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.

[Protein interaction site of Toxoplasma gondii microneme protein 6 and aldolase determined by site-directed mutagenesis].

OBJECTIVE: To identify the protein interaction site of Toxoplasma gondii microneme protein 6 (MIC6) and aldolase by using site-directed mutagenesis. METHODS: Based on Toxoplasma gondii MIC6 gene sequence (GenBank Accession No. AF110270), the specific primers were designed. Tryptophan (W)-348 of MIC6 C terminus (MIC6C) was mutated to valine (V) via site-directed mutagenesis. MIC6C W/V gene was obtained from cDNA library by PCR amplification and subcloned into pGEX-4T-1. The mutant protein GST-MIC6C W/V was expressed in E. coli, induced by 0.8 mmol/L IPTG, and purified by affinity chromatography. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with T. gondii tachyzoites lysate, and bound proteins were eluted using sample buffer. Bound products were resolved by SDS-PAGE and Western blotting. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with aldolase-His6. After incubation, the resin was washed and subjected to SDS-PAGE. RESULTS: The MIC6C W/N gene was obtained, and the recombinant plasmid MIC6C W/V/pGEX-4T-1 was successfully constructed. The mutant protein GST-MIC6C W/V was expressed and purified in vitro. SDS-PAGE analysis indicated that GST-MIC6C was co-precipitated with aldolase from T. gondii tachyzoites lysate or aldolase-His6, whereas GST-MIC6C W/V failed to precipitate aldolase from T. gondii tachyzoites lysate or aldolase-His6. Western blotting analysis using anti-aldolase antibody indicated that GST-MIC6C could pull-down aldolase from T. gondii tachyzoites lysate. CONCLUSION: Tryptophan (W348) was the interaction site of MIC6 and aldolase in T. gondii.

Cloning, expression and characterisation of a type II cystatin from Schistosoma japonicum, which could regulate macrophage activation.

Cystatin play an important role in parasite immune evasion. It is involved in many immune responses processes regulations such as inhibiting antigen presentation, modifying cytokines production and macrophage polarization. In recent years, more and more cystatins were used in treating some inflammatory diseases such as asthma and inflammation bowel diseases; however, cystatins from Schistosoma japonicum were rarely studied. In the present study, we have cloned a cystatin from the adult stage of Schistosoma japonicum, named as SjCystatin, and its sequence shares conserved domains with other type II family cystatins. It was further verified by enzyme inhibition assays. SjCystatin retained its inhibitory activity under a wide range of pH values and temperatures, can maintain its inhibitory activity at pH 6.5-7.5 and 37 °C, respectively. Then, we investigated the effects of SjCystatin on the lipopolysaccharide (LPS)-induced activated RAW264.7. Results showed that SjCystatin inhibit LPS-induced nitric oxide production in a dose-dependent manner. LPS-induced TNF-α and IL-6 production began to be inhibited at least 6 h after SjCystatin stimulation. SjCystatin significantly increased IL-10 production at 6 h after stimulation and its effect on IL-10 production diminished quickly. These results imply that SjCystatin can induce M2 macrophage polarization and can be expected to serve as a potential drug source for the medication of inflammatory disorders like other cystatins.

A field survey for Wolbchia and phage WO infections of Aedes albopictus in Guangzhou City, China.

Wolbachia are maternal endosymbiotic bacterium, which infect a diverse range of arthropods, ranging from 20 to 76% in nature. They are capable of inducing a wide range of reproductive abnormalities to their hosts, such as cytoplasmic incompatibility (CI), which has been proposed to be used as a tool to modify mosquitoes that are resistant to the development of pathogen, as an alternative vector control strategy. Here, we evaluated the prevalence of Wolbachia and phage WO infections in the field population of Aedes albopictus in Guangzhou City via polymerase chain reaction (PCR) assay using the Wolbachia specific Wolbachia surface protein (wsp) and phage WO orf7 gene primers. Based on the results of PCR and phylogeny analysis, we found that A. albopictus in Guangzhou City were infected with two Wolbachia strains, wAlbA and wAlbB. Phage WO, the virus-infected Wolbachia, was also detected in A. albopictus. One hundred and ten female individuals were screened via PCR, with 109 super-infected with Wolbachia and one sample single-infected with wAlbB strain. And 104 of 113 male individuals were both infected with wAlbA and wAlbB, and nine male samples were found to be infected with wAlbA strain only. The infection rates of phage WO in female and male individuals were 82.73 and 46.02%, respectively. These results showed that the natural Wolbachia and phage WO infections in A. albopictus population in Guangzhou were at a higher frequency at present, indicating that Wolbachia appear to be a better candidate nature resource for biological control insect vectors to reduce vector-borne diseases.

Differential proteomics of Aedes albopictus salivary gland, midgut and C6/36 cell induced by dengue virus infection.

The interaction between dengue virus (DENV) and vector mosquitoes are still poorly understood at present. In this study, 2-D DIGE combined with MS was used to analyze the differential proteomes of Aedes albopictus salivary gland, midgut and C6/36 cells induced by DENV-2. Our results indicated that the virus infection regulated several functional classes of proteins. Among them, 26 were successfully analyzed by real-time RT-PCR. The mRNA levels of 15 were the highest in salivary gland, 2 in midgut and none in C6/36 cells, however, 18 were the least in fat body compared to other organs. Interestingly, the changes of differential proteins mRNA were the most obvious in fat body post-infection. Chaperone, cytoskeleton and energy metabolism enzyme were the most down- or up- regulated proteins after DENV-2 infection. The abundant expression of these proteins in salivary gland may relate to its high susceptibility.

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.

Laccase2 is required for sclerotization and pigmentation of Aedes albopictus eggshell.

Laccase (EC 1.10.3.2) is a member of multicopper oxidases that have been found in higher plants, fungus, bacterium, and insects. Two types of laccase genes have been detected in many species of insects: laccase1 and laccase2. It has been identified that laccase2 enzyme may play a key role in sclerotization and pigmentation of insect cuticle. But few attentions were given to the biological role of laccase2 in the synthesizing of similar structures, such as oothecae, eggshell, or silk cocoons. We cloned laccase2 gene from Aedes albopictus, one main mosquito vector of dengue virus in China. An upregulation of laccase2 gene was observed after a blood meal in female adult mosquitoes, suggesting that laccase2 gene may have an involvement in the development of ovary. RNA interference experiment was performed by using adult female mosquitoes. Female mosquitoes were injected with 20 ng of double-strain RNA into the thorax. Pigmentation of mosquito eggshell was blocked that these eggs never became dark. And the incomplete sclerotization of eggshell weakened the stability and flexibility of the eggs. These eggs without enough protection were deformed and died in water. These results demonstrate that laccase2 plays a critical role in the development of eggs of A. albopictus. Laccase2 may provide a novel target for mosquito control and management.

Molecular cloning, expression, and characterization of a putative activation-associated secreted protein from Angiostrongylus cantonensis.

Activation-associated secreted protein (ASP) had been found in many helminthes, which was associated with pathogenesis and stage transition. A complementary DNA (cDNA) sequence encoding a putative two-domain ASP was obtained from an Angiostrongylus cantonensis fourth-stage larvae cDNA library, which we designated as AgASP. The cDNA of AgASP contains an open reading frame encoding 424 amino acids, the first 19 residues being a putative secretion signal. The expression pattern of this protein was investigated by real-time polymerase chain reaction and Western blot. We found that this protein expressed most highly in the brain-stage larvae (Lbr) of this parasite and existed in the excretory/secretory products of this stage. Immunofluorescence showed it existed in the lumen of the Lbr. The recombinant protein can be recognized by the infection sera from mice (nonpermissive host), while it cannot be recognized by infection sera from rats (permissive host). The infiltration of neutrophils in infected nonpermissive host can be lessened by immunizing this host with this protein (immunized vs control group, 13.7 ± 10.2 vs 65.5 ± 19.2). These findings suggest that this protein plays a role in the pathogenesis of human angiostrongyliasis and is worthy of further study.

Enzootic angiostrongyliasis in Guangzhou, China, 2008-2010.

This study was conducted to gain an understanding of the Angiostrongylus cantonensis infection status of rodent definitive host, snail intermediate host, and local residents in Guangzhou, China. A total of 430 rats were captured and 23 rats, from two species, were infected, with an average infection rate of 5.35%. A total of 795 Achatina fulica snails and 734 Pomacea canaliculata snails were collected. The average infection rates of these two species were 13.96% (111 of 795) and 1.50% (11 of 734), respectively. As for the seroprevalence of different occupations, the rate among the "general group" was significantly lower than the "occupational group." From this survey, Guangzhou is implicated to be the natural focus of Angiostrongylus cantonensis. Rattus norvegicus and Achatina fulica play important roles in spreading this nematode in Guangzhou. Residents who live in Guangzhou, especially those working in certain industries such as agriculture, food-making, and aquaculture, face a higher risk of infection.

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.

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay.

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.

Epidemiological survey of Angiostrongylus cantonensis in the west-central region of Guangdong Province, China.

The study was to understand the Angiostrongylus cantonensis infectious situation of rodent definitive host, snail intermediate host, and local residents in the west-central region of Guangdong Province in China. The snails Achatina fulica and Pomacea canaliculata collected from the survey place were digested with artificial gastric juice, and the third-stage larvae of A. cantonensis in the snails were examined under microscope. The heart and lung of rats captured from the survey place were taken to check the adult of A. cantonensis. The questionnaire surveys related to the infection of A. cantonensis were taken in local residents randomly selected, and the IgG antibody against A. cantonensis was tested in those residents with enzyme-linked immunosorbent assay (ELISA). A total of 1,391 rats including eight kinds of rats, such as Rattus norvegicus, Rattus flavipectus, Bandicota indica, Rattus sladeni, Mus musculus, Rattus rattoides, Suncus Murinus, and Rattus confucianus, were examined and 132 of them were infected by A. cantonensis, with an average infection rate of 9.49% and a mean intensity of A. cantonensis in infected rats was 9.39. A total of 3,184 snails A. fulica and 3,723 snails P. canaliculata were detected. The average infection rates of them were 25.03% (797/3,184) and 6.50% (242/3,723), respectively. There were 180 positive samples of IgG antibody against A. cantonensis in 1,800 serum samples of the residents, with a positive rate of 10.00%. The west-central region of Guangdong Province is the natural focus of A. cantonensis. In comparison with the investigation results in other regions of China, the infection rate of rat definitive host is at the middle level; in the intermediate host, the infection rate of snail A. fulica is above the middle level, and the infection rate of snail Pomacea canaliculata is below the middle level. Some local residents had already been infected by A. cantonensis or at the risk of being infected.

[Protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down].

OBJECTIVE: To identify the protein-protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down. METHODS: The aldolase and actin genes were obtained from cDNA library by PCR amplification, and subcloned respectively into pGEX-4T-1 and pET30a. The fusion protein GST-Aldolase and Actin-His6 were expressed in E. coli upon induction by 1 mmol/L IPTG and then purified with affinity chromatography. Fifteen rats were immunized intradermally with 200 microg Actin-His6 protein per rat at first time to produce the polyclonal antibodies. Then 100 microg Actin-His6 protein per rat on the 2nd-4th immunizations. Rats were immunized for 4 times with 7 days interval. The serum of rats was collected from heart at the fifth day after the final immunization. Glutathione sepharose beads were incubated with GST-Aldolase protein, then incubated with Actin-His6, and bound proteins were eluted using sample buffer. Eluants were resolved by SDS-PAGE and Western blotting. RESULTS: The aldolase and actin genes were obtained, and the recombinant plasmid aldolase/pGEX-4T-1, actin/pET30a were successfully constructed. Protein GST-Aldolase and Actin-His6 were expressed and purified in vitro. Serum samples were prepared from rats immunized with protein Actin-His6, and polyclonal antibody was purified with affinity chromatography. SDS-PAGE and Western blotting analysis of products from GST pull-down experiment showed that the protein bands on NC membrane were specifically recognized by anti-Aldolase-His6 and anti-Actin-His6 antibody. CONCLUSION: Aldolase interacts with Actin of Toxoplasma gondii.

Quantitative analysis of replication and tropisms of Dengue virus type 2 in Aedes albopictus.

Dengue virus serotype 2 (DENV-2) RNA replication profiles and tropisms were studied by using quantitative RT-PCR (q-RTPCR) in intrathoracically infected Aedes albopictus. The virus RNA replication profiles were diverse in mosquito organs. In fat body, brain, salivary gland, and malpighian tubes, it peaked at 8, 23, 23, and 27 days post-infection, respectively, and then, all declined. In midgut, it increased all the time and had no trend of decline. In ovary, it had no apparent increase. Subsequent Western blotting of DENV-2 E protein had similar results. Using ribosomal protein 7 (rpS7) as an internal control, we found that, in salivary gland, brain, fat body, and midgut, the average DENV-2 RNA levels (DENV-2 RNA/rpS7 mRNA) were 1,028, 464, 5.6, and 6.2, respectively; in malpighian tubes, it was 1, and in ovary, it was far less than 1. These results suggest that infection profiles and tropism of DENV-2 RNA in Ae. albopictus organs are significantly different.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis.

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

Cloning and functional expression of Rh50-like glycoprotein, a putative ammonia channel, in Aedes albopictus mosquitoes.

Evidence has shown that female mosquitoes can deaminate more than 80% of the ingested bloodmeal protein amino acids, and thus lead to a massive amount of ammonia production. Ammonia transport is a critical step for detoxifying ammonia in organisms. Here we characterized a putative ammonia channel gene, Rhesus (Rh) 50 glycoprotein, from Aedes albopictus (AalRh50) and determined the difference of its expression profile in different tissues at both message and protein levels as well as its response to a blood meal. We showed that AalRh50 shares a low identity with E. coli ammonia transporter (EcoAmtB), but higher identities with human RhBG and Drosophila Rh50 genes. The analysis of ammonia-conductance sites indicates that AalRh50 has residue substitutions of S237L (equivalent to S219 in AmtB) in the external vestibule, F127I (equivalent to F107 in AmtB) in the pore entrance, and S281N (equivalent to S263 in AmtB) in the internal vestibule, which could alter or reduce ammonia-conductance activity. The results from quantitative real-time-PCR and immunohistochemistry revealed that AalRh50 is expressed at significantly higher levels in the head, Malpighian tubules, and thorax of the non-blood-fed females, suggesting that AalRh50 might play roles in maintaining normal neurotransmitter metabolism, acid-base balance, and flight energy production in different tissues of mosquitoes at the non-blood-fed condition. A blood meal significantly increases AalRh50 expression in midgut, fat body, and Malpighian tubules from 3 or 6 to 24h post feeding, indicating that AalRh50 plays an important role in detoxification of excess systemic ammonia of female adults during the gonotrophic cycle.

Preliminary molecular characterization of the human pathogen Angiostrongylus cantonensis.

BACKGROUND: Human angiostrongyliasis is an emerging food-borne public health problem, with the number of cases increasing worldwide, especially in mainland China. Angiostrongylus cantonensis is the causative agent of this severe disease. However, little is known about the genetics and basic biology of A. cantonensis. RESULTS: A cDNA library of A. cantonensis fourth-stage larvae was constructed, and approximately 1,200 clones were sequenced. Bioinformatic analyses revealed 378 cDNA clusters, 54.2% of which matched known genes at a cutoff expectation value of 10(-20). Of these 378 unique cDNAs, 168 contained open reading frames encoding proteins containing an average of 238 amino acids. Characterization of the functions of these encoded proteins by Gene Ontology analysis showed enrichment in proteins with binding and catalytic activity. The observed pattern of enzymes involved in protein metabolism, lipid metabolism and glycolysis may reflect the central nervous system habitat of this pathogen. Four proteins were tested for their immunogenicity using enzyme-linked immunosorbent assays and histopathological examinations. The specificity of each of the four proteins was superior to that of crude somatic and excretory/secretory antigens of larvae, although their sensitivity was relatively low. We further showed that mice immunized with recombinant cystatin, a product of one of the four cDNA candidate genes, were partially protected from A. cantonensis infection. CONCLUSION: The data presented here substantially expand the available genetic information about the human pathogen A. cantonensis, and should be a significant resource for angiostrongyliasis researchers. As such, this work serves as a starting point for molecular approaches for diagnosing and controlling human angiostrongyliasis.