Cloning of the Heat Shock Protein 70 (HSP70) Gene of Ehrlichia sennetsu and Differential Expression of HSP70 and HSP60 mRNA after Temperature Upshift

摘要

Ehrlichia sennetsu is the causative agent of human Sennetsu ehrlichiosis. Heat shock protein 60 (HSP60) and HSP70 (DnaK) are two major bacterial HSPs, and their interaction modulates the stress response. Previously, we cloned and sequenced groEand expressed groEL of E. sennetsu. HSP60 (GroEL) was immunogenic and cross-reactive in Ehrlichiaspp. The present study was designed to (i) characterize the HSP70 gene of this organism and (ii) determine whether the expression of these two HSPs is inducible upon exposure to heat stress. A gene encoding an HSP70 homolog was isolated and sequenced from a gene library. The ehrlichial HSP70 gene encoded a 637-amino-acid protein, which had an approximate molecular mass of 68,354 Da and which was homologous to DnaK of Escherichia coli. A DNA sequence resembling ?35 and ?10 promoter sequences of E. coli dnaK was observed upstream of the ehrlichial HSP70 gene. Alignment of the predicted amino acid sequence with that of E. coli DnaK andBrucella, Salmonella, Borrelia,Chlamydia, and Mycobacterium HSP70s showed 63, 67, 63, 62, 58, and 53% identity, respectively. By reverse transcription-PCR analysis, the mRNA levels of ehrlichial HSP70 and HSP60 were examined after temperature shifts from 28 to 37°C and from 37 to 40°C. HSP70 mRNA induction levels were greater than those of HSP60 mRNA after a 37-to-40°C temperature shift, whereas the reverse was true after a 28-to-37°C temperature shift. Our data suggest that HSP60 and HSP70 may play different roles during transfer from vector temperature to human body temperature and during a febrile condition characteristic of ehrlichial disease. This study also provides a useful model system for examining mRNA expression in obligatory intracellular bacteria.