Peer review of: Mammalian histones facilitate antimicrobial synergy by disrupting the bacterial proton (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

This manuscript examines the antimicrobial activity of histones, which are now better known for 
their ability to organize DNA in mammalian chromosomes. Specifically, the authors investigate how 
histones can work together with membrane permeating antimicrobial peptides such as LL37 to 
achieve synergistic antimicrobial effects, such as disruption of PMF and DNA organization. 

 

This paper engages a long standing problem and makes exciting progress. In particular, the concept 
of synergy (such as that between different types of AMPs) have been proposed, but the 
demonstrations are not as nice as that shown here. I do have a number of questions, comments, and 
suggestions, which I hope the authors can take into consideration. 

 

This observation does not disagree or detract from the conclusions in this paper, as histones can 
contribute to antimicrobial activity in multiple ways. However, it is known that fragments of histones 
can be more antimicrobial than complete histones. Histone fragments, often the N-terminal 
fragment, where membrane permeation activity resides, may be more active (and more active at 
elevated salt levels). In a heterogeneous environment such as a lesion or the immediate vicinity of 
NETs, there may be proteases that can create such histone fragments. It may be a good idea for the 
authors to mention this just to cover the possibility. 

 

Lines 124 – 128: The authors go through a chain of logic whereby they show that unlike LL37, 
antibiotics that act via protein synthesis inhibition but do not permeate membranes are not 
synergistic with H2A, which does not permeate membranes under low divalent conditions, therefore 
these observations indicate that the antimicrobial effect of H2A requires membrane permeabilition. 
At this point of the paper, perhaps the authors mean ‘consistent with’ and not ‘indicating’? strictly 
speaking, the conclusion is not possible until the localization studies of H2A done in the next section. 

 

The use of PROPS to monitor PMF is a nice touch. It will be helpful to the reader to point out 
potential limitations of this technique for the present purpose. 

 

There are several points in the paper that can benefit from more clarifying text: For example, the 
PCA for the induced chromosome patterns, and a fuller discussion of the feedback loop model in Fig 
5, so that the reader can better evaluate assumptions etc. 

 

From the conclusions of the present paper, can we then say that an AMP like buforin, which is 
derived from H2A and can permeate membranes, self-amplifying? 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

The manuscript by Doolin et al. focuses on the synergistic anti-microbial effects of histone 2A and 
the AMP LL37 in vitro. It provides a very interesting, novel concept for immune defence and covers 
different aspects of this mechanism. However, some important questions remain unanswered and 
some of the methods should be improved for what the authors are trying to show. 

 

General concerns: 

- All experiments were conducted with H2A. Would the other histones be expected to have similar 
effects? This should at least be discussed. 

- The authors mention NET formation as a mechanism where histones have an anti-microbial role. A 
hallmark of NET formation is global citrullination of histones. Would this alter the here-shown 
mechanism? It would be good to investigate this, especially if the authors discuss NET formation as a 
main source of histones and AMPs. 

Also, other AMPs apart from LL37 should be considered. They are only very generally discussed. 

- How do the bacteria recover from LL37-H2A treatment? The growth curves indicate bacterial 
growth after approx. 15-18 hours -how is this possible? This should at least be discussed. 

- The proposed mechanism of pore stabilization is not at all clear. Is there a direct interaction 
between the molecules? In my eyes, this is the main weakness of the paper. 

- Chromatin rearrangement shown by Sytox Green staining is not convincing. 

- The importance of alterations of transcription (and translation?) at high concentrations of H2A 
remains unclear in relation to the proposed pore-formation action. 

- In vivo toxicity of histones should be considered in the introduction and discussion. 

 

In more detail: 

Introduction: 

- As mentioned above, other functions of histones in vivo and in the immune system should be 
discussed, such as the deleterious effects mediated through TLR2 and 4 (Allam et al., 2012, J Am Soc 
Nephrol., Xu et al., 2011, J Immunol.,) as well as their prominent role in sepsis (Xu et al., 2009, Nat 
Med). Also the role of histones in thrombosis should be considered (in the introduction or the 
discussion). 

 

Results: 

- PI staining of bacteria incubated with H2A under low magnesium conditions: PI-positivity could also 
be the result of the bacteria undergoing apoptosis or of the membrane becoming more permeable 
as a secondary effect. How does this experiment show that H2A is actually inhibiting growth by 
primarily increasing cell membrane permeability, as the authors suggest? 

 

- Figure 1C, figure legend: It would be helpful to indicate at what time point PI-fluorescence was 
measured in the legend. Generally, more information on treatment times in the figure legends 
would be good. 

 

- Figure 1A: E. coli appears to recover and proliferate after an initial inhibition, unlike S. aureus. How 
can this be explained? Is this difference attributable to the gram-status of the bacteria? 

 

- In Figure 1E I find the way the bacteria are attached to each other after H2A treatment very 
intriguing, however I did not understand how this would happen. Is there any way to explain this 
very specific morphology? Especially in light of figure 2, where the authors show that H2A hardly 
enters the cell without formation of pores. How can this be explained? 

 

- In Figure 1E, it might be helpful to show lower power magnifications of the bacterial cultures as 
here it looks like there are more bacteria in the picture with H2A plus LL37 – I assume this is just a 
clustering effect, though? 

Also, please check Figure numbering – there is no Figure 1G, for example but it is mentioned in the 
text. 

 

Figure 1D: 

- While there is an impressive delay in bacterial growth, after about 20 hours bacteria appear to be 
proliferating quite well again – do they develop some kind of resistance mechanism or how is the 
pore formation overcome? 

 

Figure 2A: 

- Fluorescence of bacteria treated with H2A alone should also be shown. 

To claim that H2A enhances/facilitates pore formation by LL37 I don’t think it is sufficient to measure 
cytoplasmic fluorescence of labelled LL37. Rather, it would be very helpful to actually show 
membrane localization of LL37, even if only in a semi-quantitative way, by higher resolution 
techniques. 

 

- How would H2A stabilize LL37 pores? Would the two proteins interact directly, both of them being 
cationic? Has this been shown or can this be modelled? As the authors are stressing this synergistic 
action very strongly, more information on how this would occur needs to be provided. Would this 
interaction not change upon citrullination of histones in NET formation? 

 

Figure 4B: 

- The “reorganization of the chromatin” is not convincing. How do the authors know this is truly a 
reorganization and not just steric/static interference of H2A with the binding of Sytox Green? 

- Also, both the text in the Results section and the figure legend should mention for how long the 
cells were treated with H2A before the principal component analysis was performed. 

- Finally, is there a way to perform a more functional analysis of the chromatin – as far as I 
understand, the principal component analysis mainly focuses on the staining pattern of the 
chromatin? Again, changes in this analysis might as well show altered staining behaviour instead of 
true reorganization of the chromatin. 

 

Figures 4D and E: 

- How is mCherry fluorescence a specific indicator of transcription? A decrease in fluorescence could 
also indicate less translation, could it not? 

- In Figure 4E, much higher concentrations of H2A than in the other experiments were needed to 
achieve an effect. Are these concentrations likely to be reached under physiological conditions? Do 
the authors think this is a relevant mechanism of action, if the other effects (pore stabilization, 
membrane depolarization) are seen at much lower concentrations? 

- Why was RNA synthesis not investigated after treatment with LL37 plus H2A? As all other 
experiments were conducted in this fashion, this should be added. 

 

Diuscussion: 

- The authors formulate a general defense mechanism for AMPs together with H2A. Other AMPs 
with similar effects should at least be discussed, if not shown in a proof of principle experiment. 

- As mentioned above, histones mediate significant toxicity in vivo, leading to organ damage, sepsis, 
thrombosis etc. This should be discussed here if application of histones is proposed as an 
antimicrobial therapy. Also, it should be considered that binding of histones and AMPs to NETs may 
be a mechanism to prevent generalized spread of histones (anti-inflammatory mechanism of 
aggregated NETs). 

 

 

Minor: 

In the methods section, the authors sometimes use µl and sometimes ul. 

There is no figure 1G, although it is mentioned in the text. 

Line 257: delete the “a”. 

 

 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

In this article, Doolin and collaborators explore the interactions of the histone H2A and the 
antimicrobial peptide LL-37. Co-treatment of E. coli with these two cationic proteins potentiates 
delayed bacterial growth, increased uptake of PI, and membrane blub formation. The results are 
expected since both antimicrobial proteins are well documented to act on the cytoplasmic 
membrane. The manuscript was hard to follow. Some of the supplementary figures were not 
commented, and main figures were out of order. Important experimental details were not included, 
making it difficult to interpret the data. Some of my concerns are: 

 

Fig. S1A is not commented in main text. Similar analyses should be presented as measure of 
antimicrobial activity of LL-32 and H2A. Figure 1E is wrongly referenced. Some of the primary data 
are shown for the first time in Discussion. 

 

Fig 1G is missing. SEM images don't clearly show membrane disruption. It is surprising that 
membrane blubs are not seen by SEM, when they seem to be formed in the bright fields. 

 

Not clear how cell size was calculated. 

 

Increased antimicrobial activity of H2A in low magnesium is likely due to disruption of outer 
membrane and increased uptake of H2A into periplasm. This should be tested. 

 

The experiments with kanamycin (50 ug/ml!) are hard to interpret. At these high concentrations 
kanamycin causes death. The experiments should be done with doses closer to MIC. By the way, 
aminoglycosides cause membrane disruption through mistranslation of proteins. If the proposed 
model is correct, one would expect synergism between Km and H2A. 

 

Experiments in Figure 3 and 4 are missing important controls. What are the phenotypes of cells 
treated with LL-37 or H2A by themselves? 

 

The authors claim that H2A stabilizes pores formed by LL-37. However, the data do not conclusively 
show such an mode of action. It is equally possible independent pores by both antimicrobial 
peptides work together. 

 

Most antimicrobial activity emanating from cotreatment of E. coli with LL37 and H2A appears to be 
manifested in the form of bacteriostasis, raising concerns about therapeutic potential as claim in the 
abstract. 

 

Figure 4A is unnecessary, since it is well known histones bind to DNA. The data could be moved to 
supplementary information. 

 

Line 201, Principal component analysis should be explained for people unfamiliar with the 
procedure.