Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme.

Abstract

Bordetella pertussis produces a calmodulin‐sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over‐produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti‐B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin‐dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post‐translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

“…CyaA is a 1,706-residue-long bifunctional protein organized in a modular fashion (Fig. 1A); the ATP-cyclizing, CaM-activated catalytic domain (AC) is located in the 400 amino-proximal residues, whereas the carboxyl-terminal 1,306 residues are responsible for the hemolytic phenotype of B. pertussis (17)(18)(19)(20).…”

“…Then CyaA is secreted across the bacterial envelope by a dedicated type I secretion machinery and binds to the CD11b/CD18 integrin expressed by a subset of leukocytes including neutrophils, macrophages, and dendritic cells (22,(28)(29)(30). However, CyaA can also invade a wide variety of cells that do not express this receptor, albeit with a lower efficiency (19,(31)(32)(33)(34)(35).…”

“…This biomimetic membrane will permit in vitro studies of protein translocation in precisely defined conditions. inserts its hydrophobic segments into the plasma membrane and then delivers its catalytic domain across the plasma membrane into the cell cytosol (19,31,32) (Fig. 1B).…”

“…Previous studies have shown that the translocation process is dependent on the temperature (occurring only above 15°C), the membrane potential of the target cells, and the presence of calcium ions in the mM range (32,36). Inside the cell, on binding to CaM with a subnanomolar affinity, CyaA is stimulated by more than 1,000-fold and exhibits a high catalytic rate (k cat > 2,000 s −1 ) to produce supraphysiologic levels of cAMP (12,19,37).…”

“…How the hydrophilic CyaA catalytic domain of approximately 400 residues is able to pass across the hydrophobic barrier of the plasma membrane remains largely unknown, and whether specific eukaryotic proteins and/or cell membrane components are involved in this process is also unclear (19,32,35,38,39). To characterize the molecular mechanisms of CyaA translocation across the membrane, we performed a functional in vitro assay that exploits a recently designed biomimetic membrane assembly composed of a bilayer membrane (tBLM) tethered over an amino-grafted gold surface derivatized with CaM (40).…”