Molecular characterization and transcriptional analysis of the female-enriched chondroitin proteoglycan 2 of Toxocara canis
Abstract
Toxocara canis is an important but neglected zoonotic parasite, and is the causa- tive agent of human toxocariasis. Chondroitin proteoglycans are biological macromolecules, widely distributed in extracellular matrices, with a great diver- sity of functions in mammals. However, there is limited information regarding chondroitin proteoglycans in nematode parasites. In the present study, a female-enriched chondroitin proteoglycan 2 gene of T. canis (Tc-cpg-2) was cloned and characterized. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the transcription levels of Tc-cpg-2 among tissues of male and female adult worms. A 485-amino-acid (aa) polypep- tide was predicted from a continuous 1458-nuleotide open reading frame and de- signated as TcCPG2, which contains a 21-aa signal peptide. Conserved domain searching indicated three chitin-binding peritrophin-A (CBM_14) domains in the amino acid sequence of TcCPG2. Multiple alignment with the inferred amino acid sequences of Caenorhabditis elegans and Ascaris suum showed that CBM_14 domains were well conserved among these species. Phylogenetic ana- lysis suggested that TcCPG2 was closely related to the sequence of chondroitin proteoglycan 2 of A. suum. Interestingly, a high level of Tc-cpg-2 was detected in female germline tissues, particularly in the oviduct, suggesting potential roles of this gene in reproduction (e.g. oogenesis and embryogenesis) of adult T. canis. The functional roles of Tc-cpg-2 in reproduction and development in this parasite and related parasitic nematodes warrant further functional studies.
Introduction
Toxocara canis is an important and highly prevalent in- testinal parasite of dogs and other canids (Sahu et al., 2014; Gasser et al., 2016). A wide range of animals can be infected by ingestion of embryonated eggs or infective larvae. The infective larvae develop and complete their life cycle in canid species, while they arrest developmen- tally in paratenic hosts but migrate to tissues/organs, causing the associated diseases named toxocariasis (Strube et al., 2013). Human toxocariasis is characterized by visceral larva migrans (VLM), neurological toxocaria- sis (NT), ocular larva migrans (OLM) and/or covert/com- mon toxocariasis (CT) (Rubinsky-Elefant et al., 2010). The high seroprevalence of Toxocara infection/exposure in children (Archelli et al., 2014; Cong et al., 2015) and the im- portant relationships with allergic disorders, such as asthma, chronic pruritus and urticaria, have raised public concern (Overgaauw & van Knapen, 2013; Lee et al., 2014). Although treatment with albendazole or mebendazole is effective for visceral toxocariasis, there are still many gaps in our knowledge concerning this disease, which sig- nificantly hinder the development of effective diagnostic tools and interventional strategies (Othman, 2012; Holland, 2015; Poulsen et al., 2015).
Proteoglycans are macromolecules composed of a pro-tein core and glycosaminoglycan chains, and display a great diversity of protein forms (Ruoslahti, 1988; Hardingham & Fosang, 1992). The different chains and their various lengths, as well as their pattern of sulphation, result in the switching of different chain types (Iozzo, 1998) and various functions (Laabs et al., 2007; Kwok et al., 2011). Chondroitin sulphate proteoglycans and heparan sulphate proteoglycans are widely distributed in extracellular matri- ces, and their functional roles in the development of the central nervous system and the response to central nervous system injury in vertebrates have been extensively reported (Yi et al., 2012; Dyck & Karimi-Abdolrezaee, 2015; Miller & Hsieh-Wilson, 2015). In addition, previous studies in the free-living nematode Caenorhabditis elegans indicated that chondroitin proteoglycans play crucial roles in embryonic development and vulval morphogenesis (Hwang & Horvitz, 2002; Izumikawa et al., 2004; Olson et al., 2006).Although a great number of gender-, reproduction- anddevelopment-associated genes of nematodes have been predicted through newly completed genome and tran- scriptome projects, and studied with advanced molecular technologies (Boag et al., 2003; Nisbet et al., 2004, 2008), knowledge about molecular functions and/or biological involvement of chondroitin proteoglycans in parasitic ne- matodes is scant.
Even though six female-enriched chon- droitin proteoglycan genes were identified in previous genome and transcriptome analyses of adult T. canis (Zhu et al., 2015; Zhou et al., 2017) no functions and path- ways were annotated. Hence, in the present study, we cloned and characterized the chondroitin proteoglycan 2 gene of T. canis (Tc-cpg-2), and examined its differential transcription in order to get a better understanding of its potential functional roles in this enigmatic parasite.Adult T. canis worms were collected from naturally in- fected dogs at the Rongchang Campus Animal Hospital of Southwest University, China. Male and female adults were identified based on their morphological features (Urquhart et al., 2003). The germline tissues (including tes- tis, seminal vesicle and vas deferens of male worms, and ovary, oviduct and uterus of female worms), intestine, musculature and cuticle were dissected from some male and female adult T. canis, and snap-frozen in liquid nitro-gen. All parasites and tissue samples were stored at −80° C until use.Total RNA was extracted from the whole body of male and female adult worms, respectively, using Trizol reagent (Invitrogen Corporation, Carlsbad, California, USA). The quality of total RNA was measured using a BioPhotometer (Eppendorf, Hamburg, Germany). M-MLV reverse transcriptase (Promega, Madison, USA) was used to synthesize the first-strand cDNA according to the manufacturer’s instructions. Tc-cpg-2 was amplified by conventional polymerase chain reaction (PCR) with theprimers cpg1 (5′-ATGGAGTTCAGATTCTTCATC-3′) and cpg2 (5′-TCAGTAGGCTTCACCGACCT-3′). The primer sets were designed using Primer Premier 5 software(Lalitha, 2004), based on the nucleotide sequence of Tcan_06538. The PCR reaction contained 2.5 μl of 10 ×- PCR buffer (Mg2+ free), 3.0 μl MgCl2 (25 mM), 2.0 μl deox- ynucleoside triphosphates (dNTP; 2.5 mM), 0.25 μl forward primer (10 μM), 0.25 μl reverse primer (10 μM), 2.0 μl (20–50 ng) DNA template, 0.13 μl rTaq polymerase and14.87 μl sterile water, in a total volume of 25 μl.
The PCRprotocol consisted of 95°C for 4 min; 30 cycles of 95°C for 40 s, 55°C for 30 s and 72°C for 1 min; 72°C for 10 min and holding at 4°C. The PCR products were purified using the EasyPure quick gel extraction kit (Invitrogen Corporation), ligated into the pMD 19-T (simple) vector (Takara Bio, Dalian, China) at 4°C overnight and trans- formed into Escherichia coli DH5α competent cells. The transformed clones were screened using colony PCR and the positive clones were then sequenced by Sangon Biotech (Shanghai, China).The sequences obtained were used for BlastX searching against the non-redundant protein sequences database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The signal peptide prediction was performed using SignalP 4.0 (Petersen et al., 2011). The conserved domains of the pre- dicted amino acid sequence of Tc-cpg-2 were searched against CDD v. 3.14 (Marchler-Bauer et al., 2015). The structure and function of the inferred peptide were pre- dicted using the I-TASSER server (Roy et al., 2010; Yang et al., 2015). In addition, multiple alignment was carried out by aligning the deduced amino acid sequence of Tc-cpg-2 and the predicted chondroitin proteoglycan 2 of other nematodes using MUSCLE, Clustal Omega and MAFFT software (Edgar, 2004; Sievers et al., 2011; Katoh & Standley, 2013).
The phylogenetic relationship of these sequences was analysed by constructing a phylogenetic tree using maximum likelihood in MEGA 4 software(http://www.megasoftware.net/mega4/mega.html).Total RNA was extracted from the testis, seminal vesicle, vas deferens, intestine, musculature and cuticle of male worms and from the ovary, oviduct, uterus, intestine, mus- culature and cuticle of adult female T. canis, using Trizol re- agent (Invitrogen), and measured using a BioPhotometer (Eppendorf). Total RNA was reversely transcribed using PrimeScript™ RT Reagent kit (Takara Bio, Dalian, China) to synthesize the cDNA. The primers cpg3 (5′-ACCCCGACAACGACGACTAT-3′) and cpg4(5′-CGAACGCAAACCCGTATCT-3′) were designedusing Primer Premier 5 software, based on the nucleotide sequence of Tcan_06538. The small subunit of ribosomalRNA (18S) gene was used as an internal reference control, using primers 18S1 (5′-AATTGTTGGTCTTCAACGA GGA-3′) and 18S2 (5′-AAAGGGCAGGGACGTAGTCAA-3′). The qRT-PCR reaction included 10.0 μl SYBR Premix Ex Taq II (Takara Bio), 0.8 μl of forward primer (10μM), 0.8 μl reverse primer (10 μM), 2.0 μl DNA template and6.4 μl sterile water, in a total volume of 20 μl. The qRT-PCR was performed with thermal cycling: 95°C for 30 s, and 40 cycles of 95°C for 30 s and 55°C for 30 s. Three technical re- plicates were performed, and the relative transcriptionlevel was established using the 2−ΔCt method, and pre- sented as x̄± standard deviation (SD).
Results
Molecular cloning was performed to obtain the full- length coding sequence of the Tc-cpg-2 gene. After se- quencing, a 1458-nucleotide open reading frame (ORF) was obtained. Homology searching showed high iden- tities to cpg-2 genes of other nematodes. A 485-amino-acid (aa) polypeptide containing a signal peptide was pre- dicted from the Tc-cpg-2 gene. Three chitin-binding peritrophin-A domains (CBM_14) were identified using conserved domain searching (fig. 1a). In addition, the three-dimensional structural model of predicted TcCPG2 (without signal peptide) was predicted with a TM score of 0.59 ± 0.14 and a root-mean-square deviation (RMSD) of 9.5 ± 4.6 Å (fig. 1b). The closest structural homologue to TcCPG2 was the poly-C9 component of the comple- ment membrane attack complex (PDB-5fmw). The mo- lecular function, biological process and cellular component of TcCPG2 were annotated as ion binding (GO:0043167), cytolysis (GO:0019835) and extracellular space (GO:0005615). The Tc-cpg-2 gene coding TcCPG2 was deposited in the GenBank database (accession no. KU521797).The predicted amino acid sequences of CPG2 of C. ele- gans, Caenorhabditis remanei, C. briggsae and Ascaris suum, as well as the deduced sequence of TcCPG2 were used for multiple alignment analysis. The three CBM_14 do- mains of TcCPG2 were highly conserved with those ofC. elegans, C. remanei and C. briggsae, whereas high vari-ation was found in both of the C- and N-terminal regions (fig. 2). Ten predicted amino acid sequences of CPG2 and the sequence of TcCPG2 were used for the phylogenetic analysis.
A close relationship between T. canis and A. suum, and a relatively distant relationship to species of Trichocephalida and Rhabditidae, were indicated in terms of the protein sequence similarity (fig. 3).The differential transcriptions of Tc-cpg-2 in the germ- line tissues, intestine and body wall of adult T. canis were determined using qRT-PCR. Specifically, extremely high transcription of Tc-cpg-2 was detected in the ovary and uterus, particularly in the oviduct, of the female adult (fig. 4b). Low expression of Tc-cpg-2 was observed in the testis of the male adult, compared with even lower transcription in vas deferens and cuticle of the male adult (fig. 4a), and musculature of both male and fe- male adult T. canis (fig. 4a, b). No transcription was de- tected in the intestine of both sexes. Generally, the transcriptional level of Tc-cpg-2 in the female adult worm was much higher than that in male adult T. canis, particularly in the germline tissues, suggesting its gender- related functional roles.
Discussion
Proteoglycans are versatile components of pericellular and extracellular matrices, with interactive properties with other components of eukaryotic cells. Physically, pro- teoglycans function as molecular organizers of the matrix, playing important roles in keeping the matrix hydrated, in- creasing reaction rates and regulating cell–matrix dynamics (Gallagher, 1989; Hardingham & Bayliss, 1990; Schaefer, 2014). In addition to their conventional physical effects or structural roles, chondroitin proteoglycans also have intri- guing functions in a range of biological processes, such as the development of the central nervous system and wound repair (Morgenstern et al., 2002; Im et al., 2013; Miller & Hsieh-Wilson, 2015). Recent studies in parasites indicated that cell-surface chondroitin proteoglycans play a role in the attachment of parasites to host cells; for in- stance, the adhesion of erythrocytes infected by the malaria parasite to liver cells (Pradel et al., 2002) or placenta (Gamain et al., 2002; Mardberg et al., 2002; Frick et al., 2003; Achur et al., 2008). Multiple female-enriched chondro- itin proteoglycan genes were identified in transcriptome analysis (Zhu et al., 2015; Zhou et al., 2017); however, al- most nothing is known about the roles of proteoglycans in T. canis and related parasitic nematodes.Chondroitin proteoglycans play potential roles in reproduction and/or embryotic development processes. It was reported that chondroitin proteoglycans are important components of extracellular matrices (ECMs), which are es- sential for ovulation and sperm–egg interaction (Camaioni et al., 1996; Johnston et al., 2006). In C. elegans, the possible involvement of chondroitin proteoglycans in early embryo- genesis, embryonic development and vulval morphogen- esis was indicated by disrupting genes involved in glycosaminoglycan biosynthesis (Hwang & Horvitz, 2002; Hwang et al., 2003; Mizuguchi et al., 2003; Izumikawa et al., 2004). It was also found that simultaneous CPG1 and CPG2 are crucial for embryonic cell division in C. ele- gans (Olson et al., 2006). Female-enriched chondroitin pro- teoglycan 2 of T. canis might have similar roles in reproduction and development to that of C. elegans.
In the current study, the alignment analysis showed conserved domains between the sequences of TcCPG2 and CPG2 of C. elegans, indicating conserved biological functions. In addition, high transcription levels of Tc-cpg-2 were detected in female germline tissues (fig. 4b), which can be supported by the distribution of chondroitin proteoglycans in the gonads and uterus, as well as in oocytes, spermatheca and fertilized eggs of hermaphrodite C. elegans (Mizuguchi et al., 2003). Nevertheless, the relatively high transcription of this gene in the oviduct of T. canis differed from the tissue ex- pression of CPG2 in C. elegans (Mizuguchi et al., 2003). This might be associated with the differences in structure of the reproductive tract and related biological processes of fertilization (Sato et al., 2008) between these two spe- cies. Although recent knowledge of proteoglycan biology has expanded from structural compounds to signalling molecules (Fthenou et al., 2006; Whitten & Miller, 2007; Schaefer & Schaefer, 2010; Gubbiotti & Iozzo, 2015), the molecular roles of chondroitin proteoglycans remain un- clear in T. canis and related parasitic nematodes.
In conclusion, this female-enriched chondroitin proteo- glycan 2 was predicted to be associated with reproduction and embryonic development; in particular, to be linked to oogenesis and embryogenesis. Although further function- al studies, for instance, RNA-mediated interference, are required to confirm these findings, the exciting prospects of gender-biased proteoglycans could enhance our molecular knowledge of T. canis and related parasitic nematodes, and suggest important drug targets for con- trolling this A-485 enigmatic zoonotic parasite.