Peer review of: Mode of action of teixobactins in cellular membranes (2020)

Peer Review File 

Reviewers' comments first round: 
 
Reviewer #1 (Remarks to the Author): 
 
This is an impressive paper that should be published after revision. The authors perform detailed 
NMR-based structural studies of a teixobactin analogue (Arg4,Leu10-teixobactin) and characterize 
its interactions with lipid II. Distance restraints from the NMR studies allow a molecular model for 
the interaction with the lipid sugars and pyrophosphate to be created, as well as molecular model 
for the necessary self-assembly of the teixobactin analogue. These results are interpreted in light 
of a known crystal structure and SAR studies. 
 
Additional studies with lipids derived from a model organism (M. flavus) in anionic membranes 
show weaker binding than expected. Based on this observation and additional NMR studies suggest 
a model in which teixobactin forms aggregates on the cell surface. The authors also observe its 
aggregation on vesicles through fluorescence microscopy. 
 
The title of the paper is misleading, for two reasons, claiming teixobactins and cellular conditions. 
These statements should be replaced with more specific language, such as “a teixobactin 
analogue” or “teixobactin analogues” and “with lipid membranes.” The analogues the authors 
studied, Arg4,Leu10 and Leu10-teixobactin, lack the native enduracididine at position 10. Using 
“teixobactin analogues” will clarify to the reader that the authors used analogues that lack 
enduracididine. 
 
Figure 1g: a negative control is recommended for the fluorescence microscopy experiment to 
confirm if the lipid II clusters reflect sequestration due to antibiotic action. An inactive teixobactin 
analogue, such as an N-terminally truncated teixobactin or an acyclic teixobactin, should not result 
in micron-sized clusters. 
 
Figure 3c and Lines 203, 214-215: change “D-N-Met-F1” to “D-N-Me-F1” since Met can be 
confused with methionine and since Me is a common abbreviation for methyl. 
 
Figure 4a: if possible, cell membranes from another Gram-positive bacterium should be used to 
further support 2D 15N1H ssNMR data. 
 
Line 302: specify that M. flavus is a Gram-positive bacteria 
 
Line 311: should be rationale, not rational 
 
Line 506: ideally these measurements should be done in triplicate. 
 
Line 542: specify the emission range for fluorescence detection. 
 
Morris et al. very recently reported the molecular localization of a fluorescently labeled teixobactin 
analogue in Gram-positive bacteria using fluorescence microscopy (ACS Chem. Biol. 2020, Just 
Accepted). The imaging studies therein show that the teixobactin analogue has two staining 
patterns: (1) teixobactin aggregates that result in whole-cell staining in the absence of polysorbate 
80; and (2) lateral and septal staining of cells in the presence of polysorbate 80. The whole-cell 
staining observed in the absence of polysorbate 80 is congruent with the authors’ findings that the 
teixobactin analogue forms micron-sized clusters. This paper could be cited in support of the idea 
of clustering of PG precursor molecules in the membranes of bacteria. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
Dr. Weingarth and coworkers have provided a high-resolution view of a complex in which 
teixobactins is binding either model membranes or cellular membranes. The authors have found 

that teixobactins capture lipid II in micrometer-sized clusters on membrane surfaces and proposed 
a structural model of teixobactin-lipid II complex. Another major and suprising finding is the 
weak/moderate interactions between teixobactin and lipid II interactions in cellular membranes, 
which suggests that direct binding to cell wall precursor is not the only mechanism for 
antimicrobial activity. 
 
Information such as that was unavailable and has the great potential of revising our view of the 
functional mechanism of teixobactins and their analogues. The NMR data is of high quality and 
supports the conclusions. Below are multiple minor concerns that could help improve the 
manuscript further. 
 
1) Some strong statements need re-consideration. 
First, in the abstract, the authors state that “direct interaction with cell wall precursors is not the 
principal killing mechanism.” With the current data, it will be more accurate to claim that it is “not 
the sole mechanism.” 
Second, a minor concern is the statement of “cellular condition” in the title, figure captions, and 
the text. As what is used in the study is actually membrane vesicles prepared from M. Flavus, it 
might be more specific and more appropriate to use “cellular membranes” instead. 
 
2) The manuscript needs a dedicated Discussion section to summarize our current knowledge as 
well as the ambiguous aspects of the action mode of teixobactins, which should be coupled with a 
discussion of the novelty of the findings in this study. This part of information is currently missing 
in the writing. 
 
3) The experimental data, especially the solid-state NMR data, are of high quality and are 
presented well in the maintext and the Extended dataset. The extended data will need to be 
modified to Supplementary Information and follow the format and size requirement. 
Also, there are a few technical details to be clarified: 
Line 573: it will be helpful to put down the rationale for choosing a slow spinning for the TOBSY 
experiment. 
Line 625: what is the microwave power of the gyrotron used for the DNP experiment? 
 
4) Minor format issues such as inconsistency in the use of units or journal name and page number: 
Line 353, 420, 427, 429, 434, 530, 553, 585, 909, 914, 925 (please check through the 
manuscript) 
Also, Line 123, Extended data Fig. 4 showed up before Fig. 2-3. Please Adjust the order.