Variant Surface Glycoprotein from Trypanosoma evansi Is Partially Responsible for the Cross-Reaction between Trypanosoma evansi and Trypanosoma vivax

Abstract

Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.

Isolation of two antigens from Trypanosoma evansi that are partially responsible for its cross-reactivity with Trypanosoma vivax

“…The predominant 64 kDa cross-reacting antigen, p64, was purified from the TEVA1 T. evansi isolate (Uzcanga et al, 2002) and was identified as the soluble form of a variant surface glycoprotein from this parasite (Uzcanga et al, 2004). In the present work, we have continued with the isolation and characterization of T. evansi antigens responsible for the cross-reaction between T. evansi and T. vivax.…”

“…Both resins were extensively washed in tandem and the predominant p64 cross-reacting antigen was eluted as a prominent peak in the non-adhering material (data not shown). This 64 kDa-glycosylated polypeptide was previously proven to represent the soluble form of a variant surface glycoprotein from the TEVA1 T. evansi isolate (Uzcanga et al, 2002(Uzcanga et al, , 2004.…”

“…1D). In addition, p64 is a dimer in its native state (Uzcanga et al, 2004) whereas D-II is a monomer (Fig. 3 C).…”

“…Since, the production of T. vivax antigens is a limiting factor (Desquesnes and Tresse, 1996) as this parasite is very difficult to propagate in experimental animal models (Gardiner, 1989), our aim has been to identify and isolate antigens from T. evansi that are responsible for this cross-reaction. Recently, the predominant 64 kDa glycosylated cross-reacting antigen (p64) was purified from the TEVA1 T. evansi isolate (Uzcanga et al, 2002) and identified as the soluble form of a variant surface glycoprotein (VSG) (Uzcanga et al, 2004). Here, we purified two additional proteins from the cytosolic fraction of the same T. evansi isolate, which were proven to be recognized by anti-T. vivax bovine antibodies.…”

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

“…The degree of T. evansi antigens involvement in the cross-reactions is not clear. On one side, at least one T. evansi variant surface glycoprotein (VSG), p64 induces antibody production that recognizes epitopes on the homologue T. vivax VSG (Uzcanga et al 2004). On other side the lower number of variable antigen type (VAT) diversity displayed by Trypanozoon sub-genus, to which T. evansi fits in, compared to T. vivax could be a limitation for crossreactions (Meirvenne 1996).…”