| The initial phase of Trichinella spiralis infection,known as the intestinal or adult stage,is crucial in establishing infection within the host.During this stage infectious larvae invade the intestinal epithelial cells,undergo molting and development until they reach sexual maturity,mate,and oviposition.Thus,breaching the intestinal barrier is a critical step for T.spiralis to establish successful parasitism,which is essential for the parasite’s survival and reproduction.Previous studies have demonstrated that upon infection,the T.spiralis can cause physical damage(such as penetrating the intestinal wall)and/or chemical irritation or allergic reactions induced by the excretion and secretion products(ES),reducing the integrity of the intestinal physical barrier.The composition of T.spiralis ES is complex,including certain protease molecules have been reported to hydrolyze the tight junctions(TJs)porteins forming the physical barrier of the intestine,thereby helping T.spiralis invade.However,whether there are other non-protease molecules in ES exerting similar biological functions remains unknown.Thus,this study focuses on proteins that are specifically expressed and copiously secreted during the adult or intestinal phase of T.spiralis infection,aiming to explore their roles and molecular mechanisms during this phase.T3223-8(Genbank:AAY32322.1),identified by our research group in previous studies,is specifically expressed during the adult stage.Immunofluorescence experiments have revealed that T3223-8 is found in the cuticle,stichosome of the worm and in the intestinal tissue of T.spiralis-infected mice.As a secretory protein playing a key role in T.spiralis invasion into the intestine,it’s not yet known whether T3223-8 assists T.spiralis in invading intestinal epithelial cells(IECs)by damaging the physical barrier of the intestine,and the molecular mechanism of T3223-8 causing this damage is the main investigation of the study.Initially,bioinformatics was used to analyze the gene and amino acid sequences,secondary/tertiary structure information,and predicted conserved domains of T3223-8to understand the protein better.It was found that T3223-8 belongs to the DNase II family of proteins but lacks a typical catalytic site.By comparing the gene sequences of DNase II from other species and constructing a phylogenetic tree,it was discovered that T.spiralis DNase II forms a distinct evolutionary branch,suggesting different properties and biological functions from the DNase II family proteins in other species.Immunofluorescence revealed that T3223-8 is mainly distributed on the cuticle and intestinal tissue of adult worms.RNA interference(RNAi)targeting T3223-8 leads to a decrease in the ability to invade IECs and cause cell damage,which provides evidence that T3223-8 helps T.spiralis invasion into IECs,promoting its parasitism.A subsequent experiment uses Caco-2 to create an intestinal barrier model,assessing the impact of T3223-8 on the barrier.Incorporating WN10,a protein that is also specifically expressed during the intestinal phase of T.spiralis,as a control,and using a cocktail of protease inhibitors to deactivate the enzymatic activity in ES,transepithelial resistance(TEER)values and permeability were measured across different experimental groups,establishing that,apart from protease molecules,effector molecules like T3223-8 within T.spiralis ES can participate in damaging the intestinal barrier.Results from immunofluorescence and Western blot(WB)experiments further validate this finding and confirm a connection between intestinal barrier damage and reduced expression of TJs proteins.The study then set out to dissect the molecular mechanism of T3223-8 regulating the expression of TJs protein and concluded that,as estimated,as many as 30% of genes may be under the control of miRNA.Thus,in this section of the study,a miRNA microarray associated with intestinal barrier function was utilized to analyze and screen miRNAs.Differential expression of miRNAs prior to and post T3223-8 stimulation in Caco-2 colonic cells was identified,revealing the downregulation of miR-142-3p and upregulation of miR-142-5p.By establishing cell lines that overexpress/suppress miR-142-3p/5p,it was found that miR-142 could have an impact on TEER and permeability,aggravating or alleviating the damage T3223-8 does to the intestinal barrier.Using online prediction tools like Target Scan 6.0 and miRanda as well as a dual-luciferase reporter assay,it was determined that the target gene of miR-142-3p is MYLK and the target gene of miR-142-5p is CLDND1(also known as claudin-1).In vitro and in vivo experiments also confirmed that miR-142-3p regulates expression of TJs protein via the MLCK/MLC pathway,affecting the integrity of the Caco-2 cell barrier.On the other hand,miR-142-5p regulates the function of the intestinal barrier through the negative regulation of CLDN1 expression.In summary,T3223-8 induces changes in the expression of miR-142-3p/5p in Caco-2 cells,exploiting the interaction between host cell miRNAs and target genes to disrupt the function of the intestinal epithelial barrier.Specifically,miR-142-3p activates the MLCK/p MLC pathway which reduces expression of TJs proteins(including ZO-1 and occludin),while miR-142-5p downregulates CLDN1 expression.In other words,miR-142-3p and miR-142-5p work in tandem to promote the disruption of the intestinal barrier.This study provides a new perspective on how T3223-8 uses host cell miRNA to regulate expression of TJs proteins,promoting invasion of T.spiralis into the intestines.It also suggests that miR-142-3p and miR-142-5p could be novel molecular diagnostic markers in trichinellosis,while T3223-8 could be considered a candidate molecule for developing a vaccine.These findings not only deepen researchers’ understanding of the mechanism of T.spiralis invasion into the intestines but also offer new insights for trichinellosis control and prevention. |
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