Molecular cloning, organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii

The obligate intracellular protozoan parasite Toxoplasma gondii has a singl e tubular mitochondrion. During infection, it recruits the host cell's mito chondria abutting to the intracellular vacuole, that contains the parasites . The respective contribution of host and parasitic mitochondria in the int racellular growth of T. gondii remains unknown. Heat shock protein, HSP60 h as been reported in all eukaryotes examined, as an essential chaperone requ ired for the folding and multimeric complex assembly of mitochondrial prote ins. Here, we report the isolation and molecular characterization of two cD NAs corresponding to a single T. gondii gene coding for HSP60. Using a mode l fusion protein, preHSP60-chloramphenicol acetyl transferase (CAT), we dem onstrate that the classical 22 amino acid mitochondrial presequence and the adjacent 32 amino acids of the mature protein are both required for the in vivo import into T. gondii mitochondria. The T. gondii HSP60 gene composed of five introns and six exons is transcribed into two related but differen tly spliced transcripts. Whereas the two transcripts can be detected in bot h developmental stages within the intermediate host, their levels are signi ficantly increased in bradyzoites when compared to tachyzoites. By immunobl ot analysis, the predicted 60-kDa protein corresponding to HSP60 was detect ed in both tachyzoite and bradyzoite forms. Using immunofluorescence assays , the polyclonal antibodies specific to T. gondii HSP60 recognized the mito chondrion in tachyzoites, as expected. In contrast, these antibodies reacte d against two unknown vesicular bodies which are distinct from the classica l mitochondrial pattern in bradyzoites. Taken together, these expression pa tterns of mitochondrial chaperone HSP60 suggests stage-specific induction o f the respiratory pathway in the protozoan parasite T. gondii. (C) 2000 Els evier Science B.V. All rights reserved.