Peer review of: Dead cells release a ‘necrosignal’ that activates antibiotic survival pathways in bacterial (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This is an interesting study that demonstrates a novel “necrosignaling” moonlighting function for AcrA 
released by dead cells to stimulate drug efflux in swarming cells of E. coli. This is a comprehensive 
study that not only demonstrates necrosignaling as a mechanism of drug resistance but also identifies 
the released factor and mechanism of signalling by binding to TolC. I have a few minor comments for 
consideration: 

1. The use of acronyms SR and STRIVE interchangeably makes this manuscript difficult to follow. The 
STRIVE terminology is unnecessary. The abbreviation SR is also unnecessary.“Swarming resistance” 
would be sufficient throughout. If keeping SR then this should be defined in the Main text at first 
appearance. 

2. Figure 1 legend- line 228- insert the word “Figure” so reads “…Figure S1A…” 

3. Line 132. Insert the word “of” so that sentence reads “..we tested the effect of both deleting….” 

4. Line 146- what evidence is there that FM 1-43 is specific for OM labelling? It could also be labelling 
the IM and due to the resolution limit of these images is not possible to distinguish where the Q-dot 
labelled AcrA is located on or in the cell. These images show only if there is localisation of the Qdot 
labelled AcrA to the bacterial cell. It is not necessary to over-interpret these images. The subsequent 
trypsin experiments are sufficient to demonstrate extracellular binding of AcrA. 

Reviewer #2 (Remarks to the Author): 

Swarming is a way of collective surface movement executed by several bacterial species. Adaptation 
to antibiotics can be impacted by swarming, albeit the exact molecular mechanisms remain widely 
elusive. In the present manuscript, the authors suggest that AcrA proteins from death cells can act as 
signal to trigger drug efflux in alive cells through TolC. While this finding (if true) might mark an 
interesting conceptual advance, I have several major comments and questions that require answers 
before I can recommend publication. 

In supplemental Fig. 1, the authors show a live/dead screen. However, even in the Kan25 sample the 
green fluorescent signal seems reduced to me. I do not see a convincing difference. Moreover, the 
whole figures miss error bars and statements for the statistics. 

In Fig. 1b, the authors show ‚Dead cells’/Kan250. However, the authors never show that the cells are 
indeed ‘dead’ after the kan250 treatment. I clearly miss controls here? The authors should clarify of 
whether a suspension of dead cells was applied or only the supernatant. In the next experiment, the 
only compare supernatant + proteinase K treatment. So what was done? Otherwise, the authors 
would mix apples with pears. 

Fig 1 c/d: Clearly misses the wildtype control! 

Fig 1e. How were the dead cells killed? Original data are missing. It is hard to judge the quality of the 
data from what is presented! 

Can the authors comment why they didn’t show the influence of Crp? This protein also popped up in 
both tested organisms? It is in the text just stated as “data not shown”. 

How was AcrA purified? The authors must present the quality o their purified protein. Otherwise, it 
cannot be excluded that contaminants are the cause of the effects/observations. 

I’m missing a list of the used strains and plasmids – what were the antobiotic resistances of these? 
Especially, the tolC mutant? They authors must state that – it is hard to judge the quality, 

In order to make the claim that AcrA is interacting with TolC, the authors must show a direct binding. 
This can easily be done by an in vitro approach. 

Figure S8. Again wild type control is missing! Why do some plates show inhibition of swarming at low 
Kan concentrations, but weaker at higher concentrations? e. g. Arg55Leu or N100 

Fig 2. Were the protein expression levels of the protein mutants the same? Please show a 
quantification (e.g. by western blotting). 

Fig. S9 Why are planktonic cells much smaller? Is this a general feature? Please comment. 

Figs. 2d/e . Again controls are missing! Either show Qdot705 without AcrA or AcrA without FLAG tag. 
In other words, can you exclude that Qdot705 is no-specifically interacting with the cells and/ or TolC? 

Fig. S12. Please provide the raw data. 

How can the claimed results be connected to chemotherapy-resistant cancer? That seems very 
speculative to me! At least remove that claim from the abstract. 

Reviewer #3 (Remarks to the Author): 

The manuscript describes a mechanism for the adaptive antibiotic resistance in swarming cells (called 
SR) of (mostly) Escherichia coli. Specifically, the authors demonstrate that cell death in a sub-
population of swarming bacteria leads to the release of a "necrosignal" that increases survival of the 
remaining cells in presence of lethal and elevated concentrations of antibiotics. The authors identify 
AcrA as the necrosignal released from dead cells and show that it binds to TolC to mediate SR. They 
identify the region of AcrA required for this function, show that tagged AcrA binds to the outer 
membranes of cells in a manner that depends on the presence of TolC and of specific residues on TolC. 
Last they also show that swarming cells are pre-programmed to be less permeable, have increased 
expression of efflux pumps and ROS pathways, which could contribute to increased antibiotic 
resistance. 

The manuscript is generally well written and it provides a compelling set of interesting data. However, 
there are instances where the experimental design is not clear and where some interpretations should 
be either modified, clarified or re-evaluated. 

1. The authors use the term "necrosignal" for the role of AcrA but whether it is acting as a signal 
seems to be true only for swarming cells and perhaps also, only for some antibiotics. First, unless I am 
mistaking, it appears that the antibiotics used here are effluxes from the cells using the AcrAB/D-TolC 
efflux pump. This begs the question of whether the role of AcrA as a "necrosignal" applies to 
antibiotics that would not be effluxed in the same way and whether this is a very specific effect of 
AcrA that reveals some basic structure relationship with resp[ect to its interaction with TolC. The 
structural homology with Colicin E3 is intriguing in that respect. 

2. Related to the above comment, the authors show that addition of AcrA to swarm cells induced the 
expression of a overlapping subset of genes which those which expression is modulated by the 
addition of antibiotics. However, the effect of adding AcrA to planktonic cells on these genes is not 
tested and in fact, per Fig S7, the addition of AcrA to planktonic cells only promotes a small increase 
in survival of cells treated with Isn concentrations near MIC99. If AcrA is to be a "necrosignal", 
wouldn't it be expected to also function on planktonic cells? As the authors state, (lines 126-127), the 
result in Fig. S7 suggest that the physiology of swarming cells play an important role (which they go 
on to investigate in the next paragraph). In light of the findings of the interaction with TolC and the 
presence of tagged AcrA on the OM, wouldn't this suggest that the "necro-signal" is in fact not AcrA 
but the pair AcrA-TolC? 

3. The binding of AcrA to TolC is suggested by a combination of microscopy and enzymatic treatment 
of cells with trypsin, with cellsa also expressing mutated versions of TolC. The contrast (or size) of 
images shown in Fig 2 should be increased. A scale bar should also be added to these figures. What 
was the resolution? Any possibility to have an idea of the stoichiometry of binding? 

4. The conclusion that release of AcrA by dead cells and its binding to TolC on live cells to instantly 
stimulate efflux is not a straightforward conclusion that can be drawn from the data shown in Fig. 2d 
and 3b. The data may be consistent with this model but there could also be alternative explanations 
such asthat could be missed with the resolution and methods used here- perhaps the binding is very 
weak and only (larger) cells that express more TolC are seen with binding to AcrA. How far would AcrA 
diffuse before being proteolytically cleaved? Is AcrA released by cells which have not completely lysed 
yet?These and perhaps many other considerations should be used to generate more circumspect 
conclusions. 

5. The results on ROS activation and the effect of hydrogen peroxide-mediated killing of cells are not 
discussed much but the authors emphasize them in the discussion section. Should a few comments be 
added here? Similarly, the structural homology of AcrA with colicin B3 is intriguing and only appears 
as a sentence in the discussion. 

6. Minor comments: 
Line 431- delete "," after "section" 
Line 462- what is PPB?