Characterization of the Intrinsic Phospholipase A1 Activity of Bordetella pertussis Adenylate Cyclase Toxin

Abstract

Adenylate cyclase toxin (ACT, CyaA) is one of the important virulence factors secreted by the whooping cough bacterium Bordetella pertussis, and it is essential for the colonization of the human respiratory tract by this bacterium. Cytotoxicity by ACT results from the synergy between toxin’s two main activities, production of supraphysiological cAMP levels by its N-terminal adenylate cyclase domain (AC domain), and cell membrane permeabilization, induced by its C-terminal pore-forming domain (hemolysin domain), which debilitate the host defenses. In a previous study we discovered that purified ACT is endowed with intrinsic phospholipase A1 (PLA) activity and that Ser in position 606 of the ACT polypeptide is a catalytic site for such hydrolytic activity, as part of G-X-S-X-G catalytic motif. Recently these findings and our conclusions have been directly questioned by other authors who claim that ACT-PLA activity does not exist. Here we provide new data on ACT phospholipase A1 characteristics. Based on our results we reaffirm our previous conclusions that ACT is endowed with PLA activity; that our purified ACT preparations are devoid of any impurity with phospholipase A activity; that ACT-S606A is a PLA-inactive mutant and thus, that Ser606 is a catalytic site for the toxin hydrolytic activity on phospholipids, and that ACT-PLA activity is involved in AC translocation.

The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro

“…In their reported experiments, only very small changes in fluorescent intensity of the probe (<1.1–1.4 relative ratio) were observed after the addition of CyaA. Moreover, the kinetic experiments presented in their recent paper [ 39 ] revealed odd profiles that question the validity of interpretation of the fluorescence changes as signatures of a PLA enzymatic activity. Indeed, they observed an abrupt, yet limited, change of fluorescence intensity just after addition of the CyaA sample (i.e., at the first recording point) with no further changes during the following 30 min of incubation.…”

“…Only the plateau values were different and roughly proportional to the amount of added proteins. Surprisingly, the authors were able to derive “velocities” (i.e., fluorescent change per minute) from these odd kinetic data, as shown in their plot of Figure 2 C in [ 39 ].…”

“…Recently, H. Ostolaza et al reported that CyaA exhibits a phospholipase A (PLA) activity [ 38 , 39 ] that is involved in destabilizing the lipid bilayer to facilitate ACD translocation across the plasma membrane. These results were contested by Bumba et al, who subsequently reported that CyaA has no PLA1 activity and showed that the two residues Ser606 and Asp1079 claimed to be critical for the putative PLA activity of CyaA, were not involved in CyaA translocation [ 40 ].…”

Irreversible versus repairable membrane poration: differences in permeabilization elicited by Bordetella Adenylate Cyclase Toxin and its hemolysin domain in macrophages

“…ACT is a single polypeptide of 1706 amino acids, that is unique among its relative RTX cytolysins in that, besides the characteristic pore‐forming RTX Hly domain (residues ≈ 500–1706), it has an N‐terminal enzymatically AC domain (≈ residues 1–400) which is delivered by the RTX Hly domain into the target cell cytosol. The toxin exhibits besides, intrinsic phospholipase A (PLA) activity, necessary for AC translocation . The RTX Hly domain contains, in turn, a membrane‐interacting hydrophobic region with amphipathic–hydrophobic α‐helixes (∼ 500–750 residues), two conserved Lys residues (Lys 863 and Lys 913) that are post‐translationally fatty acylated by a dedicated acyltransferase (CyaC), and a C‐terminal secretion signal recognized by a specific secretion machinery (CyaB, CyaD, and CyaE) .…”

“…ACT primarily targets host myeloid cells expressing the CD11b/CD18 integrin, which acts as specific receptor for the toxin in these cells , and upon receptor binding, the RTX Hly domain inserts into cellular membranes and mediates in AC domain translocation via membrane restructuring induced by toxin′s intrinsic PLA activity . Once inside the target cytosol, the AC domain binds calmodulin and catalyzes the conversion of ATP into cyclic adenosine monophosphate (cAMP) thereby subverting cellular signaling and leading eventually to cell death.…”