Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70 genomic locus

Abstract

Toxoplasma gondii is an important human and veterinary pathogen. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium arsenite and many of the other stressors associated with heat shock protein induction. Heat shock or stress induced activation of a set of heat shock protein genes, is characteristic of almost all eukaryotic and prokaryotic cells. Studies in other organisms indicate that heat shock proteins are developmentally regulated. We have established that increases in the expression of bag1/hsp30 and hsp70 are associated with bradyzoite development. The T. gondii hsp70 gene locus was cloned and sequenced. The regulatory regions of this gene were analysed by deletion analysis using β-galactosidase expression vectors transiently transfected into RH strain T. gondii. Expression was measured at pH 7.1 and 8.1 (i.e. pH shock) and compared to the expression obtained with similar constructs using BAG1 and SAG1 promoters. A pH-regulated region of the Tg-hsp70 gene locus was identified which has some similarities to heat shock elements described in other eukaryotic systems. Green fluorescent protein expression vectors driven by the Tg-hsp70 regulatory region were constructed and stably transfected into T. gondii. Expression of green fluorescent protein in these parasites was induced by pH shock in those lines carrying the Tghsp70 regulatory constructs. Gel shift analysis was carried out using oligomers corresponding to the pH-regulated region and a putative DNA binding protein was identified. These data support the identification of a pH responsive cis-regulatory element in the T. gondii hsp70 gene locus. A model of the interaction of hsp70 and small heat shock proteins (e.g. BAG1) in development is presented.

Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis

“…However, there was no evidence of the presence of 841 a similar element within the loci of the Hsp60 and Hsp90 genes, suggesting that 842 regulation of Hsp70 may be distinct [144]. Nonetheless, it is conceivable that 843 chaperone networks and chaperone-co-chaperone partnerships exist in this parasite.…”

“…A regulatory element that is sensitive to stressful pH conditions was located upstream 840 of the T. gondii Hsp70 gene [144]. However, there was no evidence of the presence of 841 a similar element within the loci of the Hsp60 and Hsp90 genes, suggesting that 842 regulation of Hsp70 may be distinct [144].…”

Cyclic nucleotide kinases and tachyzoite–bradyzoite transition in Toxoplasma gondii

“…PLK strain parasites were co-transfected with 25 μg TUB-CAT plasmid mixed with 100 μg BAG1 β-galactosidase (β-GAL) plasmid (Ma et al, 2004) using standard electroporation conditions with a BTX electroporater (Kim et al, 1993). Stable T. gondii transfectants bearing the chloramphenicol acetyltransferase (CAT) marker driven by the α-tubulin promoter (contained in the TUB-CAT plasmid) were selected for using 20 μM chloramphenicol as previously described (Kim et al, 1993).…”

“…The bradyzoite-specific promoter BAG1 was used to drive β-GAL gene expression (Ma et al, 2004) (Fig. 2(B)).…”

Proliferation of Toxoplasma gondii in inflammatory macrophages in vivo is associated with diminished oxygen radical production in the host cell

“…Some experiments employed modified RH strains that express either yellow fluorescent protein (YFP) behind a tubulin promoter (kind gift of Dr B. Striepen, Univ. Georgia) (Gubbels et al, 2003) or green fluorescent protein (GFP) behind a GRA1 promoter (Ma et al, 2004). For superinfection of YFP-RH-infected mice with GFP-RH, mice infected 5-6 days earlier with YFP-RH were injected i.p.…”