting that 2.4-17 million people are infected worldwide, and 91.1 million people currently living at risk of infection. A unique feature of these worms is their reliance on a family of developmentally-regulated papain-like cysteine peptidases, termed cathepsins. These proteolytic enzymes play central roles in virulence, infection, tissue migration and modulation of host innate and adaptive immune responses. The availability of a Fasciola hepatica genome, and the exploitation of transcriptomic and proteomic technologies to probe parasite growth and development, has enlightened our understanding of the cathepsin-like cysteine peptidases. Here, we clarify the structure of the cathepsin-like cysteine peptidase families and, in this context, review the phylogenetics, structure, biochemistry and function of these enzymes in the host-parasite relationship.
Assuntos
Fasciola/enzimologia , Interações Hospedeiro-Parasita/fisiologia , Peptídeo Hidrolases/metabolismo , Animais , Fasciola/genética , Genoma Helmíntico/genética , Humanos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genéticaRESUMO
Peptide vaccines constitute an interesting alternative to classical vaccines due to the possibility of selecting specific epitopes, easy of production and safety. However, an inadequate design may render these peptides poorly immunogenic or lead to undesirable outcomes (e.g., formation of B neoepitopes). As an approach to vaccine development, we evaluated the antibody response to chimeras composed of two or three known B epitopes from Trichinella and Fasciola, and several linkers (GSGSG, GPGPG and KK) in species as different as mice, sheep and turbot. All these species could mount an effective immune response to the short chimeric peptides. Nevertheless, this response depended on several factors including a favorable orientation of B-cell epitopes, adequateness of linkers and/or probability of formation of T neoepitopes. We also observed that, at least in mice, the inclusion of a decoy epitope may have favorable consequences on the antibody response to other epitopes in the chimera.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Formação de Anticorpos , Antígenos de Helmintos/imunologia , Epitopos de Linfócito B/imunologia , Fasciola/imunologia , Proteínas de Helminto/imunologia , Peptídeos/imunologia , Trichinella/imunologia , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Epitopos de Linfócito B/genética , Fasciola/genética , Feminino , Linguados , Proteínas de Helminto/genética , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/farmacologia , Ovinos , Especificidade da Espécie , Trichinella/genéticaRESUMO
Fasciola gigantica is regarded as the major liver fluke causing fasciolosis in livestock in tropical countries. Despite the significant economic and public health impacts of F. gigantica there are few studies on the pathogenesis of this parasite and our understanding is further limited by the lack of genome and transcriptome information. In this study, de novo Illumina RNA sequencing (RNA-seq) was performed to obtain a comprehensive transcriptome profile of the juvenile (42days post infection) and adult stages of F. gigantica. A total of 49,720 unigenes were produced from juvenile and adult stages of F. gigantica, with an average length of 1286 nucleotides (nt) and N50 of 2076nt. A total of 27,862 (56.03%) unigenes were annotated by BLAST similarity searches against the NCBI non-redundant protein database. Because F. gigantica needs to feed and/or digest host tissues, some proteases (including cysteine proteases and aspartic proteases), which play a role in the degradation of host tissues (protein), have been paid more attention in the present study. A total of 6511 distinct genes were found differentially expressed between juveniles and adults, of which 3993 genes were up-regulated and 2518 genes were down-regulated in adults versus juveniles, respectively. Moreover, stage-specific differentially expressed genes were identified in juvenile (17,009) and adult (6517) F. gigantica. The significantly divergent pathways of differentially expressed genes included cAMP signaling pathway (226; 4.12%), proteoglycans in cancer (256; 4.67%) and focal adhesion (199; 3.63%). The transcription pattern also revealed two egg-laying-associated pathways: cGMP-PKG signaling pathway and TGF-ß signaling pathway. This study provides the first comparative transcriptomic data concerning juvenile and adult stages of F. gigantica that will be of great value for future research efforts into understanding parasite pathogenesis and developing vaccines against this important parasite.
Assuntos
Fasciola/genética , Fasciolíase/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos , Proteínas de Helminto/genética , Redes e Vias Metabólicas/genética , Transcriptoma , Animais , Ácido Aspártico Proteases/classificação , Ácido Aspártico Proteases/genética , Búfalos , Cisteína Proteases/classificação , Cisteína Proteases/genética , Bases de Dados Genéticas , Fasciola/isolamento & purificação , Fasciola/metabolismo , Fasciolíase/parasitologia , Perfilação da Expressão Gênica , Ontologia Genética , Proteínas de Helminto/classificação , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Transdução de SinaisRESUMO
Glutaredoxins (Grxs) are small thiol-dependent proteins and key elements of redox signaling as they regulate the redox state of important cellular proteins. In the present study, the complete sequence of a glutaredoxin protein, obtained from the liver fluke Fasciola gigantica, was PCR-amplified and cloned. The 690-bp open reading frame (ORF) encodes a 230-amino acid protein with two conserved domains (FgGrxD1 and FgGrxD2) and has similarities with two monothiol Grxs of Saccharomyces cerevisiae, i.e., ScGrx3 and ScGrx4. The full-length FgGrx along with its two constituent domains were overexpressed in Escherichia coli as hexahistidyl-tagged proteins. The affinity chromatography resulted in almost pure and soluble proteins. The full-length FgGrx and the FgGrxD2 showed reddish-brown color, indicating the presence of bound iron in the second domain. In the insulin based reduction assay, both FgGrx and FgGrxD2 containing the active site motif CGFS exhibited a weak reducing activity, whereas FgGrxD1 was inactive. Additionally, FgGrx did not show any GSH-disulfide transhydrogenase activity when 2-hydroxyethyl disulfide (HED) or de-hydroascorbate (DHA) were taken as substrates. These results indicated the probable role of FgGrx in cellular iron-sulfur homeostasis. FgGrx was found to be reversibly S-glutathionylated, suggesting a potential redox regulation that is likely to take place at the active site Cys158. Since there is only one Cys in FgGrxD2, the Cys158 might be involved in FeS binding. This study is the first report on the presence of Grx in platyhelminthic parasites and provides a starting point for further characterization of the redox network in liver flukes.
Assuntos
Fasciola/fisiologia , Glutarredoxinas/genética , Proteínas de Helminto/genética , Sequência de Aminoácidos , Animais , Escherichia coli/genética , Fasciola/genética , Expressão Gênica , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Organismos Geneticamente Modificados , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Cystatins are functional as intra- and extracellular inhibitors of cysteine proteases and are expressed as single or multi-domain proteins. We have previously described two single domain type 1 cystatins in the trematode Fasciola gigantica that are released into the parasite's intestinal tract and exhibit inhibitory activity against endogenous and host cathepsin L and B proteases. In contrast, the here presented 170kDa multi-domain cystatin (FgMDC) comprises signal peptide and 12 tandem repeated cystatin-like domains with similarity to type 2 single domain cystatins. The domains show high sequence divergence with identity values often <20% and at only 26.8% between the highest matching domains 6 and 10. Several domains contain degenerated QVVAG core motifs and/or lack other important residues of active type 2 cystatins. Domain-specific antisera detected multiple forms of FgMDC ranging from <10 to >120kDa molecular mass in immunoblots of parasite crude extracts and ES product with different banding patterns for each antiserum demonstrating complex processing of the proprotein. The four domains with the highest conserved QVVAG motifs were expressed in Escherichia coli and the refolded recombinant proteins blocked cysteine protease activity in the parasite's ES product. Strikingly, immunohistochemical analysis using seven domain-specific antisera localized FgMDC in testis lobes and sperm. It is speculated that the processed cystatin-like domains have function analogous to the mammalian group of male reproductive tissue-specific type 2 cystatins and are functional in spermiogenesis and fertilization.
Assuntos
Cistatinas/química , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Fasciola/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cistatinas/genética , Cisteína Proteases , Fasciola/química , Fasciola/genética , Feminino , Proteínas de Helminto/genética , Masculino , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Espermatozoides/metabolismo , Testículo/metabolismoRESUMO
In the present study, a cDNA encoding Trx from F. gigantica (FgTrx) was cloned by polymerase chain reaction (PCR). The sequence of FgTrx, analyzed by BLAST, SignalP, and ClustralW programs, showed 315 bp of an open reading frame (ORF), 12 bp 5'UTR, 78 bp 3'UTR, and the putative FgTrx peptide comprising of 104 amino acids, with a molecular weight of 11.68 kDa, with the active site containing five amino acids (tryptophan, cysteine, glycine, proline, cysteine) with a conserved dithiol motif from the two cysteines, and pI 5.86. The peptide had no signal sequence; hence, it was not a secreted protein. The recombinant FgTrx was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTrx). The FgTrx protein expression, estimated by indirect ELISA using the rabbit anti-rFgTrx as probe, showed high levels in eggs, 2- and 4-week-old juveniles, and adult parasite. In a functional test, the rFgTrx exhibited specific activity that could be suppressed by an inhibitor (PX12). When tested by immunoblotting and immunohistochemistry, rabbit anti-rFgTrx reacted with natural FgTrx at a molecular weight of 11.68 kDa from eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica. The FgTrx protein was distributed at high levels in the tegument of 2- and 4-week-old juveniles, and the tegument, parenchyma, eggs, and reproductive organs of adult parasites. FgTrx may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or drug target.
Assuntos
Fasciola/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Helminto/metabolismo , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Fasciola/genética , Proteínas de Helminto/genética , Camundongos , Dados de Sequência Molecular , Coelhos , Tiorredoxinas/genéticaRESUMO
2-Cys peroxiredoxin (Prx) is the main antioxidant enzyme in Fasciola species for detoxifying hydrogen peroxide which is generated from the hosts' immune effector cells and the parasites' own metabolism. In this study, the recombinant Prx protein from Fasciola gigantica (rFgPrx-2) was expressed and purified in a prokaryotic expression system. This recombinant protein with molecular weight of 26 kDa was enzymatically active in reduction of hydrogen peroxide both in presence of thioredoxin and glutathione systems, and also protected the supercoiled plasmid DNA from oxidative damage in metal-catalyzed oxidation (MCO) system in a concentration-dependent manner. By immunoblotting, using antibody against rFgPrx-2 as probe, a native FgPrxs, whose MW at 25 kDa, was detected in all developmental stages of the parasite. Concentrations of native FgPrxs were increasing in all stages reaching highest level in adult stage. The antibody also showed cross reactivities with corresponding proteins in some cattle helminthes. Natural antibody to FgPrxs could be detected in the sera of mice at 3 and 4 weeks after infection with F. gigantica metacercariae. By immunofluorescence, FgPrxs was highly expressed in tegument and tegumental cells, parenchyma, moderately expressed in cecal epithelial cells in early, juvenile and adult worms. Furthermore, FgPrxs was also detected in the female reproductive organs, including eggs, ovary, vitelline cells, and testis, suggesting that FgPrxs might play an essential role in protecting parasite's tissues from free radical attack during their life cycle. Thus, FgPrxs is one potential candidate for drug therapy and vaccine development.
Assuntos
Antioxidantes/metabolismo , Fasciola/metabolismo , Proteínas de Helminto/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxirredoxinas/metabolismo , Animais , Antioxidantes/química , Bovinos , Dano ao DNA , DNA Super-Helicoidal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fasciola/genética , Fasciola/imunologia , Feminino , Regulação da Expressão Gênica , Glutationa/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Camundongos , Camundongos Endogâmicos ICR , Peso Molecular , Oxirredução , Peroxirredoxinas/química , Peroxirredoxinas/genética , Plasmídeos , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/metabolismoRESUMO
Cathepsin L proteases are a major class of endopeptidases expressed at a high level in Fasciola parasites. Several isoforms of cathepsin L were detected and they may perform different functions during the parasite development. In this study, a complete cDNA encoding a cathepsin L protease was cloned from a newly excysted juvenile (NEJ) cDNA library of Fasciola gigantica and named FgCatL1H. It encoded a 326 amino acid preproenzyme which shared 62.8-83.1% and 39.5-42.9% identity to Fasciola spp. and mammalian cathepsins L, respectively. All functionally important residues previously described for cathepsin L were conserved in FgCatL1H. Phylogenetic analysis demonstrated that FgCatL1H belonged to a distinct group, clade 4, with respect to adult and other juvenile Fasciola cathepsin L genes. FgCatL1H expression was detected by RT-PCR, using gene specific primers, in metacercariae and NEJ, and the expression gradually decreased in advanced developmental stages. A recombinant proFgCatL1H (rproFgCatL1H) was expressed in the yeast Pichia pastoris, affinity purified, and found to migrate in SDS-PAGE at approximately 47.6 and 38.3kDa in glycosylated and deglycosylated forms, respectively. The molecular mass of the activated mature rFgCatL1H in glycosylated form was approximately 40.7kDa. Immunoblotting and immunohistochemistry using rabbit antibodies against rproFgCatL1H showed that FgCatL1H was predominantly expressed in epithelial cells of the digestive tract of metacercariae, NEJs and juveniles of F. gigantica. FgCatL1H could cleave the synthetic fluorogenic substrate Z-Phe-Arg-MCA preferentially over Z-Gly-Pro-Arg-MCA at an optimum pH of 6.5. It also showed hydrolytic activity against native substrates, including type I collagen, laminin, and immunoglobulin G (IgG) in vitro, suggesting possible roles in host tissue migration and immune evasion. Therefore, the FgCatL1H is a possible target for vaccine and chemotherapy for controlling F. gigantica infection.
Assuntos
Catepsina L/genética , Catepsina L/metabolismo , Fasciola/enzimologia , Fasciola/genética , Animais , Cromatografia de Afinidade , Clonagem Molecular , Análise por Conglomerados , Sistema Digestório/enzimologia , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/enzimologia , Expressão Gênica , Perfilação da Expressão Gênica , Biblioteca Gênica , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Immunoblotting , Dados de Sequência Molecular , Peso Molecular , Filogenia , Pichia/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Cysteine proteases are important antigens in the liver fluke genus Fasciola, essential for infection, protection and nutrition. While their biochemistry, biological roles and application as vaccines have been thoroughly studied there is a lack of data concerning their regulation. In the present study we have continued our investigation of cysteine protease inhibitors in Fasciola gigantica and demonstrate, in comparison with FgStefin-1 and human cystatin C, that a second type 1 cystatin of the parasite, FgStefin-2, has been evolutionary adapted to block cathepsin B. The protein, which unusually for a type 1 cystatin carries a signal peptide, is expressed from the metacercarial to adult stage and located in the epithelial cells of the intestinal tract in all stages and in the prostate gland cells in adults. Both cell types may contribute to the released FgStefin-2 observed in the ES product of the parasite. Distinct isoforms of cathepsin B are essential for host tissue penetration during the early infection process and FgStefin-2 may act as key regulator, required to protect the minute juvenile from autoproteolysis. Expression in the prostate gland in the adult stage suggests an additional regulative role of cysteine protease activity in the reproductive system.
Assuntos
Catepsina B/antagonistas & inibidores , Cistatinas/farmacologia , Inibidores Enzimáticos/farmacologia , Fasciola/metabolismo , Proteínas de Helminto/farmacologia , Sequência de Aminoácidos , Animais , Catepsina B/metabolismo , Bovinos , Cistatina C/metabolismo , Cistatinas/química , Cistatinas/genética , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fasciola/genética , Fasciola/isolamento & purificação , Fasciolíase/parasitologia , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Concentração Inibidora 50 , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Alinhamento de SequênciaRESUMO
Fasciola gigantica fatty acid binding protein (FABP) was evaluated for evoking an immune response in mice, by delivering the gene coding for this protein with mannosylated-polyethylenimine (PEI) to peritoneal cells. Mice were immunized with 50 microg recombinant plasmid DNA (Group I) or DNA-PEI-mannose (a 22 kDa linear cationic polymer with mannose ligand) (Group II) via the intraperitoneal route. Antibody studies showed no significant humoral immune response evoked to this DNA immunization with either PEI-mannose-delivered or naked DNA. However, on protein boosting of these DNA-primed mice there was a significant enhancement of antibody titre. Flow cytometric bead array was used to measure quantities of interleukin (IL)-2, IL-4, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) cytokines. Overexpression of T-helper 1 (Th1) cytokines such as IFN-gamma and TNF, with a lower but significant expression of the T-helper 2 (Th2) cytokine IL-5 was detected. Gene delivery using polyethylenimine-mannose ligand showed significant expression of IFN-gamma and TNF (P < 0.05), but no significant difference in IL-2, IL-4 and IL-5 (P>0.05) cytokine expression was observed between naked-DNA- and mannosylated PEI-DNA-delivered mice. Naked- or PEI-delivered-DNA immunization produced insignificant levels of IL-2 and IL-4 (P>0.05) cytokines in both groups of mice.
Assuntos
Antígenos de Helmintos/imunologia , Portadores de Fármacos/farmacologia , Fasciola/imunologia , Proteínas de Ligação a Ácido Graxo/imunologia , Manose/farmacologia , Polietilenoimina/farmacologia , Vacinas de DNA/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Citocinas/metabolismo , Fasciola/genética , Proteínas de Ligação a Ácido Graxo/genética , Imunização Secundária , Injeções Intraperitoneais , Leucócitos Mononucleares/imunologia , Camundongos , Plasmídeos , Baço/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
To identify antigens that could potentially be developed as vaccines against Fasciola gigantica, somatic antigens were analyzed by immunoprecipitation using pooled sera from rats infected with F. gigantica metacercariae. A prominent antigen of the newly excysted juveniles (NEJ), cathepsin B3 protease (FgCatB3), was identified by N-terminal sequencing and PCR screening of a cDNA library. Recombinant FgCatB3 (rFgCatB3) was expressed in Pichia pastoris, and shown to catalyse the digestion of gelatin, the fluorometric substrate Z-Phe-Arg-AMC and native fibronectin, suggesting that this enzyme may be involved in digesting host connective tissues during the fluke's penetration and migration in the host. Rabbit polyclonal sera against rFgCatB3 was produced and used to determine the distribution of the native cathepsin B3 protease in various developmental stages of F. gigantica. By Western blotting, cathepsin B3 was detected in the whole body (WB) extract of metacercariae and NEJ but not in 4-week-old juveniles or adult parasites which confirmed the stage-specific characteristics of cathepsin B3. Immunolocalization of cathepsin B3 protease in each parasite stage showed that high levels of FgCatB3 were present in the caecal epithelium of the metacercariae and NEJ. The differential distribution of FgCatB3 in the different life cycle stages suggests that this protease is functionally important for the juvenile stage of F. gigantica.
Assuntos
Antígenos de Helmintos/análise , Antígenos de Helmintos/genética , Catepsinas/análise , Catepsinas/genética , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/genética , Fasciola/química , Fasciola/genética , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Catepsina H , Catepsinas/isolamento & purificação , Clonagem Molecular , Cisteína Endopeptidases/isolamento & purificação , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Fibronectinas/metabolismo , Gelatina/metabolismo , Imunoprecipitação , Pichia/genética , Reação em Cadeia da Polimerase , Coelhos , Análise de Sequência de ProteínaRESUMO
Fasciola spp. found in Asian countries are diversified in nature, and they should therefore be characterized by spermatogenesis, ploidy and genetic differentiation as well as morphology. The present study showed that spermic diploid and aspermic triploid forms of Fasciola occurred in Vietnam. The spermic diploid specimens were accurately identified as F. gigantica, while the aspermic triploids could not be identified on the basis of their morphology by the ratio of body length and width and DNA sequences of nuclear ribosomal ITS1 and mitochondrial NDI and COI genes. The molecular data also indicated that Vietnamese aspermic triploids might be hybrids and/or their offspring between Fasciola hepatica and F. gigantica, because they showed the ITS1-Fh/Fg haplotype, which had chimeric sequences of the two species. Furthermore, the aspermic triploids seem to have originated in countries other than Vietnam and to have rapidly spread to that country with infected animals.