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


 

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

The authors answered my questions to my satisfaction. 

 

The only point I want to posit is a comment in support of the authors on the relation between 
synergy/self-amplification and the role of NETs and citrullination, in questions raised by one of the 
other referees. Citrullination decreases charge on LL37 and histones and will necessarily negatively 
impact both their membrane remodeling ability and the DNA condensing ability of the latter. I don’t 
think this is necessarily detrimental to the authors’ argument: Components of NETs are widely 
implicated in autoimmunity. Having say PAD enzymes to citrullinate NETs components may be a 
natural way to modulate and control their activity. In any case, more work needs to be done in the 
field for this quite interesting and incompletely understood topic. 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

The authors have made a significant effort to answer all previously raised concerns. The manuscript 
has certainly improved by the addition of many additional aspects and better controls, such as a 
second class of AMPs and by studying the effect of histone citrullination, just to give two examples. 
Some questions still remain (including the exact mechanism of pore stabilisation), however the 
discovery and characterisation of the synergistic effect of AMPs with histones is quite striking and, in 
my eyes, merits publication in Nature Communications. 

 

The only aspect I still found somewhat unsatisfying was the novel LL37 staining. The authors here 
state that the cytoplasmatic LL37 may have been swamping the membrane-bound LL37, however, as 
far as I can see, no confocal microscopy was performed? This would allow for a better visualisation 
of membrane-bound versus intracellular LL37. If the authors are worried about a transient 
localisation of LL37 to the membrane, perhaps a time-course could be performed? With and without 
histones? Also, higher resolution imaging of the membrane-bound LL37 should be evaluated 
(STORM/STED/EM with labelling?), although I understand this might not be feasible. I am sorry to 
emphasize this point so much but I think much of the here-presented chain of arguments strongly 
relies on this pore formation. 

 

Other than that, I support publication in the journal and have no additional comments. 

 

 

Reviewer #3 (Remarks to the Author): 

 

The authors have done a very nice job at responding at my criticisms. The data provide a model for 
the apparent lack of antimicrobial activity of histones in culture at physiologic cation concentrations 
and show previously unsuspected, multifactorial interactions between histones and antimicrobial 
peptides. I still have a few minor concerns that should be considered before I endorse this paper for 
publication in Nat Communications. 

 

Combined treatment of LL-37 and H2A considerably reduces CFU counts. These insightful data 
should be shown as part of figure 1 and not supplementary information. 

 

Electroporation of H2A results in a marked lag phase. It is not clear if this is due to bacteriostasis or 
killing. Simple plating assays would not only discern between these two options but also inform 
about the relative weight membrane damage and DNA remodeling have on the antimicrobial activity 
of histones. 

 

Line 165, it is true that some inhibitors of translation such as tetracycline or chloramphenicol do not 
affect membrane permeabillity. However, some classes of aminoglycosides do, as shown in 
references 61-63. The relatively low concentrations of kanamycin together with the short incubation 
times might have contributed to the negative results. Given these concerns I am still skeptical about 
the interpretation of this set of experiments. 

 

I find the upregulation of colonic acid biosynthesis genes very interesting. The authors should 
consider that colonic acid helps Salmonella maintain the PMF (PMID 28588134). In other words, 
upregulation of colonic acid may represent an adaptation to drops in PMF.