HtrA serine proteases are highly conserved and essential ATP-independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue,Borrelia burgdorferiHtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteinsin vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN-PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn2+ > Cu2+ > Mn2+. Extending this analysis to two other HtrA proteases,E. coliDegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl2 at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases.
This study describes two distinct properties of the HtrA protease from Lyme disease bacteria known as BbHtrA. We determined that BbHtrA remains hexameric upon substrate interaction, unlike DegP, the well-characterized dodecameric homologue fromE. coli. We also demonstrate that BbHtrA, DegP, and the human orthologue HtrA1 are inhibited by physiologic concentrations of zinc. Zinc inhibition is a new tool to investigate this class of proteases and may regulate aspects of virulence of Lyme disease organisms.