Abstract
RTX (Repeats in ToXin) pore-forming toxins constitute an expanding family of exoproteins secreted by many Gram-negative bacteria and involved in infectious diseases caused by said pathogens. Despite the relevance in the host/pathogen interactions, the structure and characteristics of the lesions formed by these toxins remain enigmatic. Here, we capture the first direct nanoscale pictures of lytic pores formed by an RTX toxin, the Adenylate cyclase (ACT), secreted by the whooping cough bacterium Bordetella pertussis. We reveal that ACT associates into growing-size oligomers of variable stoichiometry and heterogeneous architecture (lines, arcs, and rings) that pierce the membrane, and that, depending on the incubation time and the toxin concentration, evolve into large enough “holes” so as to allow the flux of large molecular mass solutes, while vesicle integrity is preserved. We also resolve ACT assemblies of similar variable stoichiometry in the cell membrane of permeabilized target macrophages, proving that our model system recapitulates the process of ACT permeabilization in natural membranes. Based on our data we propose a non-concerted monomer insertion and sequential mechanism of toroidal pore formation by ACT. A size-tunable pore adds a new regulatory element to ACT-mediated cytotoxicity, with different pore sizes being putatively involved in different physiological scenarios or cell types.
Highlights
Adenylate cyclase toxin (ACT or CyaA) is crucial for colonization of the respiratory tract and disease establishment by Bordetella pertussis, the bacterium causative of whooping cough [1,2]
As compared to bulk experiments of dye release in large unilamellar vesicles (LUVs), in which the information obtained is an average of the processes of the overall liposome population, the Giant Unilamellar Vesicles (GUVs) methodology allows a more accurate estimation of the effective pore size (≈internal diameter) and on the permeabilization mechanism, because confocal microscopy allows the direct detection of individual vesicles [34,35,36]
We present an exhaustive analysis of pores formed by ACT, using the single giant unilamellar vesicle (GUV) methodology and spectroscopic and atomic force microscopy techniques
Summary
Adenylate cyclase toxin (ACT or CyaA) is crucial for colonization of the respiratory tract and disease establishment by Bordetella pertussis, the bacterium causative of whooping cough [1,2]. Cytotoxicity by ACT results from the synergy between toxin‘s two main activities, production of supraphysiological cAMP levels by its adenylate cyclase domain (AC domain), and permeabilization, induced by its pore-forming domain (hemolysin domain), which debilitate the host defenses. Maturation, and secretion of ACT are determined by the CyaCABD operon [5]. A is a 1706 amino acid polypeptide corresponding to a protoxin (pro-ACT) that matures in the bacterial cytosol to the active form by CyaC-directed acylation at two internal lysine residues (Lys 863 and Lys. 913) [6]. ACT is distinguished from the rest of RTX toxins by bearing a cell-invasive
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