The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel

Abstract

The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many Gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB₅ heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.

Introduction

The type II secretion system (T2SS) is a major protein secretion pathway found in proteobacteria.^1,2 The T2SS is a multi-protein machinery that spans both the inner and outer membranes of bacteria and consists of three subassemblies: the inner membrane platform, periplasmic pseudopilus and outer membrane complex.^3,4 The inner membrane platform consists of a cytoplasmic secretion ATPase GspE that is associated with several copies of proteins GspL, GspM, GspF and GspC. The ATPase is thought to provide the energy for the secretion process. The pseudopilus is a fiber filament composed of pseudopilins GspG, GspH, GspI, GspJ and GspK. Multiple copies of major pseudopilin GspG form the body of the pseudopilus, whereas the four minor pseudopilins form the tip of the pseudopilus.^5,6 The pseudopilus has been proposed to act as a piston and/or plug during the secretion of exoproteins.^7–9 The outer membrane complex consists of the secretin GspD, one of the largest multimeric channels in the outer membrane of bacteria. The secretins are a diverse family of proteins, with homologs in other bacterial secretion systems such as the type III secretion system (T3SS), the type IV pilus biogenesis system and the filamentous phage assembly system.^10–15

The T2SS is found in many pathogenic bacteria species, such as Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC), where it secretes, among several other proteins, the major virulence factors cholera toxin (CT) and heat-labile enterotoxin (LT), respectively. CT and LT are closely related AB₅ hetero-hexamers composed of one A and five B subunits that are exported through the secretin channel from the periplasm to the extracellular space in their folded state.¹⁶ Although the secretion signals of proteins secreted by the T2SS are still unknown, in the case of both the CT and LT toxins the secretion signal resides in the B-pentamer and not in the A-subunit.¹⁷ Interestingly, ETEC and V. cholerae are able to secrete both CT and LT.¹⁸ Due to the apparent lack of a linear secretion signal sequence it has been suggested that the T2SS secretion signal is instead a structural motif present in the native or near-native protein structure.^18–20

We recently described the 19 Å resolution structure of the T2SS secretin GspD from V. cholerae (VcGspD) determined by electron cryomicroscopy (cryoEM) and single particle reconstructions.²¹ The V. cholerae secretin forms a 200 Å tall dodecameric assembly that is ∼0.9 MDa in molecular mass. The channel contains a large periplasmic vestibule with a constriction site that is believed to be important for substrate secretion. The periplasmic vestibule is separated from a large extracellular chamber by a periplasmic gate that is closed in our reconstruction. A second gate caps the extracellular chamber (Fig. 1A). The periplasmic domain of the secretin has been implicated in exoprotein recognition⁷ and the crystal structure of a three-domain periplasmic fragment of ETEC GspD has been solved.²² Surface plasmon resonance (SPR) studies have shown that the periplasmic domain of ETEC GspD interacts with the B-pentamer of heat-labile enterotoxin.²¹ Here we used electron microscopy (EM) of negatively stained preparations to image the VcGspD complex bound to the B-pentamer of cholera toxin (CTB₅). Our studies show that CTB₅ is captured within the large periplasmic vestibule of VcGspD while the channel is in its closed state. We find that CTB₅ fits snuggly into the observed EM density compatible with a piston-driven mechanism for toxin secretion.

Figure 1.

Negative stain electron microscopy of VcGspD and the VcGspD-CTB₅ complex. (A) Slice view of the VcGspD cryoEM reconstruction (EMDB 1763).²¹ The periplasmic and outer membrane domains are labeled. (B) Electron micrograph of negatively stained VcGspD and (C) VcGspD incubated with CTB₅. (B, inset) Representative reference-free class average of VcGspD particles appears similar to the VcGspD reconstruction with a large un-obstructed periplasmic vestibule. (C, inset) Representative class average of VcGspD mixed with CTB₅ contains an additional punctate density at the bottom of the periplasmic domain of VcGspD (C, bottom inset arrow head). The punctate density is approximately 60 × 40 Å, which is consistent with the size of the CTB₅ complex.

Results and Discussion

Electron microscopy of the VcGspD-B₅ complex.

Recombinant full-length VcGspD and CTB₅ were each expressed in E. coli and purified to homogeneity for electron microscopy as described in reference ²¹. VcGspD (50 µgml⁻¹) was either applied directly to a negatively charged carbon coated copper EM grid or first incubated with CTB₅ (7.5 µgml⁻¹) for 30 minutes at 25°C. Proteins were negatively stained with uranyl formate and visualized on a 100 kV transmission electron microscope (FEI Morgagni) as previously described in reference ²³. Images were recorded on a 2k × 4k CCD camera (Gatan) at a nominal magnification of 44,000x at the specimen level (Fig. 1B and C). For image processing, micrographs were digitally binned twice to yield a final pixel size of 4.1 Åpixel⁻¹. Particles were picked manually in Ximdisp²⁴ and processed in SPIDER²⁵ to generate two-dimensional (2D) reference free class averages (Fig. 1B and C, insets).

The class averages of VcGspD particles oriented perpendicular to the channel axis (side view) reveals a protein density that is consistent with the features observed in our cryoEM reconstruction (compare Fig. 1A and B, inset). The periplasmic domain is 125 Å long and forms a large unobstructed periplasmic vestibule with a constriction located just below the periplasmic gate. The periplasmic gate appears in a closed conformation, effectively separating the periplasmic vestibule from the extracellular chamber. The extracellular chamber is capped at the top by a weak protein density corresponding to the extracellular gate that was observed in the VcGspD cryoEM reconstruction.²¹ The identification of these features suggests the channel architecture is well preserved under the conditions used for EM of negatively stained specimens.

When VcGspD was mixed with CTB₅ we observed two distinct groups of particles in the class averages. The first group (corresponding to ∼35% of all side views) appeared similar to class averages of free VcGspD, in that they contained an unobstructed periplasmic vestibule (Fig. 1B and inset). However, a second group of class averages (corresponding to ∼45% of all side views) clearly contained an additional punctate density at the entrance of the periplasmic vestibule (Fig. 1C and inset arrow head). This density has approximate dimensions 60 × 40 Å that correspond well to the size of the B-pentamer. Therefore, our EM analysis suggests that CTB₅ binds within the VcGspD periplasmic vestibule. The distribution of free and CTB₅-bound VcGspD particles is consistent with the relatively weak binding affinity observed between CTB₅ and the periplasmic domain of GspD in solution (K_D 15.5 ± 1.7 µM),²¹ and the low protein concentrations required for EM sample preparation.

Molecular model of the secretin-toxin complex.

The class averages obtained from the CTB₅-bound VcGspD particles were used to guide the placement of the complete cholera toxin AB₅ heterohexamer (PDB ID 1S5E),²⁶ within the cryoEM reconstruction of VcGspD to obtain further structural insight into the secretion mechanism. First, the atomic structure of CTB₅ was placed into the EM density obtained from the class averages of the VcGspD-CTB₅ complex (Fig. 2A). The CTB₅ structure overlays well with the punctate density located within the periplasmic vestibule of VcGspD. This placement positions the CTB₅ complex at the entrance of the VcGspD periplasmic vestibule, roughly 85 Å below the periplasmic gate. Based on this placement, we modeled the complete AB₅ cholera toxin molecule into the periplasmic vestibule of the VcGspD cryoEM reconstruction (Fig. 2B, left). The enormous 55.4 × 10⁴ ų periplasmic vestibule is capable of housing the 9.3 × 10⁴ ų AB₅ toxin. The resulting model places the cholera toxin just below the VcGspD periplasmic constriction. This placement is almost identical to what we proposed previously based on the steric consideration of the constriction diameter, which narrows the channel to 55 Å.²¹ Indeed, the B-pentamer is 65 Å wide, suggesting that the toxin could not pass through the constriction without major conformational changes occurring in the channel.

Figure 2.

Model of the VcGspD-toxin complex and implications for the T2SS toxin secretion mechanism. (A) The atomic structure of CTB₅ (gold, PDB ID 1S5E),²⁶ was fit into the CTB₅-bound VcGspD EM density. The CTB₅ structure overlays well with the punctate density located at the entrance of the VcGspD periplasmic vestibule seen in projection averages (left). The overlay was used to guide the placement of CTB₅ into the three-dimensional (3D) cryoEM reconstruction of VcGspD (EMDB 1763),²¹ placing the pentamer 85 Å below the periplasmic gate. Scale bar shown corresponds to both left and right panels. The CTB₅ structure fits snuggly within the large periplasmic vestibule, just below the channel constriction (right). (B) Key structural steps in the proposed piston driven mechanism for toxins through the T2SS.⁷,²¹ (B, left) The secretin is loaded or primed by binding large exoproteins such as the AB₅ cholera holotoxin (gold) while the channel is in its closed state. (B, middle) The pseudopilus tip (grey, PDB ID 3CI0),⁵ then binds the periplasmic domain sealing the channel entrance. As the pseudopilus (white, a pseudopilus model based on the GspG monomer structure (PDB ID 3FU1),²⁹ and a type IV pilus structure (PDB ID 2HIL),³⁰) extends via an ATP-dependent process, it would act like a piston to force the toxin into the secretin constriction. (B, right) This interaction of the toxin with the constriction could trigger a conformational change in the secretin to open the periplasmic gate, allowing the toxin to enter the extracellular chamber and be secreted. The figure was adapted from Reichow et al.²¹

We propose that the VcGspD-toxin complex that we observed in this study corresponds to the priming stage in the piston-driven secretion mechanism (Fig. 2B).^7,21 In this model, the toxin is bound within the periplasmic vestibule of the secretin with the channel gate in a closed conformation, forming a substrate loaded or primed state (Fig. 2B, left). Then, following this priming stage, a signal is somehow transferred via the inner membrane platform to the secretion ATPase GspE in the cytoplasm, leading to ATP hydrolysis and polymerization of pseudopilins and resulting in formation or extension of the pseudopilus.^27,28 Eventually, the pseudopilus tip complex GspK-GspI-GspJ reaches the secretin and possibly interacts with the periplasmic domain of the channel, sealing the channel's entrance. We have previously shown by SPR that the trimeric pseudopilus tip complex interacts with the periplasmic domain of secretin.²¹ As the pseudopilus extends further—probably through continuing ATP-driven polymerization—it would act as a piston forcing the toxin to interact with the VcGspD constriction (Fig. 2B, middle). This interaction with the secretin constriction could provide the trigger that causes a conformation change in the channel to open the periplasmic gate. This conformational change may resemble the conformational change that was observed in the T3SS secretin following needle attachment.^10,11,21 With the periplasmic gate open, the toxin may pass into the extracellular vestibule for secretion (Fig. 2B, right). Once the toxin is secreted, the pseudopilus may transiently remain extended, acting as a plug to prevent channel leakage. By an as yet unknown mechanism, the GspD periplasmic gate closes, and the pseudopilus retracts and/or disassembles partially or fully to reset the system. Whereas this model is in line with our current structural knowledge of the T2SS, a complete understanding of this mechanism will require future high-resolution structural studies of T2SS assembly and secretion.

Abbreviations

Gsp

general secretory pathway

cryoEM

electron cryomicroscopy

EM

electron microscopy

T2SS

type II secretion system

Addendum to: Reichow SL, Korotkov KV, Hol WG, Gonen T. Structure of the cholera toxin secretion channel in its closed state. Nat Struct Mol Biol. 2010;17:1226–1232. doi: 10.1038/nsmb.1910.