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. 2019 May 10;9(5):183.
doi: 10.3390/biom9050183.

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

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Free PMC article

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

David González-Bullón et al. Biomolecules. .
Free PMC article

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.

Keywords: atomic force microscopy; lipid-protein interactions; membrane permeabilization; model membranes; pore-forming proteins; protein toxins.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Calcium-dependent-permeabilization of dioleylphosphatidylcholine Giant Unilamellar Vesicles by ACT DOPC GUVs by ACT toxin. (A) Representative images of GUVs (black, if empty) in a solution of Alexa-Fluor488 (green) incubated in the absence or presence of 200 nM ACT and buffer without or with 10 mM CaCl2 (left panel). Images were taken 30 min after mixing the components. The internalization of Alexa-Fluor-488 to the lumen of GUVs (green) corresponds to permeabilized vesicles. GUV composition was DOPC. In the right panel the total percentage of permeabilization after 30′ for each condition (± ACT ± CaCl2) is depicted. In this case the threshold filling value to discriminate between permeabilized and non-permeabilized vesicles has been the 40%. (B) Distribution of the degree of filling of individual GUVs to Alexa-Fluor-488 (right panel), after treatment with 200 nM ACT for 30 min in buffer with 10 mM CaCl2. The degree of filling was calculated for each individual vesicle, from confocal images as the one shown in the left pane. In each of three experiments, a minimum of 250 vesicles were analyzed per condition. Error bars represent S.D.
Figure 2
Figure 2
Permeabilization by ACT of GUVs to fluorescently-labeled solutes with diverse molecular sizes. Representative images of GUVs (gray-black) in solutions containing Alexa-Fluor-488, 4 kDa-FITC-Dextran, 10 kDa-FITC-Dextran or 20 kDa-FITC-Dextran incubated in the absence (control), or presence of 200 nM ACT. Images were taken 30 min (sub-panels a–e) (A) or 1h (sub-panels f–j) (B) after mixing the components. The filling of the lumen of GUVs with the corresponding fluorescent probe (green) corresponds to permeabilized vesicles. GUV composition was dioleylphosphatidylcholine (DOPC). Distribution of the degree of filling of individual GUVs to Alexa-Fluor-488, 4 kDa-FITC-Dextran, 10 kDa-FITC-Dextran or 20 kDa-FITC-Dextran, after treatment with ACT for 30 min (C) or for 1 h (D). In each of three experiments ≈200 vesicles were analyzed per condition.
Figure 3
Figure 3
Permeabilization of GUVs by ACT as a function of the incubation time and the toxin concentration. Filling of DOPC GUVs with 10 kDa-FITC-Dextran was determined after incubation of the vesicles with three different ACT concentrations (50, 200, and 500 nM) and for different incubation times (30′, 60′, 90′, and 120′). The degree of filling was calculated as described in the Methods section.
Figure 4
Figure 4
Analysis by BN-PAGE of ACT multimers in LUVs. Presence of possible multimeric ACT assemblies was detected by blue-native electrophoresis (BN-PAGE) of ACT-treated DOPC large unilamellar liposomes. (A) The lipid vesicles were incubated with the toxin for 30 min at 37 °C at three lipid: protein ratios (mol ratio) 250:1 (track A), 1000:1 (track B), and 2500:1 (track C), then samples were processed to remove the unbound toxin, and then run in a blue native gel (3–12% acrylamide gradient) and the corresponding blotted membrane stained with an anti-ACT MAb (9D4). (B) Quantification by densitometry of the relative intensities of the ACT protein bands detected in the blotted membrane with the 9D4 monoclonal antibody.
Figure 5
Figure 5
Analysis by atomic force microscopy of the assemblies formed by ACT in supported lipid membranes. (A) AFM image (left upper panel) of a supported lipid bilayer (SLB) prepared from ACT-containing proteoliposomes (POPC liposomes reconstituted with ACT). The red arrowhead points to a membrane pore that has been selected for a more detailed topographic analysis; in the image there are more pores, heterogeneous in size and shape; the edges of the pores present protrusions corresponding to ACT clusters. Below this image, other ACT assemblies are shown, such as a monomer, a line and an arc, which have been selected from other AFM images. On the right-hand part of panel A, a 3D AFM topography of the pointed ACT ring is shown in a greater detail. The image reveals a circular dark hole that spans the lipid membrane (below). ACT molecules around the pore rim (yellow-white) protrude 3.97 ± 1.02 nm above the membrane plane, as confirmed by the height profile shown below the image (corresponding to the blue line in the middle of the 2D image. (B) Quantitative analysis of the structures found for ACT on SLBs. Data show the percentage of each type of structure (monomer, line, arc or ring) in all the measurements. (C) Determination of the diameter and height (in nm) of the monomeric particle in each of the different ACT assemblies (monomer, line, arc or closed ring). Mean values are depicted as box-and-whisker plots (the ends of the whiskers represent standard deviations). A total of 973 particles were measured for these determinations.
Figure 6
Figure 6
Three-dimensional atomic force microscopyAFM topography of ACT assemblies that pierce the lipid bilayer. (A) Detailed 3D topographic analysis of two types of ACT assemblies, arcs (left-hand side) and full rings (right-hand side), for which each constituent monomer has been numbered. Below, different parameters (external arc diameter, diameter, internal ring diameter, monomer diameter, monomer height and ring “hole” height) and their respective values are listed. (B) Measurement of the number of monomeric particles with a given diameter (in nm) in different ACT arc and ring assemblies.
Figure 7
Figure 7
Analysis by blue native polyacrylamide gel electrophoresis BN-PAGE of target macrophages exposed to ACT for different incubation times and PI uptake as determined by flow cytometry. (A) J774A.1 cells (1 × 106 cells/mL) were incubated with ACT (30 nM) at 37 °C for several incubation times (5–30 min) and then the separated membranes were electrophoresed by BN-PAGE, blotted into a polyvinylidene difluoride (PVDF) membrane and stained with anti-ACT MAb 9D4. Several protein bands of apparent high molecular masses were resolved, corresponding most likely to ACT oligomers of variable stoichiometry. (B) PI uptake into J774A.1 macrophages (1 × 106 cells/mL) treated with ACT (30 nM) at 37 °C for several incubation times (5–30 min) as determined by flow cytometry.

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References

    1. Carbonetti N.H. Pertussis toxin and adenylate cyclase toxin: Key virulence factors of Bordetella pertussis and cell biology tools. Future Microbiol. 2010;5:455–469. doi: 10.2217/fmb.09.133. - DOI - PMC - PubMed
    1. Mattoo S., Cherry J.D. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin. Microbiol. Rev. 2005;18:326–382. doi: 10.1128/CMR.18.2.326-382.2005. - DOI - PMC - PubMed
    1. Welch R.A. Pore-forming cytolysins of gram-negative bacteria. Mol. Microbiol. 1991;5:521–528. doi: 10.1111/j.1365-2958.1991.tb00723.x. - DOI - PubMed
    1. Welch R.A. RTX toxin structure and function: A story of numerous anomalies and few analogies in toxin biology. Curr. Top Microbiol. Immunol. 2000;257:85–111. - PubMed
    1. Ladant D., Ullmann A. Bordetella pertussis adenylate cyclase: A toxin with multiple talents. Trends Microbiol. 1999;7:172–176. doi: 10.1016/S0966-842X(99)01468-7. - DOI - PubMed

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