GRA7, AN EXCRETORY 29 KDA TOXOPLASMA-GONDII DENSE GRANULE ANTIGEN RELEASED BY INFECTED HOST-CELLS

Monoclonal antibody (mAb) TxE2, reactive with Toxoplasma gondii excret ory products, detects an acidic 29 kDa protein (p29) which, in 2D gel electrophoresis, exhibits a migration pattern distinct from those of t he toxoplasmic excretory proteins described so far. The sequence of se ven peptides from tryptic digestion of isolated p29 allowed the design of primers to obtain the coding DNA sequence. The full-length gene wa s amplified from genomic DNA of T. gondii strain BK and the sequence w as identical with that of the corresponding cDNA, providing evidence f or an intron-free gene structure. A single mRNA transcript of 1.3 kb w as detected by Northern blot analysis. The deduced 236 amino acid prot ein contains a putative N-terminal signal peptide, one site of potenti al N-linked glycosylation, and, close to the C-terminus, a further hyd rophobic, putative transmembrane domain. With synthetic peptides spann ing the sequence of p29, the epitope for mAb TxE2 was mapped adjacent to the putative signal sequence. The antigen, which represents almost 0.5% of T. gondii protein, is expressed in strains of all three intras pecies subgroups, and is associated with the parasite dense granules a s demonstrated by immunoelectron microscopy. In tachyzoite-infected ce lls, p29 accumulates within the parasitophorous vacuole and co-localiz es with its delimiting membrane. In bradyzoite-infected cells, p29 is present within the host cell cytoplasm as detected by immunofluorescen ce staining, and, furthermore, in the supernatant of cyst-bearing cell culture lacking extracellular parasites as shown by enzyme-linked imm unosorbent assay (ELISA). Thus, p29 which is named dense granule prote in (GRA)7 may indicate the presence of intracellular toxoplasma. (C) 1 998 Elsevier Science B.V. All rights reserved.