Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein

Abstract

Background: The genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico.

Methods: The 1170 bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively.

Results: The CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite.

Conclusions: The results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines.

Keywords: Apicomplexa; Cystoisosporosis; Invasion inhibition; Protozoa; Recombinant antigen; Swine.

Fig. 1

The nucleotide sequence and deduced amino acid sequence of complete coding region of CSUI_005805 gene. Vertical arrow, signal peptide cleavage site; green shaded, start codon; red shaded, stop codon; wavy underlined, putative protein kinase C phosphorylation sites; underlined, putative N-glycosylation site; gray shaded, histidine rich region

Fig. 2

Schematic representation of predicted transmembrane topology of CSUI_005805 protein. It consists of two transmembrane domains (TM) with extracellular N and C termini and a long cytoplasmic loop extending between TM1 andTM2

Fig. 3

Relative mRNA expression levels of CSUI_005805 in vitro. a Free sporozoites and merozoites. b Intracellular merozoites on day 1, 3 and 6 post-infection (p.i.). The data are displayed as mean ± standard deviation

Fig. 4

Expression of rCSUI_005805. Lane M: molecular weight marker. a Analysis of expressed recombinant protein by SDS-PAGE: uninduced culture (Lane 1); induced culture with 1 mM IPTG for 0.5 h (Lane 2) and 1 h (Lane 3), arrows indicate target protein bands of ~42 kDa. b SDS-PAGE analysis of NiNTA spin column purified rCSUI_005805: elution 1 (Lane 1); elution 2 (Lane 2), arrows indicate faint protein bands of ~25 kDa; naïve merozoite lysate (Lane 3). c Batch purified rCSUI_005805 using NiNTA agarose: stepwise elution (Lanes 1–5), arrows indicate faint protein bands of ~42 kDa. d Immunoblot of batch purified rCSUI_005805 probed with anti-His HRP conjugate (Lane 1)

Fig. 5

Immunoblot analyses of rCSUI_005805 and naïve merozoite lysate. The expressed recombinant protein and crude merozoite lysate were separated by SDS-PAGE and transferred to NC membranes. Lane M: Molecular weight marker (ordinate values describe band sizes in kDa); a Immunoblot of rCSUI_005805 probed with porcine anti-C. suis polyclonal sera (Lane 1) and with porcine pre-colostral sera (Lane 2). b Immunoblot of rCSUI_005805 probed with negative chicken serum (Lane 1) and chicken anti-rCSUI_005805 polyclonal sera (Lane 2). c Immunoblot of insoluble (Lanes 1–3) and soluble fractions (Lanes 4–6) of naïve merozoite lysate probed with porcine anti-C. suis polyclonal sera (Lanes 1 and 4), chicken anti-rCSUI_005805 polyclonal sera (Lanes 3 and 6) and negative chicken sera (Lanes 2 and 5), respectively. d Immunoblot of naïve merozoite lysate (Lanes 1 and 2) and rCSUI_005805 (Lane 3) probed with field sera from chickens vaccinated with HIPRACOX^®

Fig. 6

Localization of CSUI_005805 antigens in merozoites and sporozoites. a–c Wide-field epifluorescence microscope (×63 magnification; scale-bar: 10 μm). d–f Meta confocal laser scanning microscope (×63 magnification; scale-bar: 5 μm). a C. suis merozoites: (a1) Differential interference contrast (DIC); (a2) Localization using A488; (a3) Nuclear staining with DAPI (a4) DIC, A488 and DAPI merged. b Negative control, merozoites probed with negative chicken serum: (b1) DIC; (b2) A488; (b3) DAPI; (b4) merged. c C. suis sporozoites: (c1) DIC; (c2) A488; (c3) DAPI; (c4) merged. d and e Paraformaldehyde-fixed C. suis merozoites: (d1, e1) A488; (d2, e2) DAPI; (d3, e3) merged. f Methanol-fixed C. suis merozoites: (f1) A488; (f2) DAPI; (f3) merged