Characterisation and Recognition by Immune Hosts of a Sheep Nematode Parasite Teladorsagia circumcincta Chitinase

Characterisation and


Introduction
Teladorsagia circumcincta is a mucosal browser and resides in the abomasa of the ruminants. The parasite has a direct life-cycle where the eggs laid by the adult worms are passed on to the pasture through faeces and eggs develop into the infective stage larvae (L3), which are ingested and reside in the abomasa of the ruminants and develop into adult worms. Parasitic nematode worm infection is one of the biggest health problems for farmed ruminants worldwide. Parasitic worm infections are harmful to a host animal for many reasons and cause costly production losses and if left untreated, animals can die causing further economic loss to farmers.
The control and productivity losses caused by parasitic nematodes cost the New Zealand livestock industry ~$700 million annually. Currently, farmers rely on the use of anthelmintics to control parasitic nematodes, however resistance of parasites to one or more of these agents is now widespread. Recent industry-funded surveys in New Zealand found that 64% of sheep farms and 94% of beef farms now have parasites that are resistant to at least one of the anthelmintics [1]. It is really important to understand worm biology and look for the targets that are essential for the worm survival.
Chitinase catalyses the hydrolytic cleavage of the ß-1, 4-glycoside bonds present in biopolymers of N-acetylglucosamine, particularly in chitin. Chitinases are widely distributed in living organisms and are found in fungi, bacteria, parasites, plants and animals. The chitinolytic enzymes are also characterised based in their enzymatic action on chitin substrates. Endochitinases are the enzymes catalysing the random cleavage at internal points in the chitin chain whereas exochitinases catalyse the progressive release of acetylchitobiose from the non-reducing end of chitin.
Chitinases perform a variety of function depending on the organism they are present in, for example, in bacteria chitinases are mainly involved in the nutritional processes [2,3] whereas in yeast and fungi, these enzymes participate in morphogenesis [4,5]. In animals and plants, chitinases primarily play a role to defend the organism against infections [6][7][8] and regulate innate immunity and tissue function. In parasites, chitinase is a significant component of the eggshell and play a vital role in egg hatching [9].
Chitin is one of the most abundant polysaccharide and in nematode parasite comprises cuticles, egg-shell, pharynx and microfilarial sheath [10][11][12]. Nematode chitinases consists of multiple genes and it is believed that the enzymes have additional roles in the nematode life cycle because of the presence of stage-specific gene expression Chitinase was detected in the excretory/secretory (ES) protein of the root-knot nematode [13]. Helminth chitinase are induced during T helper-type responses and contribute to asthma, fibrosis and helminth immunity [14,15]. The chitin metabolism can provide unique targets for parasite control because chitin is not found in vertebrates. Because of being central in the chitin metabolism, chitinase has potential as a vaccine candidate. Mice vaccinated with chitinase DNA resulted in partial protection against Onchocerca volvulus [16]. Similarly, rabbits and mice vaccinated with recombinant chitinase-like proteins provided protection against mite Sacroptes scabiei and hard tick Haemaphysalis longicornis respectively [17,18].
The aim of the present experiments was to determine the fulllength sequence of chitinase in the sheep abomasal nematode parasite Teladorsagia circumcincta, express the protein, structural analysis and antigenicity of the recombinant protein.

Materials and methods
All chemicals used in these experiments were purchased from the Sigma Chemical Co. (Mo, USA) unless stated. Use of lambs for parasite culturing and harvesting adult worms for molecular biology studies has been approved in protocol # 13502 by the AgResearch Grasslands Animal Ethics Committee.

Parasite culture and collection
Pure cultures of T. circumcincta were obtained by passaging larvae through sheep and adult worms recovered as described previously [19].

RNA isolation and cDNA synthesis
RNA was isolated from adult worms and first strand cDNA synthesised from 1 ug total RNA using the iScript Select cDNA Synthesis Kit (Bio-Rad) as as described previously [19].

Cloning and expression of T. circumcincta recombinant TciCHT in E. coli
A partial T. circumcincta chitinase sequence (TELCIR_01681) was obtained from AgResearch's internal database. In order to obtain the full length chitinase gene sequence, Rapid Amplification of cDNA Ends (RACE) using the SMARTer RACE cDNA Amplification Kit (Clontech) was carried out. Both 3' end and 5' RACE primers were designed but failed to get a full-length gene. Nested PCR reactions were carried out using SL1 and SL2 primers to detect the presence of longer transcripts. The gene sequence was sent to GenScript (Hong Kong) for gene synthesis and insertion into PUC57. The TciCHT gene were cloned, using protocols described previously [20] into the expression vector AY2.4. Restriction enzymes Ndel and Notl were used in cloning and the resulting protein was N-terminal His tagged recombinant.
E. coli strain BL21 (DE3) transformed with AY2.4 TciCHT as described previously [21] was grown in Luria Broth (LB). L-Arabinose was added as inducer and the culture grown for an additional 2h at 30 oC and 250rpm. Bacteria were harvested as described before. Briefly, The pellet was weighted and resuspended in 10ml per gram of pelleted bacteria of equilibration buffer (20mM sodium biphosphate, 0.5M NaCl, 20mM Imidazole, pH 7.4). Protease inhibitors were added to the suspension, which was then passed through, the chamber of a MP110 Microfluidizer ® (Microfluidics, USA) seven times consecutively under ice, at 20,000psi to ensure the full lysis of E.coli as recommended by the manufacturer. The crude lysate was centrifuged at 15,000g for 20min at 4 o C to remove all cell debris and the supernatant collected and filtered through a 0.22µm to insure the removal of further impurities.
Recombinant TciCHT was expressed as totally insoluble protein and the protein was purified and folded as inclusion bodies as described [22]. Briefly, purified recombinant poly-histidine TciCHT was obtained by Fast protein liquid chromatography (FPLC) under native conditions using from a Ni-NTA column (Qiagen), completed with a BIO-RAD chromatography system (Bio-Rad, USA). Sodium bi-phosphate buffer was used as an equilibration buffer, sodium biphosphate containing 20mM imidazole and 8M urea as the wash buffer and sodium bi-phosphate containing 500mM imidazole and 8M urea as elution buffer. The recombinant TciCHT was purified as inclusion bodies, therefore, dialysed in the buffer containing 8M, 6M, 4M, 2M and no urea for 12h in each buffer at 4 o C. The protein concentration was determined by the Nanodrop (A280nm assay) using extinction coefficient (92610M-1cm-1) and molecular weight (34KDa).

Purification and gel Electrophoresis
Recombinant TciCHT was produced as recombinant polyhistidine protein and was obtained by FPLC under native conditions using a Ni-NTA column (Qiagen), and a Biologic DUO-FLOW BIO-RAD chromatography system (Bio-Rad, USA) as described before [22].

Protein Structure Modelling
CHT sequences from several closely related helminth species including H. contortus were collected from NCBI. Protein alignments were performed using the Muscle multiple alignment option in Geneious 8 (Biomatters Ltd) with the Blosum 62 similarity matrix. Iterative threading assembly refinement (I-TASSER) [23], was used to construct a structural model of TeciCHT. For each target, I-TASSER simulations generate a large ensemble of structural conformations, called decoys. The confidence of each model is quantitatively measured by the C-score that is calculated based on the significance of threading template alignments and the convergence parameters of the structure assembly simulations. Another important metric is TMscore which is estimated from C-score and is used for measuring the similarity of two protein structures/ Scores higher than 0.5 assumes the parent structure and modelled protein share the same fold while below 0.17 suggests a random nature to the produced model.

Chitinase assay
Recombinant chitinase activity was measured as described. The kit contains three different substrates to measure endo-and exo-chitinase activity. Each substrate was dissolved in DMSO and diluted 1:20 in the assay buffer (100 mM citric acid, 200 mM sodium phosphate, pH 5.5) prior to the assay. The assay started after the addition of recombinant TciCHT and incubated for 30 min at 37 ˚C. The reaction was stopped by the addition of stop solution (500mM sodium carbonate, 500 mM sodium bicarbonate, pH 10.5) and the liberated 4-MU was measured at 450nm.

Host Recognition
To test for the presence of antibodies in the blood and saliva that react with the recombinant enzyme, saliva and serum samples taken from parasite-exposed and -naïve sheep as described previously. The pooled serum and saliva samples used for ELISA were collected from 18 male Romney lambs 6-7 months-old and previously exposed in the field to multiple species of parasites and had developed immunity to T. circumcincta infection. TciCHT (5 µg/ml) was immobilised onto ELISA plates (Maxisorp, Thermo Scientific) overnight. Free binding sites were then blocked with Superblock (Thermo Scientific, USA) and then incubated with serial dilutions (200-to 6400-fold for serum or 20-to 160-fold for saliva) in ELISA buffer for 2h at room temperature. Bound serum immunoglobulins were then detected with rabbit antisheep Ig-HRP (Dako, Denmark), diluted 1:5000 by incubation for 1h at 37 °C and the colour developed with 3,3',5,5' tetramethylbenzidine (AppliChem, Germany). Saliva IgA was detected with rabbit antisheep IgA-HRP, which was diluted, incubated and the colour developed, as described for serum Ig.

TciCHT gene sequence and structure
The 912 bp full length T. circumcincta cDNA sequence has been The predicted 3D structure of TciCHT is shown in Figure 2. It has the highest structural similarity with 5WUP which is insect group III chitinase (CAD1) from Ostrinia furnacalis [24]. The best model predicted by I-TASSER has a C-score 1.36 and a TM-score of 0.90 ± 0.06 which indicates high quality of the prediction. Figure 2 shows the model quality in terms of Z-score by ProSA-web. The plot shows the Z-scores of all experimentally determined protein chains in current PDB and the position of the predicted TciCHT model.

Recombinant protein expression
A number of varying conditions were used in the trial expression and based on which maximal production of functional recombinant TciCHT was obtained in the E. coli strain BL21 (DE3) when expression was induced with 0.2% L-arabinose for 3h at 37 º C. The recombinant TciCHT was expressed as inclusion bodies and the inclusion bodies were treated using urea. The protein was dialysed in decreasing urea concentrations to facilitate the folding. Recombinant TciCHT was purified as N-terminal His-tagged protein with weight of about 34 kDa (Figure 3). The presence and the purity of recombinant TciCHT were confirmed by Western blotting.

TciCHT assay
No enzyme activity was detected with either substrate over pH 6-10 or enzyme concentration between 50-250 µg.

Host recognition
Recombinant TciCHT was recognised in by antibodies in saliva and serum samples from the parasite-exposed animals whereas that was not the case of the samples collected from parasite-naïve animals ( Figure 4).

Discussion
This study showed the close relationship between a T. circumcincta chitinase (TciCHT) to that from other helminth homologues.
TciCHT gene, which encoded 912 bp was cloned, expressed in E. coli.
Recombinant TciCHT consisted of 30 4 amino acid and the protein was recognised by antibodies using ELISA in the saliva and serum from the sheep that were immune to parasites, but not nematodenaïve animals.
A 912 bp full length cDNA sequence encoding T. circumcincta chitinase (TciCHT) was amplified from adult T. circumcincta cDNA, cloned and expressed in E. coli. The 304 amino acid TciCHT protein expressed in E. coli was typical of CHT identified and characterized in several helminths. The TciCHT protein had 87% homology to H. contortus homologue and 65-80% similarity to other nematode and trematode homologues ( Figure 1). Our analysis showed a high similarity of the TciCHT protein with CHT form other closely related species. We identified the most likely TciCHT model and the analysis revealed high similarity with insect group III chitinase (CAD1) from Ostrinia furnacalis.
No enzyme activity was detected with either of the three substrates or varying pH or enzyme concentrations. The recombinant TciCHT protein was expressed in E. coli as inclusion bodies and lacked biological activity. Proteins expressed as inclusion bodies may not be folded correctly and be functional enzymes [26][27][28]. Although, the TciCHT sequence appears to be truncated with both 3' and 5' ends incomplete, his doesn't seem to be the case as both 3' and 5' RACE was performed, which failed to detect any longer sequence ( Figure 1). It is interesting to note that TciCHT fully matched with the sequence available in the database indicating the possibility of a shorter sequence compared to other helminth CHT sequences.
Previous studies on the potential of fungal chitinase have shown very promising results and the enzyme has captured and completely destroyed nematode eggs [29] and animals vaccinated with a   recombinant chitinase resulted in high levels of protection [30][31][32]. Native T. circumcincta CHT is part of worm's ES products, highly antigenic and antibodies in both serum and saliva from field-immune sheep recognised recombinant TciCHT in an ELISA (Figure 4). These findings are very promising and further studies will validate the protective efficacy of recombinant TciCHT.

Summary
A 912 bp cDNA encoding Teladorsagia circumcincta chitinase was cloned and expressed in Escherichia coli. Multiple alignments of the protein sequence of TciCHT with homologues from other helminths showed good similarity to the other helminth CHT. Recombinant TciCHT was recognised by antibodies in both serum and saliva from field-immune sheep in ELISA.