Peer review of: Glycan-dependent cell adhesion mechanism of Tc toxins (2019)

Reviewers' comments: 
Reviewer #1 (Remarks to the Author): 

Tc toxins are a type of toxin found in insect and human bacterial pathogens. Tc toxins are 
composed of a pentameric membrane translocation component (TcA) and a cocoon-shaped TcB 
and TcC. The toxic enzyme that is held in the cocoon enters a translocation channel created by 
TcA. Once the holotoxin is bound to cell membrane it is endocytosed and acidification in the late 
endosome triggers the opening of the cocoon shell and the formation of a membrane channel that 
allows for the translocation of the toxin into the host cell. There have been structures of the 
holotoxin in various pre-pore and pore conformations (most determined by this laboratory); 
however, less is understood how the toxin is recruited and engaged at the cellular membrane. This 
manuscript focuses on isolating Tc holotoxin cell surface receptors and then understanding how 
the Tc toxin binds to the receptors. The authors identify heparins and Lewis antigens as receptors 
for Tc toxins from insect- and human pathogenic bacteria. They then use glycan arrays and cryo-
EM structural analyses to characterize how the identified receptors interact with the toxins. These 
analyses shows that the glycans (heparins/heparan sulfates) bind TcAs with the alpha-helical part 
of the shell domain, while the Lexis antigen (predicted to be similar to glycosylation found on 
receptors) interacts with the receptor-binding domain of the toxin of P. luminescens TcdA1. From 
these results the authors propose a two-step association model, at least for Pl TcdA1, were first 
the toxin interact with the heparin glycans then is able to bind the Lewis antigen (Lewis X). This is 
paper presents strong experiments and structures that provide new mechanistic information on 
how Tc toxins interact with cellular membranes. 

Suggestions: 
1. All the Tc toxins tested in this study appear to bind heparins, but only Pl TcdA1 appears to bind 
the Lexis-X antigen. There are a number of Tc toxin structures that have been determined and 
definitely the sequences are known. The authors should comment in the discussion on whether the 
binding pockets they have identified in this study exist (or are predicted to exist) in other Tc toxins 
(i.e. will this be a conserved mechanism for all Tc toxins or only a subset). This would provide a 
broader context to their proposed “two-step” model for Tc toxin interaction with the membrane. 

2. The authors use mammalian cell lines to test for binding to glycans. Is there a reason why the 
experiments were not done with bacteria instead – since these toxins infect bacteria? 

3. In Figure 1, the cell microscopy experiments are done at 4h and 8h time points. This would 
measure endocytosis (and intracellular degradation?) rather than the ability to interact with cell 
membrane interaction. From the methods it appears the FACs sorting is done only 15 minutes. 
Were the cells kept on ice to inhibit endocytosis? The authors should comment on how they are 
sure that their cellular assays (especially at the 4 and 8 hour time point) are accurately measuring 
the ability of the Tc toxin to interact with membrane, rather than being endocytosed and degraded 
within the cell. 


Reviewer #2 (Remarks to the Author): 

This paper sets out to determine the receptors responsible for cell recognition by ABC-type Toxin 
Complexes (Tc). Using glycan arrays, the authors determine that both Lewis antigens and sulfated 
heparans can bind to different sites on the A subunit of the Tc, and therefore propose a two-step 
mechanism, whereby the TC first binds to heparan sulfate and then to a Lewis antigen sugar. This 
subject is an important one, as the cell-surface receptors for the ABC Tcs are unknown, and their 
identification remains a key bottleneck in understanding the cell tropism of this family of bacterial 
toxins. 
The first half of the paper describes the identification of a subset of Lewis antigen oligosaccharides 
as specific ligands for the Receptor Binding Domain (RBD) D of the TcdA subunit of the Tc from 
Photorhabdus luminescens. The work is both carefully carried out and convincing, and it is clear 
that the core unit recognised is the Lewis X trisaccharide, and that the beta-4 linkage between N-
Acetylglucosamine and galactose is essential for tight binding. 

A few things would improve this section: 

1) It should be made clearer exactly which saccharide is immobilised to BSA for binding 
experiments - the Lewis X trisaccharide (compound 7). This detail is buried in the methods section 
and should be more clearly stated in the results and in Figure 2. 

2) p7, line 140 - high amounts or high concentrations? I suspect the latter is what is meant. 

3) The binding site for the trisaccharide is reasonably well described in Figure 3d and in the text, 
but I fell a great deal more information could be readily obtained by some more comparative 
analysis. For example, is the identified binding site also seen in the other RBDs of the P. 
luminescens A protein? Is the site conserved in the A subunits from other species? Lewis X sugars 
are known to be receptor binding sites for other bacterial pathogens. How does the identified 
binding site compare to other known examples of Lewis X binding? 

The second half of the paper describes the identification of heparin and heparan sulfate binding to 
several TcA subunits from different species. In comparison with the first half of the paper, this 
second section has a number of problems. To begin with, the rationale for choosing to screen 
heparan oligosaccharides is not clearly described. The initial broad screen is a self-evident place to 
start, but the rationale for focussing on heparans is not clear, especially as the initial screen 
contained heparans (compounds 183-186), but they did not show any binding. In fact, the binding 
of the P. luminescens TcA in Figure 4 seems at odds with its lack of binding to the same sugars in 
Figure 2, though it is difficult to be clear about this as the numbering scheme for the compounds 
does not seem to be consistent. 

The binding of TcA subunits is strongly electrostatic, and I wonder if enough controls have been 
put in place to be sure that it is specific? What are the pI values for BSA and WGA? Do either of 
them contain surface patches containing K or R residues? The inclusion of a known heparan 
binding protein as a positive control would help put the level of binding signal in context. 

In contrast to the data presented for the Lewis X binding, the quantitation of heparin binding by 
BLI is not reliable. The fitting of the model 1:1 binding model to the data in Supplementary Figure 
5 has failed, and the sub-nM estimate of KD is almost certainly wrong. More than that, the dose-
response curve for binding is not credible, with 63, 125 and 250 nM concentrations of heparin all 
giving the same equilibrium binding response. Would it be possible to quantitate binding (using BLI 
or SPR) with a defined heparan sulfate sugar? This might remove some of the uncertainty 
introduced by using heparin. 

The identified heparin binding site is a surprise, given that it is is the alpha-helical domain of the 
shell of TcA, rather than in any of the putative RBDs. Is the positively charged cleft identified in 
Mm-TcdA4 conserved in the other TcA subunits? Is that area also seen in Type II (chitin 
containing) Tcs? 

Given these reservations about the heparin binding, I am somewhat skeptical about the proposed 
two-step binding model. In particular, the idea that heparin binding is the initial contact with a 
sub-nM KD seems at odds with the idea that the uM binding of the Lewis antigens allows laterally 
mobile, dynamic binding of TcA to the cell surface. 

Finally, it is not made clear if the observed glycan binding patterns are congruent with, and make 
a rational explanation for, the known cell tropism of different ABC toxins for different eukaryotic 
cell types. 

Shaun Lott, University of Auckland.