Gene sequence and transcription differences in 70 kDa heat shock protein correlate with murine virulence of Toxoplasma gondii

Abstract

This study compared the genes encoding cytoplasmic 70 kDa heat shock protein in virulent and avirulent strains of Toxoplasma gondii, to determine whether differences may contribute to the variation in protein expression levels previously reported for this protozoan parasite. A T. gondii PCR probe with homology to Eimeria acervulina cytoplasmic 70 kDa heat shock protein was used to screen a genomic DNA mini-library and isolate the gene from the virulent RH strain. The entire coding region was subsequently amplified from the avirulent ME49 strain by PCR. Alignment of the gene sequences revealed that the virulent RH strain had four copies of a seven-aa repeat unit (GGMPGGM) at the 3′-end of the gene compared with five copies in the avirulent ME49 strain. Comparison of this region among other virulent and avirulent strains revealed that this difference was consistent with virulence. Copy number estimation revealed that this gene is single-copy in both the RH and the ME49 strains. Analysis of mRNA expression revealed a 1.5- to 2-fold increase in transcription of this gene in virulent strains when compared with avirulent strains. For each strain, mRNA was observed at similar levels whether grown in vivo or in vitro. Also, heat-shock treatment of tachyzoites prior to harvest did increase mRNA levels in vitro. This suggests that post-transcriptional regulation of cytoplasmic 70 kDa heat shock protein may occur in T. gondii. Differences in cytoplasmic 70 kDa heat shock protein have been demonstrated at genomic and transcriptional levels in virulent strains compared with avirulent strains, suggesting that this 70 kDa heat shock protein may play an important role in the virulence of T. gondii.