Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Abstract

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is aassociated with the whole bacteria and released into its culture media. Preparations of this enzyme invade animal cells, causing elevations in intracellular cAMP levels. Cell-associated adenylate cyclase accounted for 28% of the total adenylate cyclase activity while 72% was released into the culture supernatant. Over 90% of the cell-associated adenylate cyclase was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of the enzyme is localized on the outer surface of the bacterial cells. Enzyme activity was released from whole cells by treatment with SDS. This activity was resolved as a large form (Mr 215,000) by SDS-polyacrylamide gel electrophoresis. In contrast, the culture supernatant contained only the 45,000-dalton catalytic subunit. Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215,000) and a small form (Mr 45,000). The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation. Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with the immunopreciptated both the large and small forms of adenylate cyclase isolated from bacterial cells. Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215,000-dalton form and a concomitant increase in the amount of the smaller 45,000-dalton form. There was also a parallel increase in the ability of the cell-associated preparationto elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells. On the basis of these data, we propose that the adenylate cyclase produced by B. pertussis is synthesized as a large precursor molecule (Mr 215,000) and transported to the outer membrane of the bacteria where it is proteolytically processed to a smaller invasive form (Mr 45,000) that is released into the culture supernatant.