Molecular cloning and characterization of a novel peptidase from Trichinella spiralis and protective immunity elicited by the peptidase in BALB/c mice

Abstract

In our previous studies, a novel T. spiralis peptidase (TsP) was identified among the excretory/secretory (ES) proteins of T. spiralis intestinal infective larvae (IIL) and T. spiralis at the adult worm (AW) stage using immunoproteomics, but the biological function of TsP in the life cycle of T. spiralis is not clear. The objective of this study was to investigate the biological properties and functions of TsP in larval intrusion and protective immunity induced by immunization with rTsP. The complete TsP cDNA sequence was cloned and expressed. The results of RT-PCR, indirect immunofluorescence assay (IIFA) and western blotting revealed that TsP is a surface and secretory protein expressed in T. spiralis at different stages (muscle larvae, IIL, AWs and newborn larvae) that is principally localized at the epicuticle of the nematode. rTsP facilitated the larval intrusion of intestinal epithelial cells (IECs) and intestinal mucosa, whereas anti-rTsP antibodies suppressed larval intrusion; these facilitative and suppressive roles were dose-dependently related to rTsP or anti-rTsP antibodies. Immunization of mice with rTsP triggered an obvious humoral immune response (high levels of IgG, IgG1/ IgG2a, and sIgA) and also elicited systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune responses, as demonstrated by an evident increase in the cytokines IFN-γ and IL-4. Immunization of mice with rTsP reduced the numbers of intestinal adult worms by 38.6% and muscle larvae by 41.93%. These results demonstrate that TsP plays a vital role in the intrusion, development and survival of T. spiralis in hosts and is a promising candidate target molecule for anti-Trichinella vaccines.

Specific binding of aspartic protease and enterocytes promotes Trichinella spiralis invasion of murine intestinal epithelium cells

“…Vaccination of mice with rTsASP2 also induced the significant protection, as intestinal adult worms, NBL production and muscle larval burden in vaccinated mice was distinctly reduced. The immune protection (e.g., muscle larval burden reduction) induced by vaccination of rTsASP2 could be comparable with that of several recombinant T. spiralis proteins, such as cathepsin B (50.90%; Cui et al, 2019), serine protease (52.10%; Sun et al, 2018a), serine proteinase (52.50%; Yue et al, 2020), peptidase (41.93%; Lei et al, 2020), elastase-1 (64.06%; , and TsASP1 (60.50%; Xu et al, 2021). The reduction of the worm burden might be related with the Th1/Th2 immune response which may interrupt larval invasion of enterocytes, impair larval establishment and development in enteric epithelium (McVay et al, 2000;Song et al, 2018b).…”

“…The interaction between IECs and TsASP2 was also examined by IFT and confocal microscopy (Ren et al, 2018;Lei et al, 2020). IECs were grown on the cell culture plate until the confluence.…”

Characterization of a Novel Glutamine Synthetase From Trichinella spiralis and Its Participation in Larval Acid Resistance, Molting, and Development

“…After washes with PBS, the worms were stained using goat anti-mouse IgG-FITC conjugate (1:100; Santa Cruz, United States). Following washes again, whole worms and cross-sections were examined under a fluorescence microscope (Olympus, Tokyo, Japan) ( Lei et al, 2020 ; Yue et al, 2020 ).…”

Therapeutic Efficacy of a Trichinella Spiralis Paramyosin-Derived Peptide Modified With a Membrane-Targeting Signal in Mice With Antigen-Induced Arthritis

“…Degree of synovial hyperplasia (1–2 lines of regularly shaped synoviocytes = 0; 3–5 lines of irregularly shaped synoviocytes = 1; >5 lines of hypertrophied cells = 2); leukocyte infiltration in synovial tissue (no infiltration = 0; scattered cells = 1; focal infiltration in up to 50% of the synovial tissue = 2); percent diffusion of tissue fibrosis (no alterations = 0; 25–50% loss of the fat tissue component of synovial membranes = 1; >50% loss of the fat tissue component of synovial membranes = 2). Additionally, according to the immunofluorescence protocol, tissue deposition of MAC was assessed by immunofluorescence assay with incubating rabbit anti-mouse C9 IgG (Sigma-Aldrich) at a 1:200 dilution overnight at 4°C followed by incubation with FITC-labeled goat anti-rabbit IgG (DAKO, Glostrup, Denmark) at a 1:200 dilution for an additional 60 min at room temperature (Lei et al, 2020 ). Subsequently, the sections were observed and photographed by microscopy (Leica, Germany).…”