RESUMEN

The potentially lethal zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode larval stages of the tapeworm Echinococcus multilocularis. Metacestode growth and proliferation occurs within the inner organs of mammalian hosts, which is associated with complex molecular parasite-host interactions. The host has developed various ways to resist a parasitic infection, and the production of reactive oxygen species (ROS) is one of the most important strategies. Here, we found that scavenging of ROS reduced metacestode larval growth and germinative cell proliferation in in vivo models. Furthermore, using in vitro-cultured metacestode vesicles, we found that increased ROS levels enhanced metacestode growth and germinative cell proliferation, which was achieved by positively activating the ROS-EmERK-EmHIF1α axis. These results indicate that, beside its capacity to damage the parasite, ROS also play critical roles in metacestode growth and germinative cell proliferation. This study suggests that the effects of ROS on parasite may be bidirectional during AE infection, reflecting the parasite's adaptation to the oxidative stress microenvironment.

RESUMEN

The larval stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), one of the most lethal helminthic infections in humans. The tumor-like growth and development of the metacestode larvae within host organs are driven by a population of somatic stem cells, the germinative cells, which represent the only proliferative cells in the parasite. Host-derived factors have been shown to promote germinative cell proliferation. Since cells sense the external signal mainly in G1 phase of the cell cycle, host factors are expected to exert impacts on the machinery regulating G1/S phase of the germinative cells, which still remains largely unknown in E. multilocularis. In this study, we described the characterization of two key members of the G1/S phase cell-cycle regulation, EmCyclinD and EmCDK4/6. Our data show that EmCyclinD and EmCDK4/6 display significant sequence similarity to their respective mammalian homologs, and that EmCyclinD interacts with EmCDK4/6, forming a kinase-active complex to activate its substrate Rb1. EmCyclinD was actively expressed in the germinative cells. Addition of human EGF caused an elevated expression of EmCyclinD while inhibition of the EGFR-ERK signaling pathway in the parasite reduced the expression of EmCyclinD and downstream transcriptional factors. Treatment with Palbociclib, a specific CDK4/6 inhibitor, downregulated the expression of cell cycle-related factors and impeded germinative cell proliferation and vesicle formation from protoscoleces. Our data demonstrated that the EmCyclinD-EmCDK4/6 complex participates in the cell cycle regulation of germinative cells which is mediated by host EGF via the EGFR-ERK-EmCyclinD pathway in E. multilocularis.

RESUMEN

Alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is a lethal disease in humans. Novel therapeutic options are urgently needed since the current chemotherapy displays limited efficiency in AE treatment. In this study, we assessed the in vitro and in vivo effects of the epidermal growth factor receptor (EGFR)/MEK/extracellular signal-regulated kinase (ERK) signaling inhibitors, including BIBW2992, CI-1033, and U0126, on E. multilocularis Our data showed that BIBW2992, CI-1033, and U0126 all displayed in vitro effects on the viability of the E. multilocularis metacestode. These inhibitors also showed protoscolicidal activities and caused severe ultrastructural alterations in the parasite. Moreover, BIBW2992 and CI-1033 exhibited potent proapoptotic effects on E. multilocularis metacestodes. Strikingly, a large portion of the apoptotic cells were found to be the germinative cells. In vivo studies showed that BIBW2992 and U0126 significantly reduced parasite burden, and the parasite obtained from BIBW2992-treated mice displayed impaired structural integrity of the germinal layer. In conclusion, these findings demonstrate the potential of EGFR-mediated signaling as a target for the development of novel anti-AE agents. The EGFR inhibitor BIBW2992 represents a promising drug candidate and/or a lead compound for anti-AE chemotherapy.

Asunto(s)

Equinococosis , Echinococcus multilocularis , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Animales , Equinococosis/tratamiento farmacológico , Receptores ErbB/genética , Ratones

RESUMEN

BACKGROUND: The tumor-like growth of the metacestode larvae of the tapeworm E. multilocularis causes human alveolar echinococcosis, a severe disease mainly affecting the liver. The germinative cells, a population of adult stem cells, are crucial for the larval growth and development of the parasite within the hosts. Maintenance of the germinative cell pools relies on their abilities of extensive proliferation and self-renewal, which requires accurate control of the cell division cycle. Targeting regulators of the cell division progression may impair germinative cell populations, leading to impeded parasite growth. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we describe the characterization of EmAURKA and EmAURKB, which display significant similarity to the members of Aurora kinases that are essential mitotic kinases and play key roles in cell division. Our data suggest that EmAURKA and EmAURKB are actively expressed in the germinative cells of E. multilocularis. Treatment with low concentrations of MLN8237, a dual inhibitor of Aurora A and B, resulted in chromosomal defects in the germinative cells during mitosis, while higher concentrations of MLN8237 caused a failure in cytokinesis of the germinative cells, leading to multinucleated cells. Inhibition of the activities of Aurora kinases eventually resulted in depletion of the germinative cell populations in E. multilocularis, which in turn caused larval growth inhibition of the parasite. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the vital roles of Aurora kinases in the regulation of mitotic progression and maintenance of the germinative cells in E. multilocularis, and suggest Aurora kinases as promising druggable targets for the development of novel chemotherapeutics against human alveolar echinococcosis.

Asunto(s)

Aurora Quinasas/metabolismo , Equinococosis/parasitología , Echinococcus multilocularis/enzimología , Echinococcus multilocularis/crecimiento & desarrollo , Proteínas del Helminto/metabolismo , Secuencia de Aminoácidos , Animales , Aurora Quinasas/antagonistas & inhibidores , Aurora Quinasas/química , Aurora Quinasas/genética , Azepinas/farmacología , Clonación Molecular , Echinococcus multilocularis/efectos de los fármacos , Echinococcus multilocularis/genética , Proteínas del Helminto/antagonistas & inhibidores , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Larva/enzimología , Larva/genética , Larva/crecimiento & desarrollo , Mitosis , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Alineación de Secuencia

RESUMEN

The mechanisms involved in the fast growth of Angiostrongylus cantonensis from fifth-stage larvae (L5) to female adults and how L5 breaks through the blood-brain barrier in a permissive host remain unclear. In this work, we compared the transcriptomes of these two life stages to identify the main factors involved in the rapid growth and transition to adulthood. RNA samples from the two stages were sequenced and assembled de novo. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of 1,346 differentially expressed genes between L5 and female adults was then undertaken. Based on a combination of analytical results and developmental characteristics, we suggest that A. cantonensis synthesizes a large amount of cuticle in L5 to allow body dilatation in the rapid growth period. Products that are degraded via the lysosomal pathway may provide sufficient raw materials for cuticle production. In addition, metallopeptidases may play a key role in parasite penetration of the blood-brain barrier during migration from the brain. Overall, these results indicate that the profiles of each transcriptome are tailored to the need for survival in each developmental stage.

RESUMEN

Abstract The mechanisms involved in the fast growth of Angiostrongylus cantonensis from fifth-stage larvae (L5) to female adults and how L5 breaks through the blood-brain barrier in a permissive host remain unclear. In this work, we compared the transcriptomes of these two life stages to identify the main factors involved in the rapid growth and transition to adulthood. RNA samples from the two stages were sequenced and assembled de novo. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of 1,346 differentially expressed genes between L5 and female adults was then undertaken. Based on a combination of analytical results and developmental characteristics, we suggest that A. cantonensis synthesizes a large amount of cuticle in L5 to allow body dilatation in the rapid growth period. Products that are degraded via the lysosomal pathway may provide sufficient raw materials for cuticle production. In addition, metallopeptidases may play a key role in parasite penetration of the blood-brain barrier during migration from the brain. Overall, these results indicate that the profiles of each transcriptome are tailored to the need for survival in each developmental stage.

RESUMEN

BACKGROUND: Angiostrongyliasis caused by the rat lungworm, Angiostrongylus cantonensis (A. cantonensis), has globally spread from the traditional epidemic areas. The small intestine is the site where the third-stage larvae (L3) of this worm entered the host body, and parasite proteases are involved in this process. Ac-cathB-2, a cathepsin B-like cysteine of A. cantonensis, was formerly isolated from the insoluble part of fragmentised Escherichia coli without activity. The unplanned low activity of prokaryotic expression proteins and difficulties in genetic modification hindered understanding the function of this protein. METHODS: The recombinant Ac-cathB-2 was expressed and harvested from 293 T cells and the enzymatic property and the effects of processing on the activity of the recombinant protease were investigated in vitro. The expression of Ac-cathB-2 in response to external stimulation was assessed, and the function of this protease during host gut penetration was observed by using antiserum for inhibition. RESULTS: Of the life-cycle stages studied, L3 expressed the highest level of Ac-cathB-2 gene and released the corresponding gene product from the body. The expression of this gene was rapidly upregulated after incubating L3 in small intestine homogenate of rat. Recombinant Ac-cathB-2 was harvested from 293 T cell culture medium. This protease was activated by pepsin-HCl and the enabled Ac-cathB-2 could subsequently digest laminin and fibronectin readily. Moreover, the small intestine isolated from rat was disrupted after incubating with the activated Ac-cathB-2, resulting in the detachment of epithelial cells. Antiserum treatment inhibited the hydrolytic ability of recombinant Ac-cathB-2 by 82.7 %, and also reduced the tissue penetration of activated L3 by 41.2 %. Additionally, pre-incubation of L3 with artificial gastric acid increased the number of penetrating larvae by 53.2 %, and this alteration could be partly blocked by antiserum treatment. CONCLUSION: We believe that Ac-cathB-2 from A. cantonensis might help the worm to penetrate the rat gut, because the protease was able to degrade the tissue components of host. Nevertheless, our results further indicated that host pepsin played a beneficial role in this process by cleaving Ac-cathB-2 for activation. Thus, Ac-cathB-2 may probably represent an important target for the control of A. cantonensis infection.

Asunto(s)

Angiostrongylus cantonensis/fisiología , Catepsina B/metabolismo , Intestino Delgado/parasitología , Estadios del Ciclo de Vida , Pepsina A/metabolismo , Infecciones por Strongylida/parasitología , Angiostrongylus cantonensis/enzimología , Angiostrongylus cantonensis/crecimiento & desarrollo , Animales , Femenino , Proteínas del Helminto/metabolismo , Larva , Masculino , Ratas , Proteínas Recombinantes

RESUMEN

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.

Asunto(s)

Angiostrongylus cantonensis/enzimología , Catepsina B/fisiología , Proteínas del Helminto/fisiología , Parasitosis Intestinales/enzimología , Infecciones por Strongylida/enzimología , Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/crecimiento & desarrollo , Angiostrongylus cantonensis/fisiología , Animales , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Catepsina B/inmunología , Catepsina B/aislamiento & purificación , Línea Celular , Activación Enzimática , Células Epiteliales/parasitología , Proteínas de la Matriz Extracelular/metabolismo , Vectores Genéticos/genética , Proteínas del Helminto/antagonistas & inhibidores , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Proteínas del Helminto/aislamiento & purificación , Interacciones Huésped-Parásitos , Hidrólisis , Sueros Inmunes , Parasitosis Intestinales/parasitología , Intestinos/parasitología , Larva , Lentivirus/genética , Ratones , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Caracoles/parasitología , Infecciones por Strongylida/parasitología

RESUMEN

The cloned enolase gene of Angiostrongylus cantonensis (AcEno) comprised 1,667 bp and encoded a peptide with 434 amino acid residues which lacked of a signal peptide but contained a transmembrane region, indicating that AcEno tends to be a structural component (intracellular or membrane protein). The real-time PCR revealed a meaningful difference in the expression level of AcEno in varied development stages. By immunolocalization, native AcEno was detected mainly in the cytoplasm in most tissues, such as parietal muscle, genital tracts, nerve ring, and alimentary canal where the energy consumption is high, but not as expected on the cuticle and hypodermis layer of the nematode. This suggests that the AcEno may be involved in a host of other biological functions, rather than a receptor of plasminogen or a component of excretory-secretory antigen. In addition, AcEno expressed alike in the nucleus, indicating that AcEno also involved in regulating the continuous growth and development of A. cantonensis in hosts. Despite of living in the vasculature at a certain stage of life cycle, AcEno was not localized in the outer surface of L3 and adults, indicating that A. cantonensis may have other virulence and immune evasion mechanisms.

Asunto(s)

Angiostrongylus cantonensis/enzimología , Proteínas del Helminto/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Angiostrongylus cantonensis/genética , Animales , Clonación Molecular , Femenino , Proteínas del Helminto/genética , Estadios del Ciclo de Vida , Masculino , Fosfopiruvato Hidratasa/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN

RESUMEN

In this study we have investigated the anatomic sites of expression and developmental expression patterns of two cathepsin B-like cysteine proteases (AC-cathB-1, -2) of Angiostrongylus cantonensis. The immunolocalization results revealed that native AC-cathBs were found present in the L1 and L3 larvae, female and male adults, and the AC-cathBs were localized mainly on the digestive tract of A. cantonensis and expressed at varied levels and in different patterns in the internal tissues according to their developmental stage. Consistent with the infective stage of L3 is a much more intense staining of AC-cathBs in the esophagus compared with the intestine. In contrast to L3, more abundant signals were located to the intestine of adults, suggesting that nutrition digestion likely to be the main function of the protease at this point. AC-cathBs fluorescent signals were present in excretory pore, excretory tube in lateral cords, and muscular esophagus of larvae, further supported the AC-cathB-1, -2 likely to be released by A. cantonensis as excretory/secretory products. Additionally, only the protein AC-cathB-2 was detected in the reproductive system, especially in the wall of vas deferens, uterus, and oviduct of the parasites, whether the AC-cathB-2 has some function in germ cells development and maturation need to be further characterized. Although the anatomic sites and expression patterns were different in larvae and adults and the corresponding function might not the same, AC-cathB-1 and -2 involved in the host-parasite interaction in addition to digestive function.

Asunto(s)

Angiostrongylus cantonensis/enzimología , Catepsina B/aislamiento & purificación , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/crecimiento & desarrollo , Animales , Western Blotting , Catepsina B/genética , Catepsina B/metabolismo , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Ratones , Microscopía Fluorescente , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Caracoles

RESUMEN

Inferring evolutionary history of parasitism genes is important to understand how evolutionary mechanisms affect the occurrences of parasitism genes. In this study, we constructed multiple domain trees for parasitism genes and genes under free-living conditions. Further analyses of horizontal gene transfer (HGT)-like phylogenetic incongruences, duplications, and speciations were performed based on these trees. By comparing these analyses, the contributions of pre-adaptations were found to be more important to the evolution of parasitism genes than those of duplications, and pre-adaptations are as crucial as previously reported HGTs to parasitism. Furthermore, speciation may also affect the evolution of parasitism genes. In addition, Pristionchus pacificus was suggested to be a common model organism for studies of parasitic nematodes, including root-knot species. These analyses provided information regarding mechanisms that may have contributed to the evolution of parasitism genes.

RESUMEN

OBJECTIVE: To clone and express the full length of D-like aspartic protease gene (AsAP) of the third stage larvae of Anisakis simplex. METHODS: According to the partial information of D-like aspartic protease encoding gene of A. simplex from GenBank, specific primers were designed to amplify 3'end and 5' end of AsAP gene using rapid amplification of cDNA ends (RACE), and the full length of the D-like aspartic protease gene was obtained. Using total RNA of the third-stage larvae of A. simplex, coding sequence of the AsAP gene was amplified by reverse transcription-PCR (RT-PCR). The PCR product was digested by EcoR I and Sal I, and cloned into pET32 vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into E. coli BL21 (DE3). Expression of the protein induced by IPTG under gradient concentration and different time was conducted. RESULT: A 1 753 bp full length of AsAP was obtained, which contained 30 bp 5'UTR, 361 bp 3'UTR and a 1 362 bp open reading frame (ORF) encoding 453 amino acids with a predicted molecular mass of M(r) 50 726. It showed 65% identity with the D-like aspartic protease of Ancylostoma ceylanicum. The predicted amino acid sequence contains two conserved catalytic motif, an active site flap, an S2 subsite and an S3 subsite. A 20 amino acids signal peptide was found in the N-terminus, with significant hydrophobic property. Different concentration of the IPTG (0.2-1.6 mmol/L) showed little effect on the expression, and the production of the protein was up to maximum after 2 hours induction. CONCLUSION: The AsAP gene has been cloned and expressed.

Asunto(s)

Anisakis/enzimología , Proteasas de Ácido Aspártico/metabolismo , Proteínas del Helminto/metabolismo , Animales , Anisakis/genética , Proteasas de Ácido Aspártico/genética , Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , Expresión Génica , Proteínas del Helminto/genética , Plásmidos , Homología de Secuencia de Aminoácido

RESUMEN

Three cysteine protease genes, cathepsin B-like enzyme gene 1, 2 (AC-cathB-1, AC-cathB-2) and hemoglobin-type cysteine protease gene (AC-hem) were isolated and described from Angiostrongylus cantonensis adult. The deduced amino acid sequence of Ac-cathB-1 and AC-cathB-2 contain all of the conserved regions of cathepsin B. AC-cathB-2 is similar to a host intrusion-related cysteine protease B from Parelaphostrongylus tenuis, and the AC-hem shares high similarity to legumain from Haemonchus contortus. AC-cathB-1 was expressed significantly higher in L1 as compared with AC-hem, the AC-cathB-2 followed; AC-cathB-2 transcripts in L3 were found increased rapidly and obviously abundant, suggesting that AC-cathB-1 and AC-cathB-2 may play an important role in intermediate and final host invasion, separately. The cysteine protease genes were more or less expressed in adult stage excepted for AC-cathB-2. As the AC-cathB-1 and AC-hem highly expressed in adult worms, suggesting AC-hem may activate AC-cathB-1 which involved in the host invasion and feeding process.

Asunto(s)

Angiostrongylus cantonensis/enzimología , Proteasas de Cisteína/metabolismo , Regulación Enzimológica de la Expresión Génica , Región de Flanqueo 3'/genética , Región de Flanqueo 5'/genética , Secuencia de Aminoácidos , Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/crecimiento & desarrollo , Animales , Secuencia de Bases , Catepsina B/química , Catepsina B/genética , Catepsina B/metabolismo , Clonación Molecular , Proteasas de Cisteína/química , Proteasas de Cisteína/genética , ADN Complementario/química , ADN Complementario/genética , Femenino , Estadios del Ciclo de Vida/genética , Masculino , Datos de Secuencia Molecular , ARN de Helminto/genética , ARN de Helminto/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Caracoles , Organismos Libres de Patógenos Específicos

RESUMEN

To facilitate improved detection of the first stage larvae (L1) of Angiostrongylus cantonensis from rat faeces, a TaqMan(®) probe real-time PCR method for the detection in situ was developed targeting the second internal transcribed region of the ribosomal DNA (ITS2) of A. cantonensis. The assay was capable of detecting a single L1 in a grain of fresh faeces (weight 320 ± 125 mg) from the experimental infected Sprague-Dawley rats, and the method can also detect cell-free copro-DNA from positive faeces placed for up to 12 months at ambient environment. The present study exhibited a high level of specificity for A. cantonensis, with no fluorescence signals were observed in reference control consisting of four parasite species commonly found in the intestine of rat. This approach can overcome the limitations of DNA-based identification that faecal materials should be stored in 70% ethanol or kept as frozen samples for further tests, and thus it might be suitable and feasible for the detection of target DNA in faecal materials preserved at ambient temperature, but the detecting efficiency will depend on the amount of DNA in the samples and the time placed for the samples due to DNA degradation.

Asunto(s)

Angiostrongylus cantonensis/aislamiento & purificación , ADN de Helmintos/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de los Roedores/diagnóstico , Infecciones por Strongylida/parasitología , Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/fisiología , Animales , Sistema Libre de Células , ADN Espaciador Ribosómico/genética , Heces/parasitología , Interacciones Huésped-Parásitos , Larva/genética , Larva/fisiología , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Infecciones por Strongylida/diagnóstico

RESUMEN

To facilitate improved diagnosis and detection of the third stage larva (L3) of Anisakis pegreffii from the Minnan-Taiwan bank fishing ground in Taiwan Strait, a real-time PCR method for the detection in situ and differentiation was developed to amplify a region of the second internal transcribed spacer (ITS-2) of this parasite. The real-time PCR assay was capable of detecting 1/3 of a single L3 in 30 mg of marine fish tissue, and also exhibited a high level of specificity for A. pegreffii, no fluorescence signals were observed in other five major larval anisakid species found in commercial marine fishes caught in this fishing ground.

Asunto(s)

Anisakiasis/veterinaria , Anisakis/aislamiento & purificación , Enfermedades de los Peces/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Animales , Anisakiasis/diagnóstico , Anisakiasis/parasitología , Anisakis/genética , Cartilla de ADN , Sondas de ADN , ADN Ribosómico , ADN Espaciador Ribosómico/genética , Enfermedades de los Peces/parasitología , Peces , Colorantes Fluorescentes , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

OBJECTIVE: To establish an SYBR Green I real-time quantitative PCR method for the detection of anisakid nematodes with zoonotic potential from Taiwan Strait. METHODS: Anisakid larvae of six species (Anisakis simplex, A. physeteris, Raphidascaris trichiura, Contracaecum aduncum, C. muraenesoxi, and Contracaecum sp., a predominant species in fishes in the strait) were obtained from the guts of marine fishes and identified chiefly based on the morphological features. The ITS-2 rDNA sequences from the larvae were amplified by PCR using universal primers, then cloned and bidirectionally sequenced. According to these sequences, six specific forward primers were designed and synthesized. Specificity was determined by a series of conventional PCR respectively, the ITS-2 sequences amplified above were cloned into T vector which was subsequently transformed into E. coli DH5alpha. Following extraction and identification, the positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve. Sensitivity and reproducibility were determined. RESULTS: All the 6 standard curves established by the recombinant plasmids showed adequate linear relationship between threshold cycle (Ct) and template concentration. Melt curves were specific and all the 6 correlation coefficients were above 0.998. In the reproducibility test, the coefficients of variation (cv) of Ct values for detection of the 6 nematodes ranged between 0.18% and 2.80%, and the cv of the inter-assay ranged between 0.55% and 1.94%. The sensitivity of the real-time PCR was 1 x 10(2) copies/microl, about 100 times higher than the conventional PCR assays. The real-time quantitative PCR detection needed only 3.5 hours from the sample treatment to result report. CONCLUSION: An SYBR Green I fluorescent quantitative PCR has been developed for detecting anisakid nematodes with adequate sensitivity and specificity.

Asunto(s)

Anisakis/aislamiento & purificación , Enfermedades de los Peces/parasitología , Peces/parasitología , Reacción en Cadena de la Polimerasa/métodos , Animales , Anisakis/clasificación , Anisakis/genética , Benzotiazoles , Cartilla de ADN , Diaminas , Compuestos Orgánicos , Quinolinas , Sensibilidad y Especificidad , Análisis de Secuencia

RESUMEN

OBJECTIVE: To clone and express the full lenth of L-like cysteine protease gene of Anisakis simplex (AsCP). METHODS: According to L-like cysteine protease encoding gene of A. simplex from GenBank EST database, specific primers were designed to amplify 3'-end of AsCP gene using rapid-amplification of cDNA ends (RACE), and the full lenth of the L-like cysteine protease gene was obtained. Specific primers were designed according to the full length of the gene. Using total RNA of A. simplex third-stage larvae, coding sequence of the AsCP gene was amplified by RT-PCR. The PCR product was digested by EcoR I and Sal I, and cloned into pET32a(+) vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into Escherichia coli BL21 (DE3). Expression of the protein induced by IPTG of gradient concentration (0.2, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mmol/L) and by the same concentration (1 mmol/L) of IPTG at different time(0, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h) was conducted. The expression situation of recombinant protein was analyzed by SDS-PAGE. RESULTS: A 1211 bp of 3'-end of AsCP gene was amplified by 3'RACE, full length of the gene was 1462 bp and coding 411 amino acids. It showed 36.4% identity with the L-cysteine protease of Caenorhabditis elegans. Double enzyme digestion of the constructed recombinant plasmid pET32a(+)-AsCP showed that there was about 1150 bp fragment, the constructed recombinant plasmid was then identified by sequencing. SDS-PAGE showed that the recombinant protein (Mr 60,000) was identical with the target. IPTG showed little effect on the protein expression, and the production of protein was up to maximum after 2 hours induction. CONCLUSION: The AsCP gene has been cloned and expressed.

Asunto(s)

Anisakis/enzimología , Anisakis/genética , Cisteína/genética , Proteínas del Helminto/genética , Animales , Clonación Molecular , Cisteína/metabolismo , ADN Complementario , Vectores Genéticos , Proteínas del Helminto/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

The expressed sequence tags (ESTs) of Angiostrongylus cantonensis were analyzed in an attempt to gain further insight into its genomic expression patterns. A total of 1,277 ESTs of A. cantonensis were randomly downloaded from NCBI databank. ESTs were analyzed and annotated using Blastx. The result showed that there were 60 ESTs had no match to any of the proteins and gene sequences in the published databases, and 695 ESTs score more than 80. According to the function, the identified 695 ESTs could be grouped into 13 categories related to metabolism, cellular development, immune evasion, host-parasite interactions, and so on. Among them, 65 (9.4%) were proteases and protease inhibitors, represented 19 potential proteases and protease inhibitors genes; 42 (6.0%) were allergens or antigens, represented 15 potential antigens/allergens genes. Signal P analysis was applied to the 19 putative proteases and protease inhibitors and the 15 antigens/allergens protein sequences to identify the potential signal peptides and anchors. The result demonstrated that there were ten putative proteins had N-terminal signal peptides and three had signal anchors, these putative excretory/secretory proteins might be the products of potential parasitism genes which played an important role in the adaptation of A. cantonensis to a parasitism life. These parasitism genes and proteins identified are expected to become potential targets for future research on anti-A. cantonensis drugs; moreover, the resulting genetic information is useful in elucidating the mechanisms of parasitism of A. cantonensis.

Asunto(s)

Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/patogenicidad , Proteínas del Helminto/genética , Factores de Virulencia/genética , Animales , Etiquetas de Secuencia Expresada , Interacciones Huésped-Parásitos , Señales de Clasificación de Proteína

RESUMEN

There were six major larval anisakid species found in commercial marine fishes caught in the Minnan fishing ground in the Taiwan Strait: Anisakis physeteris, Anisakis pegreffii, Raphidascaris trichiuri, Contracaecum aduncum, Contracaecum muraenesoxi, Contracaecum sp. For rapid identification of the parasite species above, a single and a multiple primer PCR (multiplex PCR) method, using specific primers based on aligned sequences of the internal transcribed spacer ITS-1, 5.8S, and ITS-2 of nuclear ribosomal DNA, were jointly used for the rapid identification of these anisakid larvae. The primers yielded distinct PCR products for each of the anisakid nematodes, providing rapid and accurate tools for identifying anisakid nematodes with distinct geographical distribution.

Asunto(s)

Infecciones por Ascaridida/veterinaria , Ascaridoidea/aislamiento & purificación , Enfermedades de los Peces/parasitología , Reacción en Cadena de la Polimerasa/métodos , Animales , Infecciones por Ascaridida/parasitología , Ascaridoidea/clasificación , Ascaridoidea/genética , Cartilla de ADN/genética , ADN de Helmintos/química , ADN de Helmintos/aislamiento & purificación , ADN Ribosómico/química , ADN Ribosómico/aislamiento & purificación , Peces , Polimorfismo de Longitud del Fragmento de Restricción , Agua de Mar , Alineación de Secuencia , Especificidad de la Especie , Taiwán

RESUMEN

A total of 1,277 ESTs of Angiostrongylus cantonensis were downloaded from GenBank and analyzed with BlastX. SignalP V3.0 analysis was applied to predict potential putative antigen or allergen relative proteins with N-terminal secreted signal peptides or signal anchors. BlastX analysis showed that there were 614 ESTs scored more than 100, of which 14 were identical with A. cantonensis, 60 ESTs did not match any proteins in the databases. The identified 614 ESTs could be grouped into 10 categories, 80 ESTs expressed 22 antigen or allergen relative proteins, in which 12 had N-terminal secreted signal peptides and 3 had signal anchors.

Asunto(s)

Angiostrongylus cantonensis/genética , Etiquetas de Secuencia Expresada , Proteínas del Helminto/genética , Señales de Clasificación de Proteína/genética , Angiostrongylus cantonensis/metabolismo , Animales