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Characterization of a Novel Cysteine Protease in Trichinella Spiralis And

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Characterization of a Novel Cysteine Protease in Trichinella Spiralis And Hu et al. Vet Res (2021) 52:113 https://doi.org/10.1186/s13567-021-00983-1 RESEARCH ARTICLE Open Access Characterization of a novel cysteine protease in Trichinella spiralis and its role in larval intrusion, development and fecundity Yuan Yuan Hu, Ru Zhang, Shu Wei Yan, Wen Wen Yue, Jia Hang Zhang, Ruo Dan Liu, Shao Rong Long, Jing Cui* and Zhong Quan Wang* Abstract The aim of this study was to investigate the biological properties of a novel gut-specifc cysteine protease in Trich- inella spiralis (TsGSCP) and its role in larval intrusion, development and fecundity. TsGSCP has a functional C1 pepti- dase domain; C1 peptidase belongs to cathepsin B family. The TsGSCP gene cloned and expressed in Escherichia coli BL21 showed intensive immunogenicity. qPCR and Western blotting revealed that TsGSCP mRNA and protein were expressed at various T. spiralis stages, but their expression levels in intestinal infectious larvae (IIL) were clearly higher than those in muscle larvae (ML), adult worms (AWs) and new-born larvae (NBL). Indirect immunofuorescence (IIF) analysis showed that TsGSCP was primarily located at the outer cuticle and the intrauterine embryos of this parasite. rTsGSCP showed the ability to specifcally bind with IECs, and the binding site is within the IEC cytoplasm. rTsGSCP accelerated larval intrusion into host intestinal epithelial cells (IECs), whereas anti-rTsGSCP antibodies suppressed larval intrusion; the acceleration and suppression was induced by rTsGSCP and anti-rTsGSCP antibodies, respectively, in a dose-dependent manner. When ML were transfected with TsGSCP-specifc dsRNA, TsGSCP expression and enzy- matic activity were reduced by 46.82 and 37.39%, respectively, and the capacity of the larvae to intrude into IECs was also obviously impeded. Intestinal AW burden and adult female length and fecundity were signifcantly decreased in the group of mice infected with dsRNA-transfected ML compared to the control dsRNA and PBS groups. The results showed that TsGSCP plays a principal role in gut intrusion, worm development and fecundity in the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection. Keywords: Trichinella spiralis, cysteine protease, intrusion, fecundity, dsRNA Introduction trichinellosis due to infected pork or pork products Trichinellosis is a worldwide zoonotic parasitic disease were documented in China in 2004–2009 [6]. Because that is principally caused by the consumption of raw or pork from domestic pigs is consumed in high quantities undercooked meat containing Trichinella muscle lar- around the world, porcine Trichinella infection is a major vae (ML) [1]. Mammals, rodents, amphibians, reptiles risk to public health and meat safety [7]. Hence, it is nec- and birds are hosts of Trichinella [2]. Human Trichinella essary to develop preventive vaccines to interdict the infection mainly results from eating infected pork in transmission of swine Trichinella infection and eliminate developing countries [3–5]. Twelve outbreaks of human Trichinella infectious larvae in animals consumed as food [8]. Following ingestion, T. spiralis ML encapsulated in *Correspondence: [email protected]; [email protected] Department of Parasitology, Medical College, Zhengzhou University, host skeletal muscles are released from collagen cap- Zhengzhou 450052, China sules in the stomach by the action of digestive enzymes © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hu et al. Vet Res (2021) 52:113 Page 2 of 16 and activated into intestinal infective L1 larvae (IIL1) In this study, a novel gut-specifc cysteine protease of after exposure to intestinal contents or bile 0.9 h post- T. spiralis (TsGSCP, GenBank: XP_003377240.1) was infection (hpi) [9, 10]. IIL1 recognize, invade and obtained from the T. spiralis draft genome [32]. TsGSCP migrate within intestinal epithelium cells (IECs) and has a functional C1 peptidase (cathepsin B family) establish an intramulticellular niche in the intestinal domain. Te aim of this study was to assess the biological columnar epithelium, where at 10 hpi IIL1 undergo properties of TsGSCP and its role in T. spiralis invasion their frst moulting and develop into IIL2 and then of host intestinal mucosa. moult three times before becoming adult worms (AWs) 10–31 hpi. After female and male AWs mate, pregnant Materials and methods adult females generate the next generation of new-born Experimental animals, Trichinella and cells larvae (NBL), and these NBL pass into the blood cir- Four-week-old female KM mice were purchased from culation, invade skeletal muscles and are encapsulated the Henan Experimental Animal Center. Te Trichinella to complete the lifecycle [11]. Invasion of IECs by IIL1 spiralis isolate (ISS534) used in this experiment was is the most important step in T. spiralis infection and obtained from a domestic pig in Nanyang, Henan Prov- parasitism within the host intestine. Te intestinal epi- ince [33]. IECs were isolated from the small intestine of thelium is the frst natural barrier against T. spiralis normal BALB/c mice [9]. Te negative control cells were invasion and the main interaction site of the nematode C2C12 myoblasts obtained from mouse muscles [34]. and host [12, 13], but the mechanism of IIL1 penetra- Te bacterial strain used in the experiment was Escheri- tion into IECs has not been fully elucidated [14, 15]. chia coli BL21. Identifcation and characterization of IIL1 invasion- related protein molecules in T. spiralis will contribute Collection of worms at various stages and antigen to an understanding of the IEC invasion mechanism preparation and will be valuable to exploit preventive vaccines that Te carcasses of mice infected with T. spiralis were arti- block Trichinella invasion [16, 17]. fcially digested 40 days post-infection (dpi) to obtain Previous studies revealed that when they were coculti- ML [35]. Te IIL and AWs were collected from the small vated with an IEC monolayer, IIL invaded the monolayer intestine at 6 hpi, 3 dpi and 6 dpi [36, 37]. NBL were and produced additional cysteine proteases that entered acquired from 6-day-old females after culturing for 24 h the IECs [18, 19]. Cysteine proteases have been identifed [38]. Te soluble somatic worm antigens in various T. spi- in somatic soluble and excretory/secretory (ES) proteins ralis stages (ML, IIL, AW and NBL) and IIL ES antigens of T. spiralis [20]. Tese cysteine proteases might pro- were prepared as previously described [39, 40]. mote IIL1 penetration of IECs in the process of Trich- inella infection [21–23]. Our previous studies showed Bioinformatics analysis and evolutionary tree construction that several T. spiralis cysteine proteases participate in Te full-length cDNA sequence of the TsGSCP gene was IIL1 invasion, and immune serum against recombinant obtained from NCBI. Bioinformatics analysis software cysteine proteases or RNAi partially suppressed IIL1 (TargetP, TMHMM, SMART, ProtParam, Swiss-Model, intrusion [24, 25]. Recombinant protein vaccination of ProtScale, PSIPred and SignalP) was used to analyse mice led to only partial immune protection against T. spi- and predict its physicochemical properties, such as sig- ralis challenge [26, 27]. Tese results suggested that other nal peptide, subcellular localization, tertiary structure cysteine proteases participate in larval invasion of IECs. of TsGSCP and functional sites [17]. Te amino acid Terefore, it is necessary to characterize other cysteine sequences of TsGSCP were compared with the cysteine proteases and evaluate their roles in larval intrusion, protease from other organisms with BioEdit software development and fecundity in the lifecycle of T. spiralis. [30]. Te GenBank accession numbers of the cysteine Cysteine proteases constitute a superfamily of wide- proteases from other organism are as follows: T. nel- spread proteolytic enzymes in parasitic organisms and soni (KRΧ27624.1), Trichinella sp. T8 (KRZ92878.1), T. play a primary role in the lifecycle of parasites, including nativa (OUC48635.1), T. patagoniensis (KRY19675.1), worm intrusion, migration and development, exsheath- Trichinella sp. T6 (KRX85497.1), T. papuae ing, digestion and degradation of host proteins, and (KRZ80814.1), T. zimbabwensis (KRZ19202.1), T. pseu- immune evasion [28–30]. Te cysteine protease con- dospiralis (KRZ16984.1), Trichuris suis (KFD71793.1), tains cathepsin B, C, F, H, K, L, O, S, V, W and X [31]. Brugia malayi (XP_001898194.1), Caenorhabditis elegans Cathepsin
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