RNAi-mediated silencing of Trichinella spiralis glutaminase results in reduced muscle larval infectivity

Yuan Gao,Xiaoyun Li,Caixia Han,Mingxin Song,Shuang Wang,Shi Meng,Wei Li,Xiao Yang,Xiaoqing Meng

doi:10.1186/s13567-021-00921-1

Abstract

Trichinella spiralis is an important foodborne parasitic nematode distributed worldwide that infects humans and animals. Glutaminase (GLS) is an important gene in the glutamine-dependent acid resistance (AR) system; however, its role in T. spiralis muscle larvae (ML) remains unclear. The present study aimed to characterize T. spiralis GLS (TsGLS) and assess its function in T. spiralis ML AR both in vitro and in vivo using RNA interference. The results indicated that native TsGLS (72 kDa) was recognized by anti-rTsGLS serum at the muscle larvae stage; moreover, an immunofluorescence assay confirmed that TsGLS was located in the epidermis of ML. After silencing the TsGLS gene, the relative expression of TsGLS mRNA and the survival rate of T. spiralis ML were reduced by 60.11% and 16.55%, respectively, compared to those in the PBS and control groups. In vivo AR assays revealed that the worm numbers at 7 and 35 days post-infection (dpi) decreased by 61.64% and 66.71%, respectively, compared to those in the PBS group. The relative expression of TsGLS mRNA in F1 generation T. spiralis ML was reduced by 42.52%, compared to that in the PBS group. To the best of our knowledge, this is the first study to report the presence of the glutamine-dependent AR system in T. spiralis. Our results indicate that TsGLS plays a crucial role in the T. spiralis AR system; thus, it could be used as a potential candidate target molecule for producing vaccines against T. spiralis infection.

Highlights

Trichinella spiralis, a causative agent of trichinellosis, is an important foodborne parasitic nematode which is distributed worldwide [1]
We investigated the functions of T. spiralis GLS (TsGLS) in T. spiralis acid resistance (AR) systems, and TsGLS-specific small interfering RNA (siRNA) were designed to silence the expression of TsGLS in T. spiralis larvae to elucidate the functions of this gene
Effect of RNA interference (RNAi) on the culture medium of muscle larvae (ML) after TsGLS gene silencing After culturing siRNA-881-treated T. spiralis ML in media with pHs of 2.5, 4, 6.6, and 9 for 0.5, 1, and 2 h, we found that the pH values of the culture medium with the initial pH of 2.5 in the siRNA-881 group differed slightly, compared to those in the other groups

Summary

Introduction

Trichinella spiralis, a causative agent of trichinellosis, is an important foodborne parasitic nematode which is distributed worldwide [1]. Trichinellosis is an emerging/ re-emerging disease [1, 2]; T. spiralis can infect various hosts, including humans. Trichinellosis poses public health concerns, and causes economic issues in porcine animal production and food safety [4]. The acid resistance (AR) mechanism exists in several foodborne pathogenic bacteria, including Escherichia coli, and allows their survival in various acidic conditions [5]. Five distinct amino acid-dependent AR systems have been characterized in E. coli, comprising the glutamic acid-, arginine-, lysine-, ornithine-, and glutamine (Gln)-dependent AR systems [6, 7]. The Glndependent AR system is considered highly effective, and relies on L-Gln, one of the most abundant foodborne free amino acids. Upon uptake into E. coli, Gln is converted into L-glutamate (Glu) by acid-activated

Methods

Results

Conclusion