Transcriptional activation of the htrA (high-temperature requirement A) gene from Bartonella henselae

Bacterial htrA genes are typically activated as part of the periplasmic str ess response and are dependent on the extracytoplasmic sigma factor rpoE, A putative promoter region, P1, of the sigma(E)-type heat-inducible promoter s has previously been identified upstream of the htrA gene of Bartonella he nselae. Further analysis of the htrA mRNA by primer extension demonstrated that transcription initiates from pi and a second region downstream of P1, This second promoter region, termed P2, had no sequence identity to sigma(E )-type heat-inducible promoters. Promoter regions were cloned individually and in tandem into pANT3 upstream of a promoterless version of the green fl uorescent protein (GFP) gene (gfpmut3) and transformed into B. henselae by electroporation, The contiguous promoter region containing both P1 and P2 w ere necessary for the optimal transcriptional activation of the htrA gene. Promoter activity at 37 degrees C was distinctively higher than at 27 degre es C. However, thermal induction at 47 degrees C did not increase expressio n of gfpmut3. Invasion of human microvascular endothelial cells (HMEC-1) by B. henselae resulted in the formation of well-defined vacuoles containing clusters of bacteria exhibiting marked expression of gfpmut3 transcribed fr om the P1-P2 region. In addition, a moderate yet significant increase in th e ratio of bacterial GFP to DNA was detected for intracellular bacteria com pared to extracellular bacteria, indicating upregulation of htrA upon invas ion of HMEC-1. The activation of specific genes in the intracellular enviro nment may help us better understand the novel pathogenic mechanisms used by this bacterium.