Multistate kinetics of the syringe-like injection mechanism of Tc toxins

Abstract

Tc toxins are pore-forming virulence factors of many pathogenic bacteria. Following pH-induced conformational changes, they perforate the target membrane like a syringe to translocate toxic enzymes into a cell. Although this complex transformation has been structurally well studied, the reaction pathway and the resulting temporal evolution have remained elusive. We used an integrated biophysical approach to monitor prepore-to-pore transition and found a reaction time of ~30 hours for a complete transition. We show two asynchronous general steps of the process, shell opening and channel ejection, with the overall reaction pathway being a slow multistep process involving three intermediates. Liposomes, an increasingly high pH, or receptors facilitate shell opening, which is directly correlated with an increased rate of the prepore-to-pore transition. Channel ejection is a near-instantaneous process which occurs with a transition time of <60 milliseconds. Understanding the mechanism of action of Tc toxins and unveiling modulators of the kinetics are key steps toward their application as biomedical devices or biopesticides.

Fig. 1.. Schematic overview of the functional mechanism of action of Tc toxins.

The two toxin states, prepore and pore, are displayed each in two ways: as surface cartoon representations (left) and vertical cut-throughs (right). In the surface cartoon representation, the five protomers of TcA are colored differently, the TcC is violet, and TcB is in light and dark cyan. In the cut-through, TcA is orange and grey, and the HVR is highlighted in black.

Fig. 2.. EPR kinetics of TcA shell opening in the absence and presence of receptors.

(A) TcA variant (1193Cys) to probe shell opening via EPR labeled with IAP, resulting in TcA (1193-IAP). Two sketches of the different CW EPR spectra of the prepore (slower motion, 3-ns rotational correlation time) and the pore (faster motion, 1.5 ns) states simulated with EasySpin (34). (B) Three simulated spectra with the slow component interconverting in 15 hours to the fast component. The kinetics of the shell opening are obtained by plotting the peak-to-peak intensity versus time (right). a.u., arbitrary units. (C) EPR kinetics of three biological replicates at pH 11.2 with monoexponential fits (τ is the mean value). The kinetics are normalized to start at zero and to end at the asymptote of the corresponding fits. DEER modulation depths kinetics (green triangles) measured on the first batch of protein. The error bar is ±10% error. (D) Normalized CW EPR kinetics of TcA (1193-IAP) at 2.8 μM TcA pentamer concentration, pH 11.2, and 21°C alone (black) or in the presence of 71 (1:25) and 333 (1:120) μM heparin (green and magenta, respectively). Heparin was used in molar excess to mimic the situation of high heparin concentration on the membrane of the host cell. (E) CW EPR kinetics of TcA (1193-IAP) at 3 μM TcA pentamer concentration at pH 11.2 and 21°C alone (black) or in the presence of 3 μM Vsg (blue). The TcA pentamer–to–Vsg molar ratio is indicated.

Fig. 3.. Negative stain EM analyses of the effect of receptors on prepore-to-pore transition.

(A) Left: Scheme of receptor-bound TcA in the prepore and pore states with the respective representative negative stain class averages. Right: A representative negative stain EM image with particles autopicked for quantification of the prepore (orange) and pore (red) particles. Scale bar, 100 nm. (B) Quantification of autopicked pore particles of ABC WT at 70.2 nM pentamer concentration after incubation for 2 hours at pH 11.2 and 21°C, in the presence or absence of the receptors Lewis X (BSA-LeX, 5.8 μM), heparin (5.8 μM), and Vsg (70 nM). The TcA pentamer–to–receptor molar ratios are indicated. The data are presented as mean values, and the error bars represent the SEM of three independent experiments.

Fig. 4.. EPR kinetics and single-molecule fluorescence anisotropy of channel ejection.

(A) Top: Design of a TcA variant to probe channel ejection via EPR spectroscopy: TcA (914Cys/2365Cys) was labeled with IAP at both cysteines, resulting in TcA (914-IAP/2365-IAP). Bottom: EPR DEER kinetics at pH 11.2 showing a first-order reaction obtained from a global fit of two biological replicates (light and dark blue data points). For better comparison, data are normalized to the maximum modulation depth achieved. (B) Top: Design of TcA variants to probe shell opening and channel ejection for confocal single-molecule fluorescence anisotropy experiments in solution. TcA (1193Cys), TcA (2365Cys), and TcA (2384Cys) were labeled with BFL-iodacetamide, resulting in TcA (1193-BFL), TcA (2365-BFL), and TcA (2384-BFL), respectively. Bottom: Confocal single-molecule fluorescence anisotropy experiments in solution at pH 11.2. A model with a consecutive path including one intermediate state and a parallel direct path was needed to describe all three datasets with global rate constants. The three transition rates of model 4 (independent consecutive transitions in note S1, figs. S17 and S18) are shown.

Fig. 5.. Total internal reflection single-molecule fluorescence studies of shell opening and channel ejection of surface immobilized toxins.

(A) Design of TcA and TcB-TcC variants to probe single shell opening and channel ejection events. (B) Schematics of fluorescence traces probing shell opening (orange) and channel ejection (blue). After a pH jump from 7.4 to 11.2 (blue dashed line, time = 0), the toxin undergoes a transition from the prepore to pore state (shaded in gray) after a specific pretransition time (double-sided arrows). (C) Single-molecule fluorescence homoFRET experiments using biotinylated TcA (1279-At647N) to assess shell opening and channel ejection. Shell opening experiments (42 traces) revealed a distribution of the recorded transition times over 1.6 s (orange bars), whereas those of channel ejection (13 traces) showed transition times within a single frame, below 60 ms (blue bar). (D) A representative shell opening trace using biotinylated TcA (1279-At647N) which reveals transition times longer than the bin width of 29.44 ms and a change in polarization caused by a difference in homoFRET and reduction of dye mobility due to the moving RBDs. (E) A representative trace of the channel ejection heteroFRET assay using ABC with a donor dye at TcA (914-BFL) and acceptor dye at TcB (1041-At647N)-TcC. The displayed trace shows a fast transition of the signal from the prepore to pore state within one bin (bin width = 58.88 ms). (F) Global fit model 2 [see note S1 (figs. S17 and S18), only consecutive]: reaction times of immobilized, biotinylated TcA (1279-At647N) (orange) and ABC composed of TcA (914-BFL)-biotin and TcB (1041-At647N)-TcC (blue). PP, prepore; I, intermediate; P, pore.

Fig. 6.. TcA prepore-to-pore stable intermediate kinetics.

(A) Negative stain EM analysis of pore formation of the indicated K-to-W TcA variants with TcB-TcC after incubation for 2 hours at pH 11.2 and 21°C. The data are presented as mean values, and the error bars represent the SEM of three independent experiments. (B) CW EPR kinetics of TcA (1193-IAP-K567W/K2008W) and TcA (1193-IAP-K1179W) at pH 11.2. The shell opening in TcA (1193-IAP-K567W/K2008W) proceeds according to a first-order reaction with a reaction time of 3.4 ± 0.2 hours. The shell in TcA (1193-IAP-K1179W) remains closed, but we observe a small decrease in the label’s dynamics within the first hour. (C and D) Confocal multiparameter fluorescence detection (MFD) experiments analyzed by polarization-resolved fluorescence correlation spectroscopy (pFCS) and anisotropy measurements of TcA (1193-BFL-K1179W) at pH 11.2, at longer (C) and shorter incubation (D). (D) pFCS resolves the fast-wobbling dynamics of TcA (1193-BFL-K1179W). Experimental data (dots) and fit curves (full lines; see Materials and Methods) for the autocorrelation amplitude G_pp (t_c) of parallel polarized fluorescence of TcA (1193-BFL-K1179W) at different incubation times and pH conditions: incubation time of start (0 to 400 s) at pH 11 (red curve), incubation time of measurement end (4700 to 5200 s) at pH 11 (blue curve), and for comparison at pH 7 (green curve) [see note S2 (global fits in figs. S19 to S21 with results in tables S7 to S10)].

Fig. 7.. Cryo-EM structure of the TcA prepore-to-pore stable intermediate.

(A) From left to right: Cryo-EM density map of TcA WT at pH 8.0 (EMDB 10033); schematic representation of TcA (1193-BFL) incubated for 10 min at pH 11.2; cryo-EM density map of TcA (K1179W), intermediate state (SI1) after 24 hours of incubation at pH 11.2 and 21°C; cryo-EM density map of TcA (1279-At647N), intermediate state (SI1) after 6 hours of incubation at pH 11.2 and 21°C; and cryo-EM density map of TcA (K567W/K2008W), intermediate state (SI1) after 2 hours of incubation at pH 11.2 and 21°C. TcA (1279-At647N) is a WT mimic that was modified and labeled at position 1279 (see Materials and Methods). The five protomers are colored individually, and the extra density observable at the low binarization threshold (figs. S14 to S16) is shown in orange. (B) Comparison of a protomer of the TcA WT prepore (left; PBD 6RW6) and of the TcA (K1179W) (right) prepore-to-pore intermediate. (C) Bottom view of the TcA WT prepore model at pH 8.0 (PDB 6RW6) (left) compared to the TcA (1279-At647N) (middle) and TcA (K567W/K2008W) (right) intermediate state (SI1) models at pH 11.2. The panels show the average inter-residue distances between Y1188 residues at the tip of the neuraminidase-like domain (purple), which keeps the TcA shell closed. The intermediate states show higher inter-residue distances compared to the prepore, indicating a preopening of the shell. The channel (α-pore–forming domain) is colored yellow.

Fig. 8.. Consecutive model of shell destabilization, opening, and channel ejection.

Top: Scheme of the transition steps. Middle: A qualitative energy landscape depiction of prepore-to-pore transition and reaction times (τ) of the transition steps which are summarized in the table below the scheme. PP, prepore ground state; PP^#, primed prepore state; PP^*, primed prepore state after pH shift; TI = transition intermediate with flexibility at the neuraminidase domain; SI1, stable intermediate 1 with flipped-out RBDs A, B and C; SI2, stable intermediate 2 with flipped-out RBDs and an open shell; CE, channel ejected pore state with a closed channel tip; CEO, channel ejected pore state with an open channel tip. Bottom: Table showing reaction times involving stable intermediates. n.d., not determined.