CLONING AND ANALYSIS OF A BORRELIA-BURGDORFERI MEMBRANE-INTERACTIVE PROTEIN EXHIBITING HEMOLYTIC-ACTIVITY

We cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli, The haemolytic activity was erythrocyte-species specific, with progres sively decreasing activity for erythrocytes from horse, sheep, and rab bit, respectively. Genetic analysis of the haemolytic determinant reve aled two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31, blyA encodes a predic ted alpha-helical 7.4 kDa protein with a hydrophobic central region an d a positively charged C-terminus, which is structurally homologous to a large group of pore-forming toxins with cytolytic activity, blyB en codes a soluble protein which stabilized BlyA and enhanced haemolytic activity, While the majority of BlyA in E, coli was membrane-associate d, only soluble protein was haemolytically active, The haemolytic acti vity was shown to be highly protease sensitive, heat labile, independe nt of divalent cations, and extremely dependent on protein concentrati on, consistent with a requirement for oligomerization as the mechanism of action, BlyA was highly purified from E. coil in a single step uti lizing Triton X-114 phase partitioning. Genetic analysis of blyA and b lyB mutants indicated that the stability, membrane association, and ac tivity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein, The bly genes were found to be expressed at a very l ow level in cultured B. burgdorferi.