Membrane Permeabilization by Bordetella Adenylate Cyclase Toxin Involves Pores of Tunable Size

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.

Membrane Permeabilization by Pore-Forming RTX Toxins: What Kind of Lesions Do These Toxins Form?

“…In a similar line, it has been suggested the membrane destabilization effect by Aggregatibacter actinomycetemcomitans leukotoxin LtxA has similar effects [80]. Our last study with the adenylate cyclase toxin [73], which will be discussed below, supports this view that pore formation by the RTX toxins involves lipids.…”

“…It might be speculated that possible oligomeric ACT pores might be larger than 0.6–0.8 nm in diameter. This has been found in our laboratory very recently [73] and will be discussed below.…”

“…Another yet unsettled issue is the calcium-dependence of pore formation by the RTX toxins. Ca 2+ binding to the RTX repeats domain is considered essential for all biological activities of the RTX toxins, from protein folding to secretion [70,71] including binding to target membranes, and hemolysis or permeabilization of liposomes [72,73]. However, it was reported that E. coli HlyA does not need calcium in the medium for its activity towards artificial lipid bilayer membranes, even in the case of HlyA produced in calcium-depleted medium [74].…”

“…Very recently we found that the size of the ACT pore is tunable and depends on toxin concentration and on incubation time, and can evolve into a large “hole” of several nm wide, so as to allow the efflux of even small proteins from the target cytosol. Moreover, we have provided the first nanoscale pictures of ACT lytic pores in membranes [73].…”

Impact of Bacterial Toxins in the Lungs

“…B. anthracis is a Gram-positive/variable encapsulated, rectangular rod-shaped bacterium, capable of forming endospores, belonging to the genus Bacillus and family Bacillaceae [265]. It is an aerobic or facultative-anaerobic bacterium that can live everywhere in the environment [266].…”