Sequence analysis and characterization of pyruvate kinase from Clonorchis sinensis, a 53.1-kDa homopentamer, implicated immune protective efficacy against clonorchiasis

Abstract

Background: Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK), a crucial glycolytic enzyme, was characterized in this research.

Results: Differences were observed in the sequences and spatial structures of CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria), suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro.

Conclusions: The sequences and spatial structures, molecular mass, and expression profile of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.

Keywords: Clonorchis sinensis; Drug target; Excretory/secretory products; Expression profile; Immune response; Pentamer; Pyruvate kinase; Vaccine candidate.

Fig. 1

Sequence analysis of CsPK. An alignment of the protein sequence of CsPK with those of PKs from other organisms is shown. Clonorchis sinensis (C.s, GAA54498.1), Schistosoma japonicum (S.j, AAW27129.1), Eimeria tenella (E.t, AAC02529.1), Toxoplasma gondii (T.g, BAB47171.1), Plasmodium falciparum (P.f, CAD50538.1), Leishmania mexicana (L.m, CAA52898.2), Trypanosoma brucei (T.b, P30615.1), Trypanosoma cruzi (T.c, EKG02834.1), Mastigamoeba balamuthi (M.b, AAK94944.1), Cryptosporidium parvum (C.p, 4DRS_A), Homo sapiens (H.s, AAA60104.1), Mus musculus (M.m, NP_001093249.1), and Rattus norvegicus (R.n, AAA41880.1). The 3D–domains (N/A/B/C) are marked with vertical lines. The 22 predicted B cell linear epitopes with more trustworthiness are indicated by the black lines above the alignment. The rectangle indicates the PK active site signature. The triangles and open squares indicate the PEP and ADP binding sites, respectively. The binding sites of the sugar, 1-phosphate and 6-phosphate moieties of the allosteric activator fructose-1,6-bisphosphate (F16BP) are indicated by closed squares, open circles and closed circles, respectively. The black arrows and asterisks indicate monovalent cation and divalent cation binding sites, respectively

Fig. 2

Neighbour-joining phylogenetic tree of PKs. The bootstrap values are displayed at the branching point (test of phylogeny by the bootstrap method with 1000 replications). The bar indicates the substitution by p-distance method. The protein sequences were obtained from GenBank and DDBJ. The sequences are as follows: Escherichia coli (AAA24392.1, AAA24473.1), Bacillus subtilis (P80885.2), Arabidopsis thaliana (BAB10461.1), Cryptosporidium parvum (4DRS_A), Plasmodium falciparum (CAD50538.1, AAN35560), Eimeria tenella (AAC02529.1), Toxoplasma gondii (BAB47171.1, KFH06835.1), Trypanosoma brucei (P30615.1), Trypanosoma cruzi (EKG02834.1), Leishmania mexicana (CAA52898.2), Theileria parva (XP_764242.1, XP_764703.1), Saccharomyces cerevisiae (CAA24631.1), Brugia malayi (XP_001898626.1), Hymenolepis microstoma (CDS33796.1), Echinococcus granulosus (CDS23463.1), Echinococcus multilocularis (CDS43052.1), Caenorhabditis elegans (CAA93424.2), Anopheles gambiae (EAA10555.6), Drosophila melanogaster (AAC16244.1), Crassostrea gigas (CAJ28914.1), Clonorchis sinensis (GAA54498.1, GAA58090.1), Opisthorchis viverrini (KER20867.1), Schistosoma japonicum (AAW27129.1), Schistosoma haematobium (KGB40466.1), Schistosoma mansoni (CCD76479.1), Danio rerio (NP_955365.1), Xenopus laevis (NP_001084341.1), Gallus gallus (NP_990800.1), Felis catus (P11979.2), Homo sapiens (AAA60104.1), Mus musculus (NP_001093249.1), Rattus norvegicus (AAA41880.1), Ctenopharyngodon idella (AFY98078.1)

Fig. 3

Expression and apparent Mr. of rCsPK. Proteins were visualized by Coomassie Blue staining. Lane M contains protein molecular weight markers. a Expression and purification of rCsPK. Lysate of E. coli transformed with pET-28a(+)-CsPK without induction (Lane 1) and with induction (Lane 2); supernatant (Lane 3) and precipitate (Lane 4) of lysate of E. coli with pET-28a(+)-CsPK with induction; and purified rCsPK (Lane 5). b In Lane M, the protein bands with known Mr. (in descending order) are thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and BSA (67 kDa). Lane 1, Freshly purified rCsPK; Lane 2, rCsPK stored for 4 weeks at -80 °C with 4 cycles of freezing and thawing

Fig. 4

Western blotting of rCsPK after SDS-PAGE. Blots containing rCsPK were incubated with preimmune mouse serum (Lane 1), a mouse His-tagged monoclonal antibody (Lane 2), mouse anti-rCsPK sera (Lane 3), sera from mice infected with C. sinensis (Lane 4), or mouse anti-CsESPs sera (Lane 5). Blots containing CsESPs were incubated with mouse anti-rCsPK sera (Lane 6) or with preimmune mouse serum (Lane 7). Blots containing total worm extract were incubated with mouse anti-rCsPK sera (Lane 8) or with preimmune mouse serum (Lane 9)

Fig. 5

mRNA and protein levels of CsPK at various life stages of C. sinensis. a Real- time PCR. The β-actin mRNA of C. sinensis was used as an internal control. Semiquantitative analysis was conducted using the 2^-ΔΔCt method. Significant differences in the mRNA levels of CsPK in egg, adult, excysted metacercaria, and metacercaria were observed (P < 0.01). The mRNA level of CsPK in egg was higher than that in adult (56.79-fold, t ₍₂₎ = 17.392, P = 0.003), metacercaria (9.72-fold, t _(2.02) = 15.844, P = 0.004) and excysted metacercaria (5.97-fold, t ₍₄₎ = 14.477, P < 0.001). b Western blotting. Total protein (40 μg) in extracts obtained at each life stage was probed with mouse anti-rCsPK sera, revealing specific immunoreactive protein bands at approximately 53.1 kDa. No corresponding band was detected with preimmune mouse serum (data not shown). c Relative protein levels were analysed using Tanon Gis software. The protein level of CsPK was maximal in eggs, followed by excysted metacercaria, metacercaria, and adults. The protein levels were consistent with the mRNA levels. (*P < 0.05; **P < 0.01; egg vs adult: t ₍₄₎ = 12.950, P < 0.001; excysted metacercaria vs adult: t ₍₄₎ = 16.542, P < 0.001; metacercaria vs adult: t ₍₄₎ = 13.951, P < 0.001; excysted metacercaria vs metacercaria: t ₍₄₎ = -3.680, P = 0.021)

Fig. 6

Immunolocalization of CsPK in C. sinensis. Mouse anti-rCsPK sera and Cy3-conjugated goat anti-mouse IgG were used as the primary and secondary antibodies, respectively. Preimmune mouse serum was used as the primary antibody for the negative controls. Panels (c), (d), (e), and (f) show negative controls. Panels (b), (d), (f), and h are fluorescence microscopic images; the same areas of the samples photographed under white light are shown in panels (a), (c), (e), and g with scale-bars. Panel (b), localization of CsPk in adults; panel h, localization of CsPk in metacercariae. Abbreviations: tg, tegument; e, egg; v, vitellarium; os, oral sucker; vs, ventral sucker; g, genital pore; s, seminal receptacle; i, intestine; ts, testicle; u, uterus;o, ovary; p, pharynx. Scale-bars: a-d, 100 μm; e-h, 10 μm

Fig. 7

ELISA determination of antibody titres and isotypes of IgG elicited by rCsPK. Antibody titres of IgG elicited by rCsPK in rats (a) and mice (b). IgG isotypes elicited by rCsPK in rats (c). *P ≦ 0.001. 2 week: t ₍₆₎ = 6.886, P < 0.001; 4 week: t ₍₆₎ = 27.959, P < 0.001; 6 week: t ₍₆₎ = 19.829, P < 0.001; 8 week: t ₍₆₎ = 19.278, P < 0.001; 10 week: t ₍₆₎ = 6.264, P = 0.001; 12 week: t ₍₆₎ = 17.319, P < 0.001; 14 week: t ₍₆₎ = 16.977, P < 0.001; 16 week: t ₍₆₎ = 15.057, P < 0.001; 18 week: t ₍₆₎ = 37.271, P < 0.001; 20 week: t ₍₆₎ = 48.557, P < 0.001; 22 week: t ₍₆₎ = 40.796, P < 0.001; 24 week: t ₍₆₎ = 32.550, P < 0.001

Fig. 8

Rat anti-rCsPK sera inhibits C. sinensis adult subsistence in vitro. The median subsistence times of C. sinensis adults in the blank control group, the 1:40 preimmune serum group, the 1:80 preimmune serum group, the 1:160 preimmune serum group, the 1:40 anti-rCsPK serum group, the 1:80 anti-rCsPK serum group, and the 1:160 anti-rCsPK serum group were 15, 8, 8, 9, 2, 3 and 4 days, respectively. No significant difference in the rate of survival of the preimmune serum groups was observed at any serum dilution (1:40 preimmune serum group vs 1:80 preimmune serum group: χ ² = 0.01289, df = 1, P = 0.9096; 1:80 preimmune serum group vs 1:160 preimmune serum group: χ ² = 0.09872, df = 1, P = 0.7534; 1:40 preimmune serum group vs 1:160 preimmune serum group: χ ² = 0.1657, df = 1, P = 0.6839). There were significant differences among the other groups in the rate of survival (1:40 anti-rCsPK serum group vs 1:80 anti-rCsPK serum group: χ ² = 8.058, df = 1, P = 0.0045; 1:80 anti-rCsPK serum group vs 1:160 anti-rCsPK serum group: χ ² = 8.092, df = 1, P = 0.0044; blank control group vs 1:40 preimmune serum group: χ ² = 16.15, df = 1, P < 0.0001; blank control group vs 1:80 preimmune serum group: χ ² = 15.54, df = 1, P < 0.0001; blank control group vs 1:160 preimmune serum group: χ ² = 11.39, df = 1, P = 0.0007)