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A novel C-type lectin from Trichinella spiralis mediates larval invasion of host intestinal epithelial cells.

ABSTRACT: The aim of this study was to investigate the characteristics of a novel type C lectin from Trichinella spiralis (TsCTL) and its role in larval invasion of intestinal epithelial cells (IECs). TsCTL has a carbohydrate recognition domain (CRD) of C-type lectin. The full-length TsCTL cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of qPCR, Western blotting and immunofluorescence assays (IFAs) showed that TsCTL was a surface and secretory protein that was highly expressed at the T. spiralis intestinal infective larva (IIL) stages and primarily located at the cuticle, stichosome and embryos of the parasite. rTsCTL could specifically bind with IECs, and the binding site was localized in the IEC nucleus and cytoplasm. The IFA results showed that natural TsCTL was secreted and bound to the enteral epithelium at the intestinal stage of T. spiralis infection. The rTsCTL had a haemagglutinating effect on murine erythrocytes, while mannose was able to inhibit the rTsCTL agglutinating effect for mouse erythrocytes. rTsCTL accelerated larval intrusion into the IECs, whereas anti-rTsCTL antibodies and mannose significantly impeded larval intrusion in a dose-dependent manner. The results indicated that TsCTL specifically binds to IECs and promotes larval invasion of intestinal epithelium, and it might be a potential target of vaccines against T. spiralis enteral stages.

SUBMITTER: Hao HN

PROVIDER: S-EPMC9580147 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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A novel C-type lectin from Trichinella spiralis mediates larval invasion of host intestinal epithelial cells.

Hao Hui Nan HN Song Yan Yan YY Ma Kai Ning KN Wang Bo Ning BN Long Shao Rong SR Liu Ruo Dan RD Zhang Xi X Wang Zhong Quan ZQ Cui Jing J

Veterinary research 20221018 1

The aim of this study was to investigate the characteristics of a novel type C lectin from Trichinella spiralis (TsCTL) and its role in larval invasion of intestinal epithelial cells (IECs). TsCTL has a carbohydrate recognition domain (CRD) of C-type lectin. The full-length TsCTL cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of qPCR, Western blotting and immunofluorescence assays (IFAs) showed that TsCTL was a surface and secretory protein that was highly expressed ...[more]

PMID: 36258242

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Project description:BackgroundIntestinal larva invasion is a crucial step of Trichinella spiralis infection. Intestinal infective larvae (IIL) and their excretory/secretory proteins (ESP) interact with gut epithelium, which often results in gut epithelium barrier injuries. Previous studies showed when T. spiralis invaded intestinal epithelium cells, the IIL ESP disrupted the tight junctions (TJs) of Caco-2 monolayer, but the mechanism is not clear. The IIL ESP might cause gut epithelial apoptosis, weaken the gut barrier and aid the larval invasion. The aim of this study was to investigate whether T. spiralis IIL ESP participate in enterocyte apoptosis and disrupt gut epithelial barrier to promote the larval invasion.Methodology/principal findingsCell viability was assessed by CCK-8 assay and the results showed that 200 μg/ml of IIL ESP incubated with Caco-2 cells for 18 h inhibited the Caco-2 cell viability. The results of trans-epithelial electrical resistance (TEER) and FITC-dextran showed that IIL ESP decreased the TEER, increased FITC-dextran flux in Caco-2 monolayer. qPCR, Western blot and immunofluorescence test (IFT) showed that IIL ESP decreased the mRNA and protein expression of TJs (ZO-1, E-cad, Occludin and Claudin-1). The IIL ESP-induced Caco-2 cell apoptosis was observed by DAPI, Hoechst 33358, TUNEL and Annexin V/PI staining. Besides, flow cytometry revealed an increasing apoptosis rate in Caco-2 cells after the IIL ESP treatment. qPCR and Western blot analysis indicated that IIL ESP activated caspases (Caspase 3, Caspase 9 and Caspase 8), up-regulated the pro-apoptotic factors (Bax and Cytochrome c) and down-regulated the anti-apoptosis molecule Bcl-2. Interestingly, pretreatment of Caco-2 cells with apoptosis inhibitor Z-VAD-FMK abrogated and recovered the barrier function of Caco-2 monolayer destroyed by IIL ESP. Furthermore, the Z-VAD-FMK pretreatment also impeded the in vitro larva invasion of Caco-2 monolayer.ConclusionsT. spiralis IIL ESP induced gut epithelial apoptosis, reduced the TJs expression, damaged gut epithelial integrity and barrier function, and promoted larval invasion. These findings provided a basis of further understanding the interaction mechanism between T. spiralis and host gut epithelium, and they were valuable to the development new prevention and therapeutic strategy of early T. spiralis infection.

Project description:BackgroundProteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved.Methodology/principal findingTsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo.ConclusionsTsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

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A novel C-type lectin from Trichinella spiralis mediates larval invasion of host intestinal epithelial cells.

Publications

A novel C-type lectin from Trichinella spiralis mediates larval invasion of host intestinal epithelial cells.

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