Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin

1) The first one focused on the observation that lipids are released upon insertion. This issue has been discussed in the field for quite a while but has not been convincingly shown. The interpretation that this release occurs is based on the observation of density in the time resolved AFM images that appear and then disappear again. Showing conclusively that this density is indeed lipids would be a significant step ahead. Is there additional experimental evidence that this interpretation is correct? Could the authors for example provide a statistical evaluation of the size of these densities and explore if this is consistent with the expected size of the released lipids?

In addition, the process of when and how lipid ejection occurs should be discussed.

The conclusion that lipids are ejected is based on the appearance and subsequent disappearance of density in the real-time AFM images, as pointed out by the reviewers, but also on the time at which this occurs. These observations are made within minutes after injection of DTT to trigger the prepore-to-pore transition, and just before the first detection of membrane lesions within the pores.

As additional experimental evidence, we have now included a new Figure 3–figure supplement 1 that demonstrates that appearance of additional density is reproducible, after triggering the prepore-to-pore transition, in an independent time-resolved experiment carried out along the same lines as the one reported in Figure 3. It can thus be excluded that the additional density is artefactual. This leaves only two possible candidates to explain the additional high features appearing in Figure 3B-D (and in Figure 3–figure supplement 1): suilysin or lipids. While we cannot exclude the ejection of some protein, our interpretation of ejected lipids is supported (i) by the subsequent appearance of collapsed lipid vesicles on the membrane (plateaus in Figure 3D), and (ii) by the fact that after completion of pore formation, there are obviously lipids missing within the pore lumens (see holes in membrane, Figure 3G), as described in the manuscript.

We appreciate the suggestion of a statistical size evaluation in the reviewers’ comment above, but would like to point out that for such soft and clearly mobile features in AFM images (see streaky appearance of the white features in Figure 3C), reliable and accurate volume measurements are nigh impossible. For completeness, we have now also attempted confocal fluorescence and TIRF microscopy experiments with fluorescent lipids, but unfortunately with inconclusive results.

In the Discussion section, in the paragraph “Regardless of the extent of oligomerization…” we have specified that the lipid ejection occurs upon conversion of suilysin from its prepore to pore state, hence upon the unfurling of the β-hairpins into the membrane. We have now expanded the corresponding paragraph to briefly discuss how lipid ejection may occur, specifically: “The results imply that the hydrophilic inner surface of the partially or fully completed β-barrel is sufficient to destabilize the lipid membrane in the pore lumen, leading to ejection of lipid micelles from the pore.”

2) The provided structural model is of higher resolution than all other models that have been published before. Is there experimental evidence from mutational analysis that the structural model is correct? Again any evidence that the proposed interfaces indeed play a role during this process would significantly strengthen the impact of these results.

In response to this question, we have analysed the electrostatic properties of the surfaces of domain 1 that form the major interfaces in the prepore and pore oligomers. The results show that both models have extended regions of complementary charge on the predicted interacting surfaces. Although the extent of complementary charge is less in the pore model, in this case the oligomer is stabilized by the beta barrel of the pore. This analysis lends support to both models, is discussed in the Results section on the Cryo-EM data, and is shown in the new Figure 1–figure supplement 3.

Further issues that should be addressed are:

3) “Negative stain EM and rotational symmetry analysis of complete rings of disulphide locked (Gly52Cys/Ser187Cys) suilysin prepores and wild-type pores formed on lipid monolayers revealed that most rings contain 37 subunits.” This is not documented properly: it should be, at least in the supplemental data. Cryo-EM reaches a resolution of 15 Å where the subunits are just visible. Negative stain is not likely to produce the same resolution; usually 25 Å is good. So how sure then is the 37-fold symmetry?

The subunit repeat is clearly resolved around the outer edge of the ring in negative stain end views on membrane monolayers. End view class averages and rotational correlation plots documenting the presence of 36-39-mers are presented in a new Figure 1–figure supplement 1.

4) “The pore structure is similar to that observed with pneumolysin, but with improved resolution, resolving the β-barrel and defining hinge bending and domain movements.” There is no evidence that the β-barrel and defining hinge bending are resolved in the 15 Å map. It is that a convincing model has been developed to demonstrate this. Convincing is the height change seen by AFM - the 2 observation together make this work exciting. Please reformulate.

This sentence has now been reformulated to: “The pore structure is similar to that observed with pneumolysin, but with improved resolution, and showing significant differences in the hinge bending and domain movements.”

5) In the title and in some parts of the manuscript suilysin is named as a cholesterol-dependent cytolysin. Does cholesterol play a role in the assembly and insertion process? What is the role of cholesterol?

We have now specified in the introduction that CDCs, exemplified by perfringolysin O, require >∼30% for membrane binding (with the new reference Johnson et al., 2012). Consistent with that observation on perfringolysin O, our experiments with suilysin on membranes with 10% and 20% cholesterol content did not show any significant sign of membrane binding and pore formation (data not shown, since this point is well established in the CDC literature). As for the dependency on cholesterol above the ∼30% threshold, we acquired data mostly at 33% and 55% cholesterol content, but did not observe large changes in oligomeric populations in this range (compare, e.g., Figure 4 and Figure 4–figure supplement 1).

6) The attachment to the membrane is assumed to be irreversible. What is the basis for this assumption? Is there a conformational change that occurs upon membrane binding? And a second upon oligomerization followed by the final conformational change when the pore is formed?

Firstly, the assumption of irreversible oligomerization is dictated by the presence of a broad maximum for incomplete (but oligomeric) assemblies in the prepore state (Figure 4B): Because the chemical interface between all subunits is identical, this maximum cannot be explained as an equilibrium result, and must thus represent kinetically trapped assemblies, hence irreversible oligomerization.

As for monomer attachment to the membrane, it will eventually become irreversible because of the irreversibility of the subsequent oligomerization. The monomer attachment to the membrane can therefore be approximately described by an effective irreversible rate equation. The above-mentioned assumption of irreversible membrane binding was thus made because it simplifies the model (reducing the number of free parameters) while it is not critical for the modelling results.

7) The assembly itself is assumed not to be cooperative. What about the conformational change from the pre-pore to the pore? Is this a cooperative process?

Our experiments in Figure 4–figure supplement 3 suggest that the prepore-to-pore transition is indeed a cooperative process, as we have now made more explicit in the Discussion (“The activity of wild-type suilysin was greatly impaired…”).