Molecular cloning of the 82-kDa heat shock protein (HSP90) of Toxoplasma gondii associated with the entry into and growth in host cells

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Abstract

Among the monoclonal antibodies (mAb) against Toxoplasma gondii, mAb Tg485 specifically reacted with an 82-kDa cytoplasmic protein of tachyzoites. The protein was secreted from extracellular tachyzoites, but was not released into the parasitophorous vacuole after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg485. The full-length cDNA was amplified by the 5′-RACE method and sequenced. The deduced amino acid sequence of the 82 kDa protein reacting with Tg485 revealed a polypeptide of 708 amino acids showing significant homology to the heat shock protein 90 (HSP90) family of other organisms, especially to those of apicomplexan species. Treatment with geldanamycin, a drug known to interfere with HSP90 function, did not affect the secretion of TgHSP90 from extracellular tachyzoites, but the entry of the tachyzoites into host cells and the intracellular growth of the parasite were significantly disturbed.

Section snippets

Materials and methods

Parasite and preparation of excretory/secretory protein. The RH strain of T. gondii was maintained by peritoneal passage in Balb/c mice. Prior to use, tachyzoites were purified by centrifugation over 40% Percoll (Amersham–Phamacia Biotech, Uppsala, Sweden) in PBS solution [26]. Purified tachyzoites (3 × 108) were incubated at 37 °C for 1 h under mild agitation in 1.0 ml Hank’s balanced salt solution (Gibco-BRL, Rockville, MD). After centrifugation for 5 min at 6000 rpm, the supernatant was stored as

Antigen recognized by mAb Tg485

While screening mAbs against crude extracts of T. gondii tachyzoites, we identified a mAb (Tg485) reacting with an 82 kDa antigen. Tg485 also blotted the antigen in the excretory/secretory proteins, which implied that the antigen was secreted from the tachyzoites before entry into host cells (Fig. 1).

By immunofluorescence microscopy with Tg485, fluorescence dispersed in the cytoplasm of the extracellular tachyzoites (Fig. 2A), and a similar pattern was observed in the intracellularly growing

Discussion

We report here the identification and molecular characterization of TgHSP90, a novel cytoplasmic heat shock protein of T. gondii. TgHSP90 was identified using a mAb Tg485, which recognized an 82 kDa tachyzoite protein on Western blot and localized to the cytoplasm by immunofluorescence microscopy. TgHSP90 was secreted before the entry of tachyzoites into host cells but not into the parasitophorous vacuole (PV) or PV membrane (PVM). The sequence of TgHSP90 contained a highly hydrophobic and

Acknowledgements

This study was supported by Grant No. R01-2002-000-00422-0 from the Basic Research Program of the Korea Science and engineering Foundation.

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