Four Cholesterol-Recognition Motifs in the Pore-Forming and Translocation Domains of Adenylate Cyclase Toxin Are Essential for Invasion of Eukaryotic Cells and Lysis of Erythrocytes

Abstract

Adenylate Cyclase Toxin (ACT or CyaA) is one of the important virulence factors secreted by Bordetella pertussis, the bacterium causative of whooping cough. ACT debilitates host defenses by production of unregulated levels of cAMP into the cell cytosol upon delivery of its N-terminal domain with adenylate cyclase activity (AC domain) and by forming pores in the plasma membrane of macrophages. Binding of soluble toxin monomers to the plasma membrane of target cells and conversion into membrane-integrated proteins are the first and last step for these toxin activities; however, the molecular determinants in the protein or the target membrane that govern this conversion to an active toxin form are fully unknown. It was previously reported that cytotoxic and cytolytic activities of ACT depend on membrane cholesterol. Here we show that ACT specifically interacts with membrane cholesterol, and find in two membrane-interacting ACT domains, four cholesterol-binding motifs that are essential for AC domain translocation and lytic activities. We hypothesize that direct ACT interaction with membrane cholesterol through those four cholesterol-binding motifs drives insertion and stabilizes the transmembrane topology of several helical elements that ultimately build the ACT structure for AC delivery and pore-formation, thereby explaining the cholesterol-dependence of the ACT activities. The requirement for lipid-mediated stabilization of transmembrane helices appears to be a unifying mechanism to modulate toxicity in pore-forming toxins.

Toxin-Triggered Liposomes for the Controlled Release of Antibiotics to Treat Infections Associated with Gram-Negative Bacteria

“…In addition to this shared process of membrane disruption, LtxA and many of the RTX toxins possess a CRAC domain that enables strong binding to cholesterol. 47,[77][78][79][80][81] For this reason, we incorporated a large proportion of cholesterol in our liposomal formulation and observed that the incorporation of cholesterol in the liposomes greatly enhanced LtxA-mediated leakage. Prior work has used a similar approach, using natural or synthetic cell membranes to sequester toxins produced by Gram-positive bacteria due to their affinity for specific lipid components.…”

Membrane Interaction Characteristics of the RTX Toxins and the Cholesterol-Dependence of Their Cytolytic/Cytotoxic Activity

“…For a number of RTX toxins, it has been documented that the cholesterol dependence for the biological activity is sterol-specific, and it is due to direct toxin binding to the sterol molecules in the membrane and not to indirect effects of this lipid on the physical state of the phospholipid bilayer [ 69 , 79 , 100 , 108 ]. In 2014, Vazquez and colleagues, using different biochemical and biophysical assays, demonstrated the direct interaction of UPEC HlyA with cholesterol but not with sphingomyelin [ 100 ].…”

“…Moreover, RtxA bound with 2–3-fold higher efficacy to the wells of an ELISA plate coated with cholesterol-BSA than to the wells coated with free BSA, showing that the toxin is able to interact with cholesterol independently of the presence of other membrane components [ 66 ]. Our group has recently shown that pre-incubation of B. pertussis CyaA with free cholesterol notably diminishes the toxin-induced hemolysis, and that binding of CyaA to pure lipid vesicles is notably increased proportionally to the cholesterol concentration present in the lipid bilayer (10–50%) and that ergosterol (ergosta-5,7,22-trien-3β-ol), an analog of cholesterol, cannot reproduce this effect [ 108 ].…”

“…In the last ten years, the presence of a variable number of both CRAC and CARC motifs in the primary structure has been documented for an increasing number of RTX toxins [ 69 , 79 , 100 , 108 ] (see Table 2 ). The first RTX toxin in which CRAC/CARC cholesterol-binding sites were identified was A. actinomycetemcomitans LtxA [ 66 ].…”

“…In 2017, Masin and colleagues predicted five CRAC motifs in the pore-forming domain of the CyaA sequence, the CRAC 627–638 ( 627 VQQSH Y ADQLDK 638 ), CRAC 654–661 (LAQL Y RDK), CRAC 721–728 ( 721 LAND Y ARK 728 ), and CRAC 732–741 ( 732 LGGPQA Y FEK 741 ) sites [ 129 ]. More recently, our laboratory has identified four additional CRAC/CARC sites in the B. pertussis CyaA primary structure: two of them in two helices of the pore-forming domain, the CRAC 521 and CARC 532 motifs, and the other two sites, CARC 415 and CRAC 485 , in two helices of the translocation region [ 108 ] (see Table 3 and Table 4 ).…”