Upregulated galectin-1 in Angiostrongylus cantonensis L5 reduces body fat and increases oxidative stress tolerance.

BACKGROUND: Angiostrongylus cantonensis L5, parasitizing human cerebrospinal fluid, causes eosinophilic meningitis, which is attributed to tissue inflammatory responses caused primarily by the high percentage of eosinophils. Eosinophils are also involved in killing helminths, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival. In previous study, we demonstrated the extracellular function of Acan-Gal-1 in inducing the apoptosis of macrophages. Here, the intracellular functions of Acan-Gal-1 were investigated, aiming to further reveal the mechanism involved in A. cantonensis L5 worms surviving inflammatory responses in the human central nervous system. METHODS: In this study, a model organism, Caenorhabditis elegans, was used as a surrogate to investigate the intracellular functions of Acan-Gal-1 in protecting the worm from its host's immune attacks. First, structural characterization of Acan-Gal-1 was analyzed using bioinformatics; second, qRT-PCR was used to monitor the stage specificity of Acan-gal-1 expression in A. cantonensis. Microinjections were performed to detect the tissue specificity of lec-1 expression, the homolog of Acan-gal-1 in C. elegans. Third, microinjection was performed to develop Acan-gal-1::rfp transgenic worms. Then, oxidative stress assay and Oil Red O fat staining were used to determine the functions of Acan-Gal-1 in C. elegans. RESULTS: The results of detecting the stage specificity of Acan-gal-1 expression showed that Acan-Gal-1 was upregulated in both L5 and adult worms. Detection of the tissue specificity showed that the homolog of Acan-gal-1 in C. elegans, lec-1 was expressed ubiquitously and mainly localized in cuticle. Investigating the intracellular functions of Acan-Gal-1 in the surrogate C. elegans showed that N2 worms expressing pCe-lec-1::Acan-gal-1::rfp, with lipid deposition reduced, were significantly resistant to oxidative stress; lec-1 mutant worms, where lipid deposition increased, showed susceptible to oxidative stress, and this phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp. Expressing pCe-lec-1::Acan-gal-1::rfp or lec-1 RNAi in fat-6;fat-7 double-mutant worms, where fat stores were reduced, had no significant effect on the oxidative stress tolerance. CONCLUSION: In C. elegans worms, upregulated Acan-Gal-1 plays a defensive role against damage due to oxidative stress for worm survival by reducing fat deposition. This might indicate the mechanism by which A. cantonensis L5 worms, with upregulated Acan-Gal-1, survive the immune attack of eosinophils in the human central nervous system.

Downregulated RPS-30 in Angiostrongylus cantonensis L5 plays a defensive role against damage due to oxidative stress.

BACKGROUND: Eosinophilic meningitis, caused by fifth-stage larvae of the nematode (roundworm) Angiostrongylus cantonensis, is mainly attributed to the contribution of eosinophils to tissue inflammatory responses in helminthic infections. Eosinophils are associated with the killing of helminths via peroxidative oxidation and hydrogen peroxide generated by the dismutation of superoxide produced during respiratory bursts. In contrast, when residing in the host with high level of eosinophils, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival in the hosts. In a previous study we demonstrated that the expression of the A. cantonensis RPS 30 gene (Acan-rps-30) was significantly downregulated in A. cantonensis L5 roundworms residing in cerebrospinal fluid with a high level of eosinophils. Acan-RPS-30 is a protein homologous to the human Fau protein that plays a pro-apoptotic regulatory role and may function in protecting worms from oxidative stress. METHODS: The isolation and structural characterization of Acan-RPS-30 were performed using rapid amplification of cDNA ends (RACE), genome walking and bioinformatics. Quantitative real-time-PCR and microinjection were used to detect the expression patterns of Acan-rps-30. Feeding RNA interference (RNAi) was used to knockdown the apoptosis gene ced-3. Microinjection was performed to construct transgenic worms. An oxidative stress assay was used to determine the functions of Acan-RPS-30. RESULTS: Our results showed that Acan-RPS-30 consisted of 130 amino acids. It was grouped into clade V with C. elegans in the phylogenetic analysis. It was expressed ubiquitously in worms and was downregulated in both L5 larvae and adult A. cantonensis. Worms expressing pCe-rps30::Acan-rps-30::rfp, with the refractile "button-like" apoptotic corpses, were susceptible to oxidative stress. Apoptosis genes ced-3 and ced-4 were both upregulated in the transgenic worms. The phenotype susceptible to oxidative stress could be converted with a ced-3 defective mutation and RNAi. rps-30-/- mutant worms were resistant to oxidative stress, with ced-3 and ced-4 both downregulated. The oxidative stress-resistant phenotype could be rescued and inhibited by through the expression of pCe-rps30::Acan-rps-30::rfp in rps-3-/- mutant worms. CONCLUSION: In C. elegans worms, downregulated RPS-30 plays a defensive role against damage due to oxidative stress, facilitating worm survival by regulating downregulated ced-3. This observation may indicate the mechanism by which A. cantonensis L5 worms, with downregulated Acan-RPS-30, survive in the central nervous system of humans from the immune response of eosinophils.

The opposite roles of PAS-5 and Galectin-1 in immune response during the early infection of Angiostrongylus cantonensis.

BACKGROUND: Angiostrongylus cantonensis is a human zoonotic nematode parasite. Our previous studies found that PAS-5 and Galectin-1 (Gal-1) proteins of A. cantonensis could be strongly recognized by sera from mice infected with A. cantonensis. In this study, we further evaluated the potential roles of these two proteins in the induction of immune response in mice. METHODS: Mice were immunized with recombinant PAS-5 or Gal-1 and then challenged with 30 infective A. cantonensis larvae following the last immunization. We then examined the infected mice for changes in serum antibodies and cytokines by ELISA, CD4+ T cells and CD4+CD25+FoxP3+ regulatory T cells (Tregs) by flow cytometry, and tissue damage severity by hematoxylin-eosin (H&E) staining. RESULTS: Compared with control mice, the PAS-5-immunized mice exhibited increased levels of serum antibodies and cytokines (except for IL-10) at different time points post-infection. PAS-5 immunization promoted significant proliferation of CD4+ T cells, and caused more damage in the brain tissue. Vaccination with Gal-1 inhibited the production of antibodies (except for IgG1) and IFN-γ, but promoted the expression of IL-4 and IL-10. Gal-1 immunization results in significant increases in the levels of CD4+CD25+FoxP3+ Tregs, and mild inflammatory changes. CONCLUSIONS: Taken together, our findings show that PAS-5 enhances, but Gal-1 inhibits the immune response in the early stage of A. cantonensis infections.

[Cloning of galectin-1 gene of Angiostrongylus cantonensis and testing the agglutination property of the galectin-1 protein].

Objective: To clone and express the galectin-1 gene of Angiostrongylus cantonensis, and test the agglutination property of its protein. Methods: The three-dimensional structure of galectin-1 was analyzed with Swiss Model. Total RNA was extracted from male worms of A. cantonensis. Primers were designed for galectin-1 based on its coding region (GenBank Accession No. JN133961.1). RT-PCR was performed, and the PCR products were subcloned to pCold Ⅲ plasmid and transduced into Escherichia coli BL21 strain. The recombinant plasmid was extracted from positive clones on LB plate containing 100 µg/ml Kanamycin, and validated with double digestion, PCR identification and sequencing. The confirmed positive clones of E. coli BL21 with the recombinant plasmid were grown in LB medium containing ampicillin (100 µg/ml, 100 µl). IPTG was added to induce expression of the plasmid. The galectin-1 recombinant protein was purified with Ni-NTA beads, and analyzed with SDS-PAGE and Western blotting using anti-serum of mouse immunized with whole worms of A. cantonensis. The agglutination reaction with red blood cells in fresh blood of ICR mouse was observed for the 10-fold serial dilutions of recombinant proteins (5.55 × 10(-1)-5.55 × 10(-5) ng/µl). Results: The Swiss Model analysis showed that the functional galectin-1 had a non-dimeric form. As was expected, the RT-PCR products had a size of 850 bp. Results of double digestion, PCR and sequencing showed successful construction of the pCold Ⅲ-galectin-1 plasmid. SDS-PAGE revealed expression of soluble recombinant fusion protein with molecular weight of ~36 000. Western blotting showed that the galectin-1 protein was recognized by mouse anti-serum. In addition, the minimun concentration of galectin-1 that showed significant agglutination reactions with mouse red blood cells was 5.55 × 10(-4) ng/µl. Conclusion: The galectin-1 clone can be expressed in the pCold Ⅲ plasmid, and its protein product has agglutination property.

Profiling of differentially expressed genes in sheep T lymphocytes response to an artificial primary Haemonchus contortus infection.

BACKGROUND: Haemonchus contortus is a common bloodsucking nematode causing widespread economic loss in agriculture. Upon H. contortus infection, a series of host responses is elicited, especially those related to T lymphocyte immunity. Existing studies mainly focus on the general immune responses of sheep T lymphocyte to H. contortus, lacking investigations at the molecular level. The objective of this study was to obtain a systematic transcriptional profiling of the T lymphocytes in H. contortus primary-infected sheep. METHODS: Nematode-free sheep were orally infected once with H. contortus L3s. T lymphocyte samples were collected from the peripheral blood of 0, 3, 30 and 60 days post infection (dpi) infected sheep. Microarrays were used to compare gene transcription levels between samples. Quantitative RT-PCR was employed to validate the microarray data. Gene Ontology and KEGG pathway analysis were utilized for the annotation of differentially expressed genes. RESULTS: Our microarray data was consistent with qPCR results. From microarrays, 853, 242 and 42 differentially expressed genes were obtained in the 3d vs. 0d, 30d vs. 0d and 60d vs. 0d comparison groups, respectively. Gene Ontology and KEGG pathway analysis indicated that these genes were involved in metabolism, signaling, cell growth and immune system processes. Functional analysis of significant differentially expressed genes, such as SLC9A3R2, ABCB9, COMMD4, SUGT1, FCER1G, GSK3A, PAK4 and FCER2, revealed a crucial association with cellular homeostasis maintenance and immune response. Our data suggested that maintaining both effective immunological response and natural cellular activity are important for T lymphocytes in fighting against H. contortus infection. CONCLUSIONS: Our results provide a substantial list of candidate genes in sheep T lymphocytes response to H. contortus infection, and contribute novel insights into a general immune response upon infection.

Expression of Caenorhabditis elegans-expressed Trans-HPS, partial aminopeptidase H11 from Haemonchus contortus.

Aminopeptidase H11 present in the surface of intestine microvilli in Haemonchus contortus was identified as the most effective antigen candidate. However, its recombinant forms produced in Escherichiacoli, insect cells and yeast could not provide promising protection against H. contortus challenge, probably due to the inappropriate glycosylation and/or conformational folding. Herein, partial H11 containing the potential zinc-binding domain and two predicted glycosylation sites (nt 1 bp-1710 bp, Trans-HPS) was subcloned downstream of 5' flanking region of Caenorhabditis elegans cpr-1 gene in pPD95.77 vector, with the deletion of GFP gene. The recombinant was expressed in C. elegans and verified by blotting with anti-H11 and anti-Trans-HPS rabbit polyclonal antibodies and anti-His monoclonal antibody. Stably inherited Trans-HPS in worm descendants was achieved by integration using UV irradiation. Immunization with the crude Trans-HPS extracted from transgenic worms resulted in 37.71% reduction in faecal egg counts (FEC) (P<0.05) and 24.91% reduction in worm burden, but an upward curve with moderate rate of daily FEC in goats. These results suggested an apparent delay against H. contortus egg-laying in goats, which differed from that with bacteria-origin form of partial H11 (nt 670 bp-1710 bp, HPS) (26.04% reduction in FEC and 18.46% reduction in worm burden). These findings indicate the feasibility of sufficient C. elegans-expressed H11 for the immunological research and vaccine development.