Bordetella CyaA toxinaren eta kolesterolaren arteko ezagutza molekularra

Abstract

Insertion and assembly of pore-forming toxins have been the subject of growing investigations to understand protein conversion from water-soluble forms to stable membrane-integrated structures. A great variety of pore-forming toxins requires lipid-mediated stabilization of transmembrane helices to modulate toxicity. For example, cumulative evidences show that membrane cholesterol is essential for the mechanism of action of several RTX toxins secreted by pathogenic bacteria. In fact, increasing number of RTX toxins present various functional cholesterol-recognition motifs of the CRAC and CARC type, which appears to be another common feature shared in the family. Amongst others, it was documented that the cytotoxic and cytolytic activities of the adenylate cyclase toxin-hemolysin (CyaA) secreted by the whooping cough bacterium Bordetella pertussis are modulated by the membrane cholesterol concentration. Yet, it was still unclear whether this was due to direct binding to cholesterol, or to indirect effects of the sterol on the physical state of the phospholipid bilayer. We provide here an ample set of evidences clarifying that cholesterol enhances the membrane binding of CyaA, primarily due to preferential toxin-cholesterol interaction. We report herein that such cholesterol sensitivity is provided by at least four cholesterol-recognition motifs (CARC415, CRAC485, CRAC521 and CARC532) in key membrane-interacting domains. Moreover, we use a synthetic peptide to demonstrate that at least one of those motifs, the CRAC485, is responsible for the cholesterol binding by CyaA and we stablish the membrane-interacting characteristics of such peptide. Importantly, we show that the peptide does not itself exhibit lytic properties in the range of concentrations in which inhibits CyaA activity on cells, all of which allows affirming that the use of this peptide may represent a valid non-toxic method for blocking binding of CyaA to host cells and minimize Cya-induced cytotoxicity. Hence, this indicates that it may have a potential clinical applicability in controlling infection of Bordetella.