Stable transfection of Trypanosoma cruzi epimastigotes with the trypomastigote-specific complement regulatory protein cDNA confers complement resistance

Abstract

Trypanosoma cruzi blood stage trypomastigotes are highly resistant to complement-mediated killing in normal serum. A previously described trypomastigote surface glycoprotein was shown to have binding affinity for human complement components C3b and C4b and restrict activation of the complement cascade, thus preventing lysis of the parasites. Insect stage epimastigotes do not produce detectable levels of this 160-kDa complement regulatory protein (CRP) and are highly sensitive to the lytic effects of complement. Epimastigotes were stably transfected with a T. cruzi expression vector carrying the trypomastigote CRP cDNA and produced fully functional recombinant CRP. The recombinant CRP had binding affinity for C3b, and the transfected epimastigotes were protected from complement-mediated lysis. These results demonstrate for the first time that a developmentally regulated gene of T. cruzi trypomastigotes can be expressed in noninfectious epimastigotes and that production of CRP by epimastigotes is sufficient to confer a virulence-associated trait. Furthermore, these studies demonstrate the critical role that trypomastigote CRP plays in the protection of parasites from the deleterious effects of complement, thus establishing the protein as a virulence factor of T. cruzi.

FIG. 1

Construction of pTEX-BX23 and pTEX-CRP expression plasmids. (A) pTEX-BX23 was constructed by ligating a fragment containing the entire crp coding region and the 53-bp upstream (5′ UT) and 19-bp downstream (3′ UT) sequences into pTEX at the BamHI and XhoI sites. (B) pTEX-CRP was constructed by flipping the orientation of the SacII-KpnI fragment of pTEX-BX23 containing the crp gene and removing the 5′ UT crp sequence. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Neo^R, neomycin phosphotransferase gene.

FIG. 2

Ethidium bromide-stained gel and Southern blot of RT-PCR products derived from transfected T. cruzi epimastigote RNA. (A) Lanes 2 to 4 contain the RT-PCR products from the CRP 2.3 cell line without RT in the reaction mixture (lane 2), the vector-transfected cell line (lane 3), and the CRP 2.3 cell line (lane 4). Lanes 5 to 7 are an autoradiograph of a Southern blot of RT-PCR samples that are the same as those in lanes 2 to 4 but probed with a CRP-specific oligomer. Lane 1 contains ethidium bromide-stained DNA size markers. Sizes in base pairs are indicated at the left. (B) Schematic representation of the full-length CRP-10 cDNA (approximately 3,300 bp), illustrating the positions of the oligomers used in the RT-PCR and the Southern blot in panel A. The RT primer was the antisense sequence of the CRP-10 cDNA at nucleotide 1767. PCR amplification was carried out with a sense oligomer derived from the last 25 nucleotides of the T. cruzi mini-exon sequence and an antisense oligomer beginning at nucleotide 1337 of the CRP-10 cDNA (CRP-20), as described in Materials and Methods.

FIG. 3

Western blot of membrane protein extracts derived from transfected T. cruzi epimastigotes. Protein extracts were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with mouse anti-CRP serum (1:400) (lanes 1 to 3) or normal mouse serum (NMS; 1:400) (lanes 4 to 6). Lanes contained 5 μg of protein derived from epimastigote cell lines transfected with CRP 2.3 (lanes 1 and 4), CRP 2.14 (lanes 2 and 5), or the pTEX vector (lanes 3 and 6). Molecular mass standards (kilodaltons) are shown on the left.

FIG. 4

C3b affinity chromatography of membrane protein extracts derived from transfected T. cruzi epimastigotes. Epimastigotes were metabolically labeled with [³⁵S]methionine, and detergent-solubilized membrane protein extracts were subjected to C3b affinity chromatography. Eluted proteins were fractionated by SDS-PAGE, and the gel was prepared for fluorography and exposed to X-ray film for 24 h. Lanes: 1, C3b-eluted protein from tissue culture-derived T. cruzi trypomastigotes; 2, transfected CRP 2.3 epimastigotes; 3, pTEX-transfected epimastigotes. Molecular mass standards (kilodaltons) are shown at the left.