Characterization of Schistosoma japonicum tetraspanning orphan receptor and its role in binding to complement C2 and immunoprotection against murine schistosomiasis

Abstract

Background: Schistosomiasis remains an important global public health problem, as millions of people are at risk of acquiring infection. An ideal method for sustainable control of schistosomiasis would be to develop an efficient vaccine. Schistosomes can survive in the host vascular system by immune evasion, regulating the host complement cascade. Schistosoma japonicum tetraspanning orphan receptor (SjTOR) is a complement regulator, which is a tegument membrane protein. To date there is no experimental evidence to explain the function of SjTOR.

Results: We cloned the first extracellular domain of the SjTOR (SjTOR-ed1) gene and expressed the gene in Escherichia coli. The expression level of SjTOR in different developmental stages of S. japonicum was assessed by quantitative real-time RT-PCR. Western blotting showed that recombinant SjTOR-ed1 (rSjTOR-ed1) could be recognised by schistosome-infected mouse serum. Immunolocalization indicated that the protein was mainly distributed on the tegument of the parasite. Haemolytic assays and ELISA revealed that rSjTOR-ed1 could inhibit complement hemolysis and bind to complement C2. Purified rSjTOR-ed1 emulsified with ISA206 adjuvant could induce a significant reduction of worm burden from 24.51 to 26.51%, and liver egg numbers from 32.92 to 39.62% in two independent trials in mice.

Conclusions: The results of this study indicated that rSjTOR-ed1 could inhibit complement hemolysis and bind to complement C2, and it is a potential vaccine candidate that protects against S. japonicum infection.

Keywords: C2-binding receptor; Schistosoma japonicum; Tetraspanning orphan receptor; Vaccine.

Fig. 1

ClustalX alignment of the amino acid sequences of SjTOR (Q5DC12), SmTOR (C4QM85), ShTOR (Q9BLM6), and TcTOR (Q5J7P3). The peptide sequences shown to bind C2 are boxed. Regions with high identity and similarity among TOR sequences are indicated by black and grey columns. The first extracellular domain is shaded in green (AA 49–167)

Fig. 2

Analysis of SjTOR expression in different development stages. Samples throughout the 11 developmental stages of S. japonicum and between male and female adult worms were analysed by real-time PCR. Data were normalised against level of an internal housekeeping control gene (α-tubulin)

Fig. 3

Analysis of expression and antigenicity of rSjTOR-ed1. a Expression and purification of SjTOR-ed1 in E. coli. Cell extracts and fractions from E. coli BL21 (DE3) transformed with pET28-SjTOR-ed1 were separated by 12% SDS-PAGE. Lanes A and B: total extract of a clone before and after induction with one mM IPTG; Lanes C and D: supernatant and inclusion bodies, respectively, after lysis; Lane E: rSjTOR-ed1 protein purified from inclusion bodies through Ni-NTA His-binding resin. b Western blot analysis of the antigenicity of rSjTOR-ed1. Lane M: molecular mass markers; Lanes A, B and C: purified rSjTOR-ed1 was probed with anti-rSjTOR-ed1 mouse serum (positive control), serum from mice infected with S. japonicum (experiment group), and normal mouse serum (negative control), respectively

Fig. 4

Localisation of SjTOR in 35-day-old S. japonicum and Cercariae. a-d Sections and cercariae were probed with serum from naive mice (a and c, negative control) or mice serum against rSjTOR-ed1 (b and d)

Fig. 5

Immunohistochemical localisation of SjTOR in 35-day-old S. japonicum and Eggs. a-d The sections were detected by DAB, the positive staining of DAB is brown. Parasite slides were probed with serum from naive mice (a, negative control) or mice serum against rSjTOR-ed1 (b). The positive liver slides were probed with serum from naive mice (c, negative control) or mice serum against rSjTOR-ed1 (d)

Fig. 6

Haemolytic assays to determine the anticomplementary activity of rSjTOR-ed1. a The inhibition rate of hemolysis with increasing protein concentration. b The ability of pre-challenge anti-SjTOR-ed1 serum to inhibit the action of rSjTOR-ed1

Fig. 7

An ELISA assay for detecting the binding of rSjTOR-ed1 to C2. Plates were coated with rSjTOR-ed1 or Albumin Bovine V

Fig. 8

Kinetics of specific anti-rSjTOR-ed1 IgG induced in mice immunised with rSjTOR-ed1. Mice were injected with rSjTOR-ed1 and 206 adjuvant in PBS. Serum samples were obtained from each group at weeks 0, 2, 4, 6 and 12, and were analysed by ELISA. Asterisks indicate significant difference between the rSjTOR-ed1 immunised group and the 206 adjuvant control group (**P < 0.01)