Schistosome japonicum Proteins that Interact with the Gynecophoral Canal Protein Identied Using a Yeast Two-hybrid System

Background: The large amount of eggs produced by mature female worms not only induce major pathological damage to hosts of schistosomes but also lead to disease transmission. Mature female schistosome worms need constant pairing contact with a male partner as male signaling is indispensable to female growth, development, and reproduction. The gynecophoral canal protein (GCP), a cell-surface glycoprotein that is gender-specic to the male worm, plays a potential role in the interaction between males and females and in stimulating female development and maturation. Methods: In this study, a yeast two-hybrid cDNA library of Schistosome japonicum (Sj) parasites 18 days post-infection was constructed; the SjGCP gene was inserted into a pGBKT7-BD bait plasmid and used as a bait protein to screen for its molecular interactions using a yeast mating procedure. Results: Twenty-four prey proteins that interacted with the SjGCP were selected after excluding false positives; the interactions between two of these, SjLGL and SjColV, and SjGCP were veried by co-immunoprecipitation. Conclusions: The proteins that interacted with SjGCP were identied as being associated with growth, development, and reproductive functionality in S. japonicum.


Introduction
Schistosomiasis is an acute and chronic disease caused by blood ukes (trematode worms) of the genus Schistosoma, which results in severe pathological damage to both human and domestic animal hosts and causes substantial economic losses in many developing countries. Estimates show that at least 290.8 million people required preventive treatment for schistosomiasis in 2018 [1].
Unlike other trematode parasites, the blood ukes of the family Schistosomatidae are gonochorous; they have evolved separate sexes or dioecy, and the sex of an individual is determined in the zygote through a chromosomal mechanism [2]. Male schistosome worms are larger than the females, more muscular, and have a ventral groove called the gynecophoral canal in which females reside for maturation, mating, and egg production [3]. These dioecious worms exhibit unique reproductive biology: the female worms need to continuously pair with males to maintain sexual maturation [4,5]. The stimuli from the male worms play important roles in the growth and reproductive development of female worms [6], and these stimuli are independent of sperm transfer and fertilization [4,[7][8][9]. The male worms are proposed to deliver speci c molecules to female worms during pairing; the molecules associated this "magic male factor" have been unveiled in studies of the gynecophoral canal protein (GCP) [10].
GCP is a cell-surface glycoprotein that is speci cally expressed in the gynecophoric canal of male schistosomes (the site of direct interaction between the mating pair) and widely distributed on the surface of mated females following transfer from the male [11][12][13]. Structurally, GCP lacks a transmembrane domain, but has short, conserved repeat regions with sequence similarity to Fasciclin I, a developmentally regulated neural cell adhesion protein [13][14][15][16]. Fasciclin I-like proteins play important roles in cells, mediating adhesion, migration and differentiation [17][18][19][20]. Periostin, the mammalian homologue of Fasciclin I, also activates the Akt/PKB-and FAK-mediated signaling pathways [21,22].
Interestingly, the transcription pro le of GCP in schistosomes exhibits peak expression at a time that coincides with worm pairing [2]. Moreover, GCP is regulated via transforming growth factor-β (TGF-β)dependent signaling in Schistosoma mansoni (Sm), providing evidence for a role of GCP in male-female interactions and the male-stimulated reproductive maturation of the female schistosome [23]. Studies of S. japonicum have revealed that GCP is necessary for pairing to occur between male and female worms [24]. Although the biochemical activity of GCP has not yet been clearly de ned, there is accumulating evidence for its participation in male-female interaction in these worms [10].
Protein-protein interactions regulate many key biological processes, including cell-cell interactions, cell motility, signal transduction and vesicular tra cking and are fundamental to cell function [25,26]. The yeast two hybrid (Y2H) assay is a technique that enables the rapid identi cation of several putative interacting proteins for a given protein of interest [27]. The great advantage of the Y2H assay, which is performed in vivo, is that the proteins being tested are more likely to be in their native conformations, leading to increased sensitivity and accuracy of detection [27,28]. The Y2H system is both simple and highly e cient, and commonly used to screen for interacting proteins.
In the present study, we performed a Y2H screening procedure to identify proteins that interact with SjGCP, and the interactions were veri ed through yeast mating and co-immunoprecipitation.

Parasites and animals
The Chinese strain of S. japonicum was maintained in Oncomelania hupensis at the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (SVRI, CAAS). Freshly shed cercariae were obtained by exposing infected snails to light. The numbers and viability of the cercariae were determined prior to infection using a light microscope. Male New Zealand rabbits (6 weeks old and 2.0-2.5 kg, purchased from the Shanghai Experimental Animal Center, Chinese Academy of Sciences) were used to collect S. japonicum worms. To obtain the worms, the coverglass method of shaving the abdominal skin was used. The rabbits were each exposed to approximately 5,000 cercariae. The worms were collected from the infected rabbits at 18 days post-infection through hepatic-portal perfusion. This technique has been fully described previously [29]. The worms were washed three times in phosphate buffered saline Construction and identi cation of the pGBKT7-BD-SjGCP bait plasmid The pGBKT7-BD-SjGCP bait plasmid was constructed and identi ed as previously described [30]. Brie y, total RNA was extracted from S. japonicum worms using TRIzol reagent (Thermo Fisher Scienti c, Waltham, MA, USA) and cDNA was reverse transcribed from the total RNA using a PrimeScript RT reagent kit (Takara, Japan). With reference to the SjGCP sequence in GenBank (accession no: AF519183), the open reading frame of the SjGCP gene was ampli ed by PCR from the cDNA, based on the speci c primers (forward) 5′-ATCCATGGGCATGGAATCAATTCGGAATCACTCC-3′ and (reverse) 5′-GTCCTGCAGCGTTTATATTCATCTTGAAATGGTG − 3′(the Nco I and Pst I restriction sites are underlined). The PCR product was ligated into the pGBKT7-BD plasmid digested with the same restriction enzymes. The recombinant pGBKT7-BD-SjGCP plasmid was con rmed by double restriction enzyme digestion and sequencing (Sangon Biotech, Shanghai, China) and retransformed into the yeast strain Y187 with the aid of the Yeastmaker Yeast Transformation System 2 kit according to the manufacturer's protocol (Clontech, USA). The recombinant pGBKT7-BD-SjGCP plasmid in the Y187 culture on SD/-Trp agar plates was extracted using an EasyYeast Plasmid Extraction kit (Clontech, USA) and resequenced.
The expression of BD-SjGCP fusion proteins in Y187 was analyzed by western blotting with Gal4 DNA-BD monoclonal antibodies. The auto-activation of the BD-SjGCP fusion protein was tested in Y187 and AH109 yeast cells on SD/-Trp/X-α-Gal, SD/-His/-Trp/ X-α-Gal and SD/-Ade/-Trp/X-α-Gal agar plates, and the toxicity of the BD-SjGCP fusion protein was analyzed by comparing the growth curve of the Y187 yeast cells with the pGBKT7-BD-SjGCP plasmid and an empty pGBKT7-BD vector.
Construction of a Y2H cDNA library for S. japonicum 18 days post-infection Total RNA from S. japonicum 18 days post-infection was extracted using TRIzol reagent (Invitrogen, USA) and treated with DNaseI (Takara, Japan) to eliminate any contaminating genomic DNA. Then, a Y2H cDNA library was constructed using a homologous recombination method according to the instructions of the Matchmaker™ Library Construction and Screening Kit. Brie y, single stranded cDNA was synthesized from the 18 day total RNA using oligo dT primers, and double-stranded cDNA (ds cDNA) was obtained by LD-PCR ampli cation with 5′and 3′amplimers using an Advantage® 2 PCR Kit. The ds cDNA was further puri ed using a CHROMA-SPIN TE-400 column to limit very small inserts in the library. The puri ed ds cDNA and linearized pGADT7-Rec AD cloning vector with denatured Herring Testes Carrier DNA were transformed into competent AH109 yeast cells. The positive transformants cultured on SD/-Leu plates at 30 °C for 3-6 days were harvested to form the Y2H library. At the same time, transformants at dilutions of 10 1 , 10 2 , 10 3 and 10 4 were seeded on 100 mm SD/-Leu plates to calculate the transformation e ciency according to the following equation: transformation e ciency (cfu/ug DNA) = [clone number (cfu) × total suspension volume (µL)] / [plating volume (µL) × dilution factor × amount of DNA used (ug)]. The harvested libraries from the 10 1 , 10 2 , 10 3 and 10 4 dilutions were then seeded on 100 mm SD/-Leu plates to calculate the library titer as follows: library titer (cfu/ml) = (colonies on SD/-Leu × dilution factor) / volume plated (ml).
To calculate the recombination rate of the library and analyze the average insert size, 96 colonies were randomly selected from the library and ampli ed by PCR with a Matchmaker LD-insert screening amplimer. The cycling conditions were as follows: 94 °C for 15 min, followed by 35 cycles at 94 °C for 1 min, 60 °C for 55 s, and 72 °C for 3 min. Speci c primers (Table 1) for six known S. japonicum genes [SjCyclophilin C (GQ403664.1), SjMagonashi (GQ403668.1), SjGCP (AF519183), SjCyclophilin B (GQ403665.1), SjZFP1 (GQ901167.1) and Sj32 (AY429343.1)] were used to sh the target genes from the library to check the representativeness of the library. Table 1 Primer sequences for the ampli cation of six S. japonicum genes SjGCP interactive protein screening The yeast mating method was used to screen for proteins that interact with SjGCP. Brie y, the AH109 yeast strain containing the cDNA library from 18 day S. japonicum was co-cultured with a Y187 yeast strain with the pGBKT7-BD-SjGCP bait plasmid. The mated culture was plated on SD/-Ade/-His/-Leu/-Trp (QDO) agar plates and incubated for 3-5 days at 30 °C. Simultaneously, 100 µl of the transformants at dilutions of 10 1 , 10 2 , 10 3 and 10 4 were seeded on SD/-Leu, SD/-Trp and SD/-Leu/-Trp 100 mm agar plates to calculate the mating e ciency and screened clones as follows: mating e ciency = (cfu/mL of diploids × 100)/(cfu/mL of AH109 partner); screened clones = cfu/mL of diploids × resuspension volume (mL). Then, the director clone on QDO agar plates was passaged on QDO-supplemented X-α-Gal (QDO/Xα-Gal) agar plates three times for screening.
The monoclonal clones selected by repeated passages on the QDO-supplemented X-α-Gal agar plates were picked out, and the bait and prey protein expression plasmids were extracted using the yeast plasmid extraction kit. Yeast plasmids were identi ed by PCR using pGADT7 universal vector primers, and those that met the requirements were further transformed into E. coli DH5α competent cells and then selected on LB agar plates (containing 100 µg/ml ampicillin) to enrich the prey plasmids for sequencing.
The obtained sequences were subjected to a BLAST search against the National Center for Biotechnology Information (NCBI) databases to annotate the functions of the corresponding genes.

Retesting the interactions in yeast by yeast mating
Reverse-hybridization was used to con rm the interaction between SjGCP and the potential prey proteins.
Competent cells of yeast Y187 and AH109 were prepared according to the Clontech protocol, and the bait plasmid pGBKT7-BD-SjGCP, unrelated protein plasmid pGBKT7-BD-Lamin and empty vector plasmids pGBKT7-BD and pGBKT7-BD-53 were transferred into yeast Y187 cells, plated on SD-Trp culture plates, and cultured at 30 °C for 3-6 days. Each potential prey protein plasmid (pGADT7-AD-prey protein) as well as the empty vectors pGADT7-AD, pGADT7-AD-Lamin C and pGADT7-AD-RecT were transformed into yeast AH109 cells, plated on SD-Leu culture plates, and cultured at 30 °C for 3-6 days. Monoclonal AH109 and Y187 yeast cells grown on culture plates were mated in a 0.5 ml culture medium of 2 × YPDA, shaken at 30 °C for 20-24 h and the mating mixtures were cultured on SD/-Leu/-Trp plates for 3-6 days ( Table 2). The diploid clones grown on the SD/-Leu/-Trp plates were then streaked on SQDO/X-α-Gal and cultured at 30 °C for 5 ~ 6 days.

Retesting the interaction in mammalian cells by coimmunoprecipitation
To further validate the interaction of SjGCP with selected target proteins, the prey protein's gene sequence was subcloned into pCMV-Myc using the enzymes S I and Xho I from the pGADT7-AD-Prey to construct pCMV-Myc-Prey. The open reading frame of SjGCP was cloned into the p3xFLAG-CMV plasmid to obtain p3xFLAG-CMV-SjGCP. The pGADT7-AD-Prey and p3xFLAG-CMV-SjGCP plasmids were then co-transferred into HEK293 cells. Meanwhile, pCMV-Myc-Prey and p3xFLAG-CMV, and p3xFLAG-CMV-GCP and pCMV-Myc, were co-transferred into HEK293 as negative controls. The expression of the prey protein and SjGCP (and the interaction between them) in the HEK293 cells was detected by co-immunoprecipitation (Table 2). Brie y, after 48 h of culture, the HEK293 cells were harvested using non-denaturing cell lysis buffer. The supernatant was transferred to new tubes and the concentration of the total protein was determined using a BCA assay. The total protein (500 µg) was mixed with 10 µg of primary Red anti-Flag M2 beads and shaken on a rotating shaker at 4 °C overnight. Finally, the beads and immunocomplexes were washed with PBS, eluted by boiling in sample buffer and detected by western blotting using anti-Flag monoclonal mouse antibody (1:1000) and anti-Myc monoclonal mouse antibody (1:1000). Twenty micrograms of total protein were used as the input.

Construction and identi cation of the pGBKT7-BD-SjGCP bait plasmid
The complete coding sequence of SjGCP (1,933 bp) was obtained by PCR ampli cation. Subsequently, the ampli ed fragments were subcloned into the vector pGBKT7 using Nco I and Pst I. The recombinant plasmid pGBKT7-BD-SjGCP was retransformed into Y187 and cultured on defective culture plates with SD/-Trp. Western blot analysis indicated that the BD-SjGCP fusion proteins were successfully expressed in Y187 yeast cells, did not exhibit auto-activity in Y187 and AH109 yeast cells and were not harmful to the yeast cells [31].

cDNA library construction S. japonicum at 18 days postinfection
The high integrity total RNA (A 260/A 280: 1.86A) extracted from 18-day post-infection S. japonicum were used to construct the Y2H cDNA library. The transformation e ciency of this library was 1.07 × 10 5 cfu/ug PGADT7-Rec and the library capacity was 2.5 × 10 6 pfu/mL. Ninety-six positive clones were randomly selected and identi ed by PCR using the Matchmaker LD-insert screening amplimer. The results indicated that the majority of the inserts ranged from 0.25 to 2.0 kb in size (Fig. 1). After puri cation using a Chroma Spin TE-400 column, the fragments smaller than 200 bp were eliminated, which effectively avoided the presence of very small inserts and non-recombinant clones in the library. All six gene-speci c primers were able to amplify the target genes from the library (Fig. 2). One S. japonicum eggshell protein gene (the messenger RNA of which should be in mature female worms) was not ampli ed from this library (data not shown). These results showed that the library was well represented and met the requirements for the subsequent Y2H library screening.

SjGCP interactive protein screening
The cultures mated with the library strain (AH109) and reporter strain (Y187) were plated on 100 mm QDO plates and SD/-Leu, SD/-Trp and SD/-Leu/-Trp plates at dilutions of 10 1 , 10 2 , 10 3 and 10 4 . The colonies were calculated, and the mating e ciency was found to be 20.92%; the number of screened clones was 4.1 × 10 7 cfu. A total of 522 colonies from the QDO plates were repeatedly passaged on QDO/X-α-Gal agar plates, and 383 mated clones were obtained. The yeast mating e ciency was determined to be 20.92% and the number of screened clones was 4.1 × 10 7 cfu.
The pGADT7-AD-prey plasmids from the 383 mated clones were extracted and identi ed by PCR using pGADT7 universal vector primers. They were re-transfected into DH5 and the 131 prey plasmids enriched in E. coli were sequenced and revealed exogenous insertion sequences. A total of 45 unique gene sequences were obtained after bioinformatic analysis.

Retesting the protein interactions in yeast by yeast mating
In order to exclude false positives, the pGADT7-AD-prey plasmid corresponding with these 45 unique gene sequences were re-transfected into AH109 yeast cells to reverse-hybridize with Y187 yeast cells that included pGBKT7-BD-SjGCP to verify their interactions. The interactions between SjLGL and SjGCP are shown in Fig. 3. Finally, 24 proteins that interacted with SjGCP in the yeast system were identi ed (Table 3).

Retesting the protein interactions in mammalian cells by coimmunoprecipitation
To further determine whether the interaction between the prey and SjGCP occurred in vitro, coimmunoprecipitation was performed in HEK293T cells co-expressing SjGCP-FLAG and prey-Myc. The recombinant plasmid pCMV-Myc-prey was constructed using S I and Xho I, and SjGCP was cloned into the p3xFLAG-CMV plasmid (Fig. 4A). Two prey proteins, SJCHGC06122 (SjLGL) and SJCHGC09290 (SjCol V) were selected to test their interaction with SjGCP. The results showed that SjGCP-FLAG could be co-immunoprecipitated with anti-FLAG antibodies or anti-Myc when co-expressed with SjLGL-Myc, demonstrating an interaction between SjGCP-FLAG and SjLGL-Myc in the cells (Fig. 4B).

Discussion
Schistosomiasis is one of the most important parasitic diseases in the world in terms of public health impact, second only to malaria. There are ve schistosome species most often associated with human infections; the three clinically most important and highly studied species are S. mansoni, S. haematobium and S. japonicum [32,33]. The major pathological damage induced by schistosomiasis is caused by the large amount eggs deposited in the host's tissues. These eggs can induce severe granulomatous and cause transmission of schistosomiasis following their release into the environment [34]. Therefore, blocking the maturation and oviposition of female worms is particularly important to the control of schistosomiasis.
As dioecious parasites, female schistosomes must maintain constant pairing contact with a male partner to induce differentiation of their reproductive organs. The co-clasping of male and female schistosomes is key to the sexual maturation of females, and male stimuli are indispensable to the growth and development of females [35]. Female worms that have been recovered from unisexual infections display stunted growth and undeveloped reproductive systems when compared with their counterparts from bisexual infections [8,36]. Separation of established mating pairs results in the rapid degeneration of the vitelline gland and ovary, a decline in DNA synthesis associated with vitelline cell production and a reduction in the size of the female parasite [7], suggesting that schistosomes have developed a system by which male signals either directly or indirectly control female development and maturation [35,[37][38][39][40][41]. All of these phenomena indicate that male and female cohabitation is extremely important to sexual reproduction. Therefore, exploring the regulatory factors affecting the pairing of male and female schistosomes and their mechanisms of action is of great interest. Although the mechanisms underlying the action of GCP, a key molecule transferred between males and females, are not well understood, there is evidence for its participation in male-female interactions [2, 10-13, 23, 24]. In the present study, SjGCP was constructed as a bait protein, which was applied in a Y2H system. A Y2H cDNA library of S. japonicum at 18 days post-infection was also successfully constructed and the screening of proteins that interacted with SjGCP was performed using a yeast mating procedure. A total of 45 unique gene sequences were identi ed within 131 sequenced prey plasmids. Eventually, 24 proteins were found to interact with SjGCP after veri cation through yeast mating to exclude false positives. The interactions between two of them and SjGCP were then veri ed by co-immunoprecipitation.
Yeast two-hybrid technology has been widely used in recent years as a technical platform for discovering and studying the interactions between proteins in living cells. Co-immunoprecipitation is a powerful and simple method for detecting protein-protein interactions, and as a form of protein a nity chromatography, co-immunoprecipitation has su cient sensitivity to detect weak interactions and provide a reliable means of identifying protein-protein interactions when used in combination with a Y2H system. In this study, among the 24 proteins that interacted with SjGCP in the yeast system, the interactions between SjGCP and SjLGL (SJCHGC06122) and SjCol-V(SJCHGC09290) were veri ed using co-immunoprecipitation. These experiments indicated that the interactive proteins screened out in this study were reliable.
Among the 24 interactive proteins, four (SJCHGC09290, SJCHGC05265, SJCHGC09378 and SJCHGC00545) belonged to the collagen family of proteins and were constituents of the extracellular matrix structure. SJCHGC04226 has procollagen-lysine 5-dioxygenase activity, SJCHGC05668 is a membrane-associated protein, SJCHGC00472 is an enolase, SJCHGC00694 is an actin, SJCHGC06072 is twin lin, an actin-binding protein, SJCHGC00443 is a cytoplasmic fatty acid-binding protein (FABP), SJCHGC06074 is a UDP-glucose 4-epimerase, and SJCHGC06122 is a lethal giant larvae homolog protein (LGL). These proteins were found at the tegument of S. japonicum [42][43][44][45]. SJCHGC09129 (heat shock protein HSP60) and SJCHGC05222 (ubiquitin-conjugating enzyme E) are intra-cellular chaperones for other proteins, and they can covalently attach to other cellular proteins. These proteins have the basis for interacting with SjGCP proteins either at a speci c location or while functioning. Studies have shown that some interacting SjGCP molecules such as SjFABP, SjLGL, and SjColV affect S. japonicum growth, development, and embryonic activity [44,46].
In this study, the Y2H system was used to select proteins that interact with SjGCP, and the results indicated that the ndings were reliable. Four proteins that were found to interact with SjGCP remain unknown or unannotated, even though genome sequencing and functional analysis of S. japonicum started in 2009 [47]. As the functions of these unannotated proteins are further elucidated, the mechanisms imparted by SjGCP on the growth and development of female S. japonicum worms will gradually be revealed, but the effects of Schistosome males on females remain unclear and require further study. Abbreviations S. japonicum: Schistosoma japonicum; GCP: gynecophoral canal protein Sm: Schistosoma mansoni; Y2H: yeast two hybrid; TGF: transforming growth factor; PBS: phosphate-bufered saline; HSP: heat shock protein; LGL: lethal giant larvae; FABP: fatty acid-binding protein; ColV: collagen alpha 1 (V).

Declarations
Ethics approval and consent to participate Not applicable

Consent to publish
The author con rms: the work described has not been published before; it is not under consideration for publication elsewhere; its publication has been approved by all co-authors; its publication has been approved by the responsible authorities at the institution where the work is carried out.

Availability of data and materials
All data generated or analyzed during this study are included in this article.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by grants from the Chinese National Natural Science Foundation (no.