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.