Pore formation by the CDTb component of the Clostridioides difficile binary toxin is Ca2+-dependent

Abstract

Clostridioides difficile infection (CDI) is one of the five most urgent bacterial threats in the United States. Furthermore, hypervirulent CDI strains express a third toxin termed the C. difficile binary toxin (CDT), and its molecular mechanism for entering host cells is not fully elucidated. Like other AB-type binary toxins, CDT enters host cells via endosomes. Here we show via surface plasmon resonance and electrochemical impedance spectroscopy that the cell-binding component of CDT, termed CDTb, binds and form pores in lipid bilayers in the absence of its enzymatic component, CDTa. This occurs upon lowering free Ca²⁺ ion concentration, and not by decreasing pH, as found for other binary toxins (i.e., anthrax). Cryogenic electron microscopy (CryoEM), X-ray crystallography, and nuclear magnetic resonance (NMR) studies show that dissociation of Ca²⁺ from a single site in receptor binding domain 1 (RBD1) of CDTb triggers conformational exchange in CDTb. These and structure/function studies of a Ca²⁺-binding double mutant targeting RBD1 (i.e., D623A/D734A) support a model in which dissociation of Ca²⁺ from RBD1 induces dynamic properties in CDTb that enable it to bind and form pores in lipid bilayers.

Fig. 1. CDTb binds and forms pores in lipid bilayers in a Ca²⁺-dependent manner.

Surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) data are shown as red triangles or black circles, respectively. The SPR/EIS data were collected in the (A) absence or (B) presence of the Ca²⁺ ion chelator EGTA (see section “Methods”).

Fig. 2. Local resolution in electron density maps for two classes of Ca²⁺-depleted CDTb conformations detected by cryoEM (termed CDTb^(-Ca)).

(Top) The RBD2 distorted structure (class 1) and (Bottom) the heptamer structure with the RBD2 domains intact (class 2). The difference in the degree of RBD2 resolution is distinguishable by side and top projections of each class.

Fig. 3. Mutation of the Ca²⁺-coordinating residues D623A and D734A of RBD1 abolish Ca²⁺ binding.

Ca²⁺ binding to RBD1^WT (red filled circles) and RBD1^D623A/D734A (blue filled circles) was examined by fluorescence spectroscopy at 350 nM (n = 3). The fluorescence emission intensity of the single tryptophan residue in each RBD1 construct (Trp632) was monitored as a function of increasing Ca²⁺ concentration. Ca²⁺ binding was observed only for RBD1^WT (^CaK_D = 40 ± 10 µM) upon fitting a model with noncooperative binding of Ca²⁺ to a single site in the RBD1^WT. No Ca²⁺ binding was detected up to 10 mM Ca²⁺ for RBD1^D623A/D734A (^CaK_D > 10 mM).

Fig. 4. In the presence of Ca²⁺, RBD1^WT retains a single structure regardless of pH.

A Overlay of NMR data for RBD1^WT at neutral (black) and acidic (red) pH values in 10 mM Ca²⁺. ¹⁵N-HSQC NMR data were collected using 0.1 mM RBD1^WT at 25 °C and pH 7.0 (black) or pH 5.0 (red). Residues of RBD1^WT were assigned sequence-specifically (residues 616-748), and those marked by blue asterisks are associated with the His-tag. The contour marked “X” shifted significantly but could not be assigned unambiguously as correlations to this H^N were very weak in 3D NMR data sets. The contour labeled G645^* appeared in the noise at pH 7.0 and grew stronger at pH 5.0. Correlations connected by horizontal lines correspond to sidechain NH₂ groups. B Graphed chemical shift perturbations (CSPs) of Ca²⁺-bound RBD1^WT residues upon lowering pH from 7.0 to 5.0. The average CSP for all residues is indicated by a red dashed line. The solid red line indicates one standard deviation (1σ; 0.03) above the average CSP, and residues are indicated that were above the average CSP. Blue bars are shown above residues that are in beta-strand regions of RBD1 as determined by X-ray crystallography and NMR chemical shift values. White bars represent random coils.

Fig. 5. Ca²⁺ is necessary to stabilize RBD1 from a molten globule state to single stable structure.

Overlay of 2D ¹⁵N-edited HSQC NMR spectra of RBD1^D623A/D734A (black) and RBD1^WT (red) after incubation with excess Ca²⁺ (10 mM Ca²⁺). Residues of RBD1^WT were assigned sequence-specifically (residues 616-748), and those marked by blue asterisks are associated with the His-tag. A correlation for G645 appeared in the noise of the RBD1^WT sample and is indicated by a box. The spectrum for RBD1^D623A/D734A remained in conformational exchange even with Ca²⁺ added, so the remaining few detectable correlations (in black) could not be sequence-specifically assigned. Correlations connected by horizontal lines correspond to sidechain NH₂ groups. RBD1^WT data were collected with 0.25 mM RBD1^WT at 25 °C and pH 7.0; RBD1^D623A/D734A data were collected with 0.1 mM RBD1^D623A/D734A at 25 °C and pH 7.0.

Fig. 6. Comparisons of the structural architecture for CDTb^WT and CDTb^D623A/D734A.

A Side and top projections of the diheptamer structure in the “asymmetric form” of CDTb (PDB: 6UWR). The heptamer with the β-barrel extension is illustrated in different colors; heptamerization domain 1 (HD1; residues 212-297) in red, β-barrel domain (βBD; residues 298-401) in green, heptamerization domain 2 (HD2; residues 402-486) in violet, linker region 1 (L1; residues 487-513) in gray, heptamerization domain 3 (HD3; residues 514-615) in yellow, receptor binding domain 1 (RBD1; residues 616-748) in blue, linker region 2 (L2; residues 745-756) in gray, and receptor binding domain 2 (RBD2; residues 757-876) in cyan. Dual Ca²⁺ ions are bound in HD1 and the single Ca²⁺ ion bound in RBD1 are shown as green spheres. B The single heptamer extracted from CDTb is superimposed with the electron mesh map of the CDTb^D623A/D734A structure. C The electron density map of CDTb^D623A/D734A is colored by local resolution with C7 symmetry imposed. Increased flexibility is observed in the outer regions of the core heptamer, and it is most pronounced for the RBD1 domain and the tip of the β-barrel extension.

Fig. 7. Ca²⁺ stabilizes the global fold of RBD1.

A X-ray crystal structure of Ca²⁺-bound RBD1^WT (residues 616-748; 2.3 Å; PDB = 9MUI) with β-sheets 1–10, and N and C-termini labeled. The inset shows backbone and sidechain atoms of residues in the Ca²⁺-binding site of RBD1^WT. Ca²⁺ is represented by a green sphere. B CD spectra of RBD1^WT without Ca²⁺ (black), in the presence of Ca²⁺ (2.4 mM; red, 10 mM; blue), and in 8 M urea (without Ca²⁺; gray, with 2.4 mM Ca²⁺; dark gray). C CD spectra of RBD1^D623A/D734A without Ca²⁺ (black) and in the presence of Ca²⁺ (2.4 mM, red; 10 mM, blue), and 8 M urea (without Ca²⁺; gray, 2.4 mM Ca²⁺; dark gray). All CD experiments were performed in triplicate (n = 3).

Fig. 8. Model for membrane association and pore-formation by CDTb.

The change in orientation of the β-barrel domain (purple) within a single monomer of the CDTb heptamer is presented as CDTb transitions from an extracellular (A) to an early endosomal environment (B) and mature endosomal environment (C). In the extracellular environment, Ca²⁺ is bound to RBD1 and HD1. In a Ca²⁺-bound state, CDTb alone is unable to bind lipid bilayers or form pores. Following its uptake, dissociation of Ca²⁺ from the RBD1 domain of CDTb (^CaK_D = 40 ± 10 µM) occurs as Ca²⁺ is exported out of the endosome, resulting in the destabilization of the RBD1 domain as it enters conformational exchange. At this point (i), residues associated with the β-barrel domain begin extending into a “pre-pore” conformation of heptameric CDTb. (ii) The β-barrel domain extends fully, creating pores through the endosomal membrane at low micromolar [Ca²⁺] (i.e., <40 µM). (iii) Pore formation can begin to serve as channels for CDTa to enter the cytosol, leading to cytotoxic effects and the disruption of the intestinal epithelium that facilitate the increased severity of CDT-associated CDI. However, a more complete mechanism of action for CDTa delivery by CDTb requires further examination.