RNAi-mediated silencing of Trichinella spiralis serpin-type serine protease inhibitors results in a reduction in larval infectivity

Nana Yi,Mingxu Liu,Pengcheng Yu,Yixin Lu,Zhaokun Liu,Chang Liu,Lijia Wu,Jingzhe Guan

doi:10.1186/s13567-020-00860-3

Abstract

Trichinella spiralis serpin-type serine protease inhibitors (TsSPIs) are expressed in adult worms (AW), newborn larvae (NBL) and muscle larvae (ML) of T. spiralis, with the ML stage demonstrating the highest expression level. This study aims to determine TsSPI functions in larval viability and invasion of intestinal epithelial cells in vitro, as well as their development, survival, and fecundity in vivo via RNAi. TsSPI-specific siRNAs and dsRNA were transfected into ML by incubation. The silencing effect of TsSPI transcription and expression was determined using qPCR and western blot, respectively. After incubation in 60 ng/μL dsRNA–TsSPI for 3 days, larval TsSPI mRNA and protein expression levels were reduced by 68.7% and 68.4% (P < 0.05), respectively. dsRNA-mediated silencing of TsSPI significantly impacted larval invasion into intestinal epithelial cells in vitro but did not affect the survival rate of larvae. After challenge with dsRNA–TsSPI-treated ML, mice exhibited a 56.0% reduction in intestinal AW burden and 56.9% reduction in ML burden (P < 0.05), but NBL production of female AW remained the same (P > 0.05). Our results revealed that RNAi-mediated silencing of TsSPI expression in T. spiralis significantly reduced larval infectivity and survival in the host but had no effect on the survival rate and fecundity. Furthermore, TsSPIs have no effect on the growth and reproduction of parasites but may be directly involved in regulating the interaction of T. spiralis and the host. Therefore, TsSPIs are crucial in the process of T. spiralis larval invasion and parasite survival in the host.

Highlights

Trichinellosis is a widespread food-borne parasitic zoonosis whose route of infection is through the ingestion of raw or undercooked meat containing infective muscle larvae (ML) of Trichinella [1]
Specific small interfering RNA (siRNA)‐ or Double-stranded RNA (dsRNA)‐mediated suppression of T. spiralis Serine protease inhibitors (TsSPI) mRNA expression The qPCR results showed that the TsSPI gene was 52.6%, 28.1%, and 18.2% of the relative transcription level compared with the control group in ML treated with 1, 2 and 3 μM siRNA-986, respectively (P < 0.05) (Figure 2A)
RNA interference (RNAi) has been widely used in various fields of eukaryotic biology because of its simple operation and strong repeatability in C. elegans [37, 38], especially when organisms are unsuitable for classical genetic methods that directly evaluate gene function

Summary

Introduction

Trichinellosis is a widespread food-borne parasitic zoonosis whose route of infection is through the ingestion of raw or undercooked meat containing infective muscle larvae (ML) of Trichinella [1]. 11 million people have been infected by this nematode worldwide [2, 3]. Trichinellosis is considered an emerging/re-emerging disease [4]. Trichinellosis is not under control in endemic areas because this disease is widely distributed worldwide [5]. Trichinella spiralis is an intestinal nematode that infects more than 150 mammalian species [6]. When contaminated meat is ingested, the encapsulation of T. spiralis ML is broken down by gastric juice, ML is liberated in the host’s stomach, and bile activates ML to change to intestinal infectious larvae (IIL). The evading mechanism has attracted considerable attention from researchers because T. spiralis can escape

Objectives

Methods

Results

Conclusion