Development of a recombinase polymerase amplification (RPA-EXO) and lateral flow assay (RPA-LFA) based on the ITS1 gene for the detection of Angiostrongylus cantonensis in gastropod intermediate hosts.

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai'i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies µL-1 to ~1 copy µL-1. All three assays consistently detected 50-100 copies µL-1 in triplicate and qPCR was able to detect ~13 copies µL-1 in triplicate. RPA-EXO was able to detect 25 copies µL-1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies µL-1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.

Molecular cloning and characterization of a cathepsin L-like cysteine protease of Angiostrongylus cantonensis.

Angiostrongylus cantonensis is a parasitic nematode dwelling in the heart and pulmonary arteries of rats, which can cause angiostrongyliasis in human by accidental infections, manifested as eosinophilic meningitis or meningoencephalitis. Cysteine proteases are the major class of endopeptidases that are expressed at a high level in A. cantonensis, which suggests it may play key roles in pathogenesis of the disease. In this study, the biological properties of the cathepsin L-like peptidase (Ac-cathL) of A. cantonensis were investigated. The Ac-cathL gene was identified from the fourth stage cDNA library of A. cantonensis, and then cloned and characterized by bioinformatics analysis and heterologous expression. The open reading frame (ORF) of Ac-cathL (1068 bp) encodes a protein of 355 amino acids with an estimated molecular weight of 58.0 kDa. Sequence analysis and multiple sequence alignment demonstrated that Ac-cathL resembles members of cathepsin L family of other parasites and mammals. Stage-dependent mRNA expression analysis showed that Ac-cathL transcripts were expressed in all stages of A. cantonensis, with the highest expression in female stage. The recombinant Ac-cathL (rAc-cathL) expressed in Escherichia coli exhibited protease activity in acidic pH as demonstrated by gelatin zymography, as well as hydrolytic activity against natural substrates, including BSA, human IgG and human fibrinogen. Immunolocalization revealed that Ac-cathL is localized in tegument of the 18 days post infection stage and uterus of the female adult stage. Therefore, these results implied that the Ac-cathL plays important roles in host tissue migration, nutrition uptake and immune evasion.

Regulatory effect of host miR-101b-3p on parasitism of nematode Angiostrongylus cantonensis via superoxide dismutase 3.

MicroRNA plays a vital role in the regulation of host-parasite interaction. In recent years, genomic and transcriptomic resources have become increasingly available for many helminths, but only a limited number of reports in this area are on the regulatory effects of host microRNAs on parasitic nematodes. In this work, we screened increased expression of host microRNAs after nematode infection from miRNA-seq data and predicted target genes by combined bioinformatics analysis and transcriptional profiling. We elucidated regulatory effects of one host miRNA on nematode infection using miRNA inhibitor and adeno-associated virus (AAV)-based TuD miRNA inhibitor. Using AAV-based TuD miRNA inhibitor, we showed that stable blockade of mmu-miR-101b-3p could alleviate the pathological damages of Angiostrongylus cantonensis, a parasitic nematode. Data from a luciferase report assay showed that mmu-miR-101b-3p targeted the extracellular superoxide dismutase 3 (Acsod3). Increased Acsod3 expression in larvae and alleviated oxidative damages were seen in the groups receiving mmu-miR-101b-3p inhibitor treatment in vitro and AAV-based TuD miRNA inhibitor injection in vivo. Results of this study demonstrate that murine miR-101b-3p inhibits the expression of antioxidant enzyme in A. cantonensis to strengthen host oxidative responses to nematodes. This work expands our knowledge of interspecies regulation of nematode gene expression by of host miRNAs.

Cytochrome c oxidase subunit I haplotype diversity of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae).

The rat lungworm Angiostrongylus cantonensis is a food-borne zoonotic parasite of public health importance worldwide. It is the primary etiologic agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans in many countries. It is highly endemic in Thailand especially in the northeast region. In this study, A. cantonensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed three additional COI haplotypes of A. cantonensis. The geographical isolates of A. cantonensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. malaysiensis. In the present study, distinct haplotypes were identified in seven regions of Thailand - AC10 in Phitsanulok (northern region), AC11 in Nakhon Phanom (northeastern region), AC15 in Trat (eastern region), AC16 in Chantaburi (eastern region), AC4 in Samut Prakan (central region), AC14 in Kanchanaburi (western region), and AC13 in Ranong (southern region). Phylogenetic analysis revealed that these haplotypes formed distinct lineages. In general, the COI sequences did not differentiate the worldwide geographical isolates of A. cantonensis. This study has further confirmed the presence of COI haplotype diversity in various geographical isolates of A. cantonensis. The COI gene sequence will be a suitable marker for studying population structure, phylogeography and genetic diversity of the rat lungworm.

Pepsin is a positive regulator of Ac-cathB-2 involved in the rat gut penetration of Angiostrongylus cantonensis.

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.

Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1) is involved in host gut penetration.

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.

[Substitution saturation analysis of mitochondrial cytochrome C oxidase subunit 1 (COX1) gene of Angiostrongylus cantonensis].

OBJECTIVE: To determine the substitution saturation of mitochondrial cytochrome c oxidase subunit I (COX1) gene of Angiostrongylus cantonensis. METHODS: One hundred and thirty A. cantonensis adult worms were collected from 33 sampling sites and used to amplify the complete sequence of COX1 gene. The nucleotide diversity, the number of haplotypes and the number of mutations were calculated by DnaSP software after sequence alignment. The distribution of base substitutions was characterized. The level of nucleotide substitution saturation was evaluated by plotting transitions and transversions against pairwise genetic distance. RESULTS: A total of 130 complete COX1 sequences of 1 577 bp were obtained. One hundred seventy-one nucleotides were found to be variable, resulting in 39 haplotypes with a diversity of 0.8114. The nucleotide diversity was 0.02841. Mutations were evenly distributed along the COX1 gene and did not show significant clustering. The analysis of COX1 gene showed that the amount of substitutions was increasing with the extension of genetic distance, and transitions outnumbered transversions; the increase rate was 0.76, and 0.16, respectively. The substitution rate was markedly different among the three codon positions: the maximum rate was found at the third codon position, followed by the first position, and the second position. The analysis of COX1 gene among 7 members of Metastrongyloidea showed that transversions outnumbered transitions when the genetic distance was more than 0.15. When the number of transitions exceeded 6%, a plateau was reached; while the transvertions increased linearly. CONCLUSION: No substitution saturation is found in mitochondrial COX1 gene of A. cantonensis. Therefore, the substitutions at each codon position could be considered in phylogeny analysis.

Enolase of Angiostrongylus cantonensis: more likely a structural component?

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.

Matrix metalloproteinase-9 leads to blood-brain barrier leakage in mice with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis.

Blood-brain barrier (BBB) disruption is associated with tight junction protein degradation, basal membrane disruption, and astrocyte damage. This study aims to investigate the role of matrix metalloproteinase (MMP)-9 in BBB disruption during Angiostrongylus cantonensis infection. We used mice infected with A. cantonensis, in which parasite-induced eosinophilia and inflammation might induce MMP-9 elevation. MMP-9 could cause claudin-5 degradation in endothelium tight junction, collagen type IV degradation in basal membranes, and S100B degradation in astrocytes of wild-type mice. BBB permeability was significantly attenuated in MMP-9 knockout mice than in wild-type mice in angiostrongyliasis meningoencephalitis. Immune cell aggregates were also more attenuated in the brains of MMP-9 knockout mice than in the brains of wild-type mice. Results suggest that MMP-9 activities are significant in BBB disruption in angiostrongyliasis meningoencephalitis. This study improves understanding of molecular mechanisms that underlie brain invasion by A. cantonensis, which is a key step in the pathogenesis of meningoencephalitis, and can offer a new strategy to reduce mortality.

Immunolocalization and developmental expression patterns of two cathepsin B proteases (AC-cathB-1, -2) of Angiostrongylus cantonensis.

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.

Identification and characterization of an asparaginyl endopeptidase from Angiostrongylus cantonensis.

Asparaginyl endopeptidase, also known as legumain, is a family of cysteine proteases in many organisms. In this study, an asparaginyl endopeptidase (Ac-AEP) was identified from the cDNA library of Angiostrongylus cantonensis. The full-length of Ac-AEP was determined to be 1,472 bp with an open reading frame of 1,341 bp encoding a putative protein with 446 amino acids. This putative protein was determined to have 37-65% identity in the amino acid sequences of the asparaginyl endopeptidases of other parasitic helminths. By real-time quantitative PCR analysis, Ac-AEP was revealed to be more abundantly expressed in the female adult worms than in other development stages. A recombinant asparaginyl endopeptidase (rAc-AEP) was then produced by a Pichia pastoris expression system. Posttranslational modification was shown to occur via N-linked glycosylation in this recombinant enzyme. The proteolytic activity of rAc-AEP was inhibited by iodoacetamide but not affected by E64, pepatain A, AEBSF, and EDTA. Moreover, the purified rAc-AEP was recognized by IgG in serum samples from BALB/c or ICR mice with A. cantonensis infection and patients with eosinophilic meningitis. These findings indicate that the rAc-AEP may have the potential for detecting A. cantonensis infection.

Genetic diversity of the rat lungworm, Angiostrongylus cantonensis, the major cause of eosinophilic meningitis.

Various aspects of the genetics of the rat lungworm, Angiostrongylus cantonensis, are reviewed. This nematode has an XX/XO sex-determination mechanism, with the female having a 2n = 12 (XX) and the male 2n = 11 (XO) chromosome constitution. Allozymes (12 loci) exhibit a low proportion of polymorphic loci (P = .08) and low mean heterozygosity (H = 0.43) in specimens from Hawa'i, and no polymorphism or heterozygosity in specimens from Thailand. The phosphoglucomutase-2 (PGM-2) locus exhibits sex-limited expression, with no detectable enzyme activity in the male worms from either location. Based on the 12 allozyme loci, Nei's genetic distance between the Hawa'i and Thailand isolates is D = 0.03. The p-distance (proportion of nucleotide sites) based on cytochrome c oxidase subunit I (COI) is 3.61% between the Thailand and China isolates as well as between Thailand and Hawa'i isolates, and 0.83% between China and Hawa'i isolates. The partial DNA sequences of the 66 kDa protein gene show a great diversity of haplotypes, indicating both inter- and intra-population variation. Intra-specific sequence variation is also found in the internal transcribed spacer regions. For the small-subunit ribosomal RNA (SSU rRNA) gene, two distinct genotypes have been recorded.

Characterization and immunolocalization of mutated ornithine decarboxylase antizyme from Angiostrongylus cantonensis.

Ornithine decarboxylase antizyme (OAZ), a prominent regulator of cell proliferation, DNA/RNA transformation and tumorigenesis, can bind to ornithine decarboxylase (ODC) and facilitate its degradation. Expression of OAZ requires a unique ribosomal frame shift that is regulated by levels of polyamine in the cell. In this study, we cloned an OAZ gene with the +1 ribosomal frame-shift from a fourth-stage larvae cDNA library of Angiostrongylus cantonensis. We removed one nucleotide to express the gene without polyamine. The sequence analysis showed that the deleted-mutation ornithine decarboxylase antizyme (DM-AcOAZ) contained a conservative domain related to other species OAZ. Quantitative real-time PCR revealed that DM-AcOAZ was expressed in L3 and L4 stages and adult female worms. More notably the expression level is the highest in the adult female stage. Immunohistochemical studies indicated that DM-AcOAZ was specifically localized in the uterus, oocyte and intestine in adult female worms. MTT assays showed that in DM-AcOAZ transfected HeLa cells, cell proliferation is inhibited. In conclusion, DM-AcOAZ may be a female-enriched protein and may involved in the cell proliferation in A. cantonensis.

Cathepsin B-like and hemoglobin-type cysteine proteases: stage-specific gene expression in Angiostrongy cantonensis.

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.

Cloning and characterization of a novel cathepsin B-like cysteine proteinase from Angiostrongylus cantonensis.

Cysteine protease plays a key role in host-parasite interactions. In this study, we identified a novel gene encoding a cathepsin B-like cysteine protease (AcCBL1) from the cDNA library of Angiostrongysus cantonensis fourth-stage larvae (L4) and characterized its biological role in the parasite. Sequence and phylogeny analysis showed that AcCBL1 is related to other cathepsin B family members with the conserved catalytic triad (Cys, His, Asn) and diagnostic occluding loop. In addition, the sequence contains a specific "hemoglobinase motif" and might have a hemoglobinase (Hb)-degrading function. The recombinant AcCBL1 (rAcCBL1) exhibited the protease activity by gelation SDS/PAGE assay; rAcCBL1 can cleave the fluorogenic substrate Z-Arg-Arg-AMC, and the optimum pH was 5.5. The enzyme can hydrolyse several host proteins including Hb and human IgG in acidic pH, but low levels of hydrolysis were observed in neutral pH. Reverse transcription polymerase chain reaction revealed that AcCBL1 expression was detected throughout various developmental stages, L3, L4, adult male and female worms. Western blotting analysis indicated that AcCBL1 was an excretory/secretory product of L4 in mature form of protease. Immunolocalization demonstrated that AcCBL1 was mainly localized in the intestine of L4. These results suggest that rAcCBL1 may play an important role in the parasite nutrition uptake.

Molecular characterization and immunolocalization of a protein disulfide isomerase from Angiostrongylus cantonensis.

Protein disulfide isomerases (PDIs), belonging to the thioredoxin superfamily, are oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds among cysteine residues of proteins. In this study, we report the cloning and characterization of a cDNA encoding a protein disulfide isomerase (AcPDI) from a cDNA library of fourth-stage larvae of Angiostrongylus cantonensis. The deduced amino acid sequence contains two thioredoxin domains and exhibits high identity to the homologues from other species. Quantitative real-time PCR (qRT-PCR) was performed at the third-stage larvae, fourth-stage larvae, and adult stage of A. cantonensis, and the results revealed that the AcPDI mRNA, while expressed at all three stages, is expressed at a significantly higher level in female adult worms. Results of immunohistochemical studies indicated that the AcPDI expression was specifically localized in the tegument and uterus wall of female adult worms. Biochemical analysis showed that recombinant AcPDI was biologically active in vitro and exhibited the typical biochemical functions of PDIs: oxidase/isomerase and reductase activities. Collectively, these results implied that AcPDI may be a female-enriched protein and associated with the reproductive development of A. cantonensis. In addition, considering its biochemical properties, AcPDI may be involved in the formation of the cuticle of A. cantonensis.

Molecular cloning and characterization of a matrix metalloproteinase, from Caenorhabditis elegans: employed to identify homologous protein from Angiostrongylus cantonensis.

Matrix metalloproteinases (MMPs) are a class of zinc-binding endopeptidases mainly responsible for degrading extracellular matrix constituent components, which also serve as effectors of leukocyte recruitment, cytotoxicity, cytokine and chemokine processing, and defensin activation involved in multiple mechanisms of immunomodulation. MMPs are a conserved proteolytic enzyme family participating in normal physiological function. In the present study, we have cloned a gene named CEMMP62 from Caenorhabditis elegans, the putative 62-kDa protein that contained 579 residues with MMP-conserved catalytic domain known as ZnMc_MMP and showed high identities with MMPs from other nematodes, and demonstrated that the recombinant CEMMP62 (rCEMMP62) expressed and purified from Escherichia coli could have weak proteolytic activity on swine gelatin; Western blot analysis revealed that sera from BALB/c mice immunized by recombinant protein could recognize excretory-secretary antigens from Angiostrongylus cantonensis third-stage larvae (L3). Also, the antiserum can recognize larval soluble antigens of L4 coming from mice (nonpermissive host) infected with A. cantonensis while it cannot recognize larval soluble antigens of L4 coming from rats (permissive host) infected with A. cantonensis. The results implied that probably CEMMP62 has homologous proteins which exist in A. cantonensis, and the potential MMP may play an important role in A. cantonensis infection and pathogenic process.

Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis.

Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43-53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood-brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite-host interactions.

Detection of anti-oxidant enzymatic activities and purification of glutathione transferases from Angiostrongylus cantonensis.

There are several anti-oxidant enzyme families that play pivotal roles in facilitating the survival of parasites. Glutathione transferases (GSTs) are members of the anti-oxidant family that can detoxify a broad range of exogenous or endogenous compounds including reactive oxidative species. GSTs have been studied as vaccine candidates, immunodiagnostic markers and as treatment targets. Helminths of the genus Angiostrongylus live inside arteries of vertebrates and two main species are associated with accidental human infections: Angiostrongylus costaricensis adult worms live inside the mesenteric arteries and larvae of Angiostrongylus cantonensis become trapped in the central nervous system vasculature. Since the interactions between angiostrongylid nematodes and their vertebrate hosts are poorly understood, this study characterized the anti-oxidant enzymatic activities of A. cantonensis from female worms by collecting excreted and secreted (ES) and total extract (TE) molecules. Catalase (CAT) and superoxide dismutase (SOD) activities were found both in the ES and TE while glutathione peroxidase (GPX) and GST were found only in the TE. GSTs were purified by glutathione agarose affinity column (AcGST) and the pool of eluted GSTs was analyzed by mass spectrometry (LC-MS/MS) and de novo sequencing (Masslynx software). Sequences from two peptides (AcGSTpep1 and AcGSTpep2) present high identity to the N-terminal and C-terminal from sigma class GSTs of nematodes. It is known that these GST enzymes are associated with host immune regulation. Furthermore, understanding the role of parasite-derived anti-oxidant molecules is important in understanding host-parasite interactions.

Uptake and utilization of arachidonic acid in infective larvae of Angiostrongylus cantonensis.

Infective larvae of Angiostrongylus cantonensis may take up and incorporate exogenous arachidonic acid into their lipid pool. By scintillation counting, uptake and incorporation were determined to be time dependent. Arachidonic acid was mainly incorporated into phospholipid (56.8%) and neutral lipid (22.4%) pools. In the neutral lipids, 64.0% was diglyceride and 36.0% triglyceride. Phosphatidylcholine was the predominant fatty acid in the phospholipid pool. In addition to the release of leukotriene B4, the parasite was found to generate radiolabelled CO2 after incubation with [U-14C]arachidonate. Moreover, enzymatic analysis of crude extracts revealed the presence of acyl-CoA dehydrogenase (short and long chain), thiolase, enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. These findings suggest that infective larvae of A. cantonensis not only take up and incorporate exogenous arachidonic acid into their lipid pool, but may also utilize the fatty acid through a functional ß-oxidation pathway.