Identification of the interaction between MAPK1 and Eimeria acervulina serine protease inhibitor: a preliminary functional study

Abstract

Eimeria spp. can invade different intestines of chickens. Among them, Eimeria acervulina (E. acervulina, Ea) is the most virulent, and it is characterized by disruption of the intestinal nutrient uptake mechanism, leading to weight loss and even death. As a result, coccidia have caused a heavy burden on the poultry industry [1]. Currently, understanding the invasion mechanism of E. acervulina in host cells is the basis for developing the most effective preventive method for coccidiosis. However, the specific mechanism of E. acervulina invasion is unclear, so the interactions between parasite and host cells need to be studied in depth. Serine thiol proteinase inhibitors widely exist in bacteria, viruses and parasites. It can protect against the dissolution of host proteins and the development of pathogens in the process of pathogen invasion [2]. The serine protease inhibitor (SERPIN) of Toxoplasma gondii, which has the ability to inhibit trypsin activity, was the first SERPIN to be studied in parasites. SERPIN in T. gondii may play an important role in protecting against the degradation of host intestinal proteases and reducing the ability of host proteins to hydrolyse T. gondii [3]. SERPIN in T. gondii can also promote the growth of tachyzoites in the host. Subsequently, a 79-amino acid protease inhibitor, SERPIN, which can completely inhibit the activity of bacterial subtilisin, was found in Neospora caninum [4]. SERPIN plays an important role not only in parasite physiology but also in the interactions with the host. Real-time quantitative PCR analysis at all developmental stages of E. tenella showed that SERPIN1 is highly expressed in sporozoites [5]. Previous studies have shown that the secreted SERPIN protein in sporozoites may also regulate the host immune pathway. In Schistosoma mansoni, SERPIN can inhibit neutrophil proteases and regulate the degradation of tissues to promote the migration of parasites in the host [6]. SERPIN secreted by parasites can also be used to protect themselves from degradation by host proteases, thus manipulating the host response to parasites. Studies have shown that they are mainly involved in physiological processes such as blood sucking, digestion, reproduction and immune response and affect the interaction between parasites and hosts [7]. SERPIN from Trichinella spiralis (TsSPI) is not directly involved in the growth and reproduction of parasites but regulates the interaction between T. spiralis and its host to a certain extent. TsSPIs can regulate the polarization of macrophages and subsequently affect the balance among host inflammatory factors to regulate the host immune response and create a favorable environment for the colonization of Trichinella spiralis in the host [8]. Taeniasis solium SERPIN can play a biological role by participating in the inflammatory and apoptotic pathways of the host [9]. Previous studies have shown that SERPIN plays important roles during host-cell invasion, and 66 related proteins that interact with E. tenella SERPIN on the host have been preliminarily screened [10]. However, few studies have focused on the binding partner of Ea-SERPIN. To identify the ligand-binding partner molecules that may play an important role in the invasion process of E. acervulina, a yeast two-hybrid system was used to screen the associated proteins from the yeast complementary DNA (cDNA) library of chicken duodenal epithelium cells using Ea-SERPIN as bait. In this study, a close genetic relationship was identified between E. acervulina and E. maxima and between E. acervulina and T. gondii. The protein homology of SERPIN between E. acervulina and E. maxima was 87%, and that between E. acervulina and Toxoplasma gondii was 43% (Supplementary Figure S1). The yeast cDNA library of chicken duodenal epithelium cells in the pGADT7 vector was constructed using a Matchmaker Library Construction and Screening kit (Clontech, Palo Alto, USA) as the prey. Preys containing a Gal4 activation domain (AD prey) were transformed into the yeast strain Y187. The efficiency of transformation and the size of the insert fragment satisfied the quality requirements of the yeast library. For yeast two-hybrid screening, SERPIN was amplified by polymerase chain reaction (PCR) using the forward primer 5′-CCC CATATGATGGCATTATTAAGTAAATTAACTCG-3′ and the reverse primer 5′-CCCCTGCAGTTACTGCTGTGCAGCTGTCGGGTCAG-3′ from E. acervulina cDNA and then ligated into the NdeI-PstI sites of pGBKT7 as a bait. The recombinant plasmid was transformed into Y2H GOLD yeast cells, and the transformants were separately grown on plates containing minimal yeast medium without tryptoph

1. Introduction

Eimeria spp. can invade different intestines of chickens. Among them, Eimeria acervulina (E. acervulina, Ea) is the most virulent, and it is characterized by disruption of the intestinal nutrient uptake mechanism, leading to weight loss and even death. As a result, coccidia have caused a heavy burden on the poultry industry [1]. Currently, understanding the invasion mechanism of E. acervulina in host cells is the basis for developing the most effective preventive method for coccidiosis. However, the specific mechanism of E. acervulina invasion is unclear, so the interactions between parasite and host cells need to be studied in depth.

Serine thiol proteinase inhibitors widely exist in bacteria, viruses and parasites. It can protect against the dissolution of host proteins and the development of pathogens in the process of pathogen invasion [2]. The serine protease inhibitor (SERPIN) of Toxoplasma gondii, which has the ability to inhibit trypsin activity, was the first SERPIN to be studied in parasites. SERPIN in T. gondii may play an important role in protecting against the degradation of host intestinal proteases and reducing the ability of host proteins to hydrolyse T. gondii [3]. SERPIN in T. gondii can also promote the growth of tachyzoites in the host. Subsequently, a 79-amino acid protease inhibitor, SERPIN, which can completely inhibit the activity of bacterial subtilisin, was found in Neospora caninum [4]. SERPIN plays an important role not only in parasite physiology but also in the interactions with the host. Real-time quantitative PCR analysis at all developmental stages of E. tenella showed that SERPIN1 is highly expressed in sporozoites [5]. Previous studies have shown that the secreted SERPIN protein in sporozoites may also regulate the host immune pathway. In Schistosoma mansoni, SERPIN can inhibit neutrophil proteases and regulate the degradation of tissues to promote the migration of parasites in the host [6]. SERPIN secreted by parasites can also be used to protect themselves from degradation by host proteases, thus manipulating the host response to parasites. Studies have shown that they are mainly involved in physiological processes such as blood sucking, digestion, reproduction and immune response and affect the interaction between parasites and hosts [7]. SERPIN from Trichinella spiralis (TsSPI) is not directly involved in the growth and reproduction of parasites but regulates the interaction between T. spiralis and its host to a certain extent. TsSPIs can regulate the polarization of macrophages and subsequently affect the balance among host inflammatory factors to regulate the host immune response and create a favorable environment for the colonization of Trichinella spiralis in the host [8]. Taeniasis solium SERPIN can play a biological role by participating in the inflammatory and apoptotic pathways of the host [9].

Previous studies have shown that SERPIN plays important roles during host-cell invasion, and 66 related proteins that interact with E. tenella SERPIN on the host have been preliminarily screened [10]. However, few studies have focused on the binding partner of Ea-SERPIN. To identify the ligand-binding partner molecules that may play an important role in the invasion process of E. acervulina, a yeast two-hybrid system was used to screen the associated proteins from the yeast complementary DNA (cDNA) library of chicken duodenal epithelium cells using Ea-SERPIN as bait.