Peer review of: Bacterial polyphosphates interfere with the innate host defense to infection (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

 

In this manuscript, Roewe, et al. investigate the relationship between bacterial polyphosphate 
(polyP) and the mammalian immune response following infection. In particular study addresses the 
important question of how bacteria might be able to counteract the antimicrobial response via 
production of long polyP chains. 

 

The work begins with phenotypic analyses using a sepsis model. The authors demonstrate the ability 
of long-polyphosphates, characteristic of those made by bacteria, to impair survival of mice by 
decreasing myeloid cell response post-sepsis. The reverse also appears to be true -mice infected 
with E. coli that can’t make any polyP (∆ppk) show increased survival. The number of myeloid cells 
and their associated immune by-products (such as iNOS) also are also impacted by exogenous long-
chain polyP and by the ppk status of the E. coli used for infection. 

 

The second part of the workinvolves an RNA-seq analysis of the response to LPS treatment of 
macrophages in the presence or absence of exogenous long-chain polyP. The authors use this 
analysis as a staging point to describe long-chain polyP-mediated inhibition of M1 macrophage gene 
expression and an increase in M2 gene expression, antagonism of type I interferon response and 
decreased MHCII-mediated antigen presentation. 

 

The overall model seems to be that bacteria make long-chain polyphosphates during infection and 
that these polyP chains interfere with various myeloid cell functions to promote E. coli survival at the 
expense of the host. 

 

Overall this is a nice study. The work is generally well controlled, the experiments convincing, and 
conclusions solid. The manuscript is descriptive rather than pinpointing a specific mechanism. 
However, more than anything else, this reflect the state of the field and the inherent difficulties in 
studying polyphosphates in vivo. Despite the lack of mechanism, I do feel that the work represents 
an important contribution. However, there a number of concerns I would like to see addressed 
before the manuscript is suitable for publication. 

 

 

1. The authors find polyphosphate in the peritoneal cavity after infection (extended data 1g). How 
does this compare with un infected mice, and are the chain lengths representative of those 
presumed to be found in bacteria? 

 

Is the model that bacterial polyphosphate is interacting with receptors on the outside of the myeloid 
cells? If so, how do the authors suppose that the polyP is released from the bacteria? 

 

2. The authors should clarify in the main text if polyP concentrations are measured in terms of free 
phosphate units. 

 

3. Figure 1: 

a) There seem to be some inconsistencies in Fig 1 with regards to the survival in response to wild-
type polyP. Can the authors comment on this? 

b) Figure 1B should ideally have a control where long-chain polyP is assayed along-side of the short 
chain. 

c) Can the authors comment further on the drastic impact of PPX (survival 100 %) – Why should this 
effect be any greater than that of the ∆ppx mutant strain? 

 

4. RNA-seq analyses: 

a) Are ‘significant ‘genes, GO-terms and other analyses corrected for multiple testing? 

b) Figure 2a (and other figures similar): Why are the non-significant genes being shown here? What 
would the trends look like if they just showed the significant hits? 

c) Did the genes Arg1, Ym1, Fizz1 appear ‘regulated’ in the RNA-seq? 

 

5. Article format seems too condensed: The relevance and impact of the paper could be made 
clearer by expanding the introduction and discussion. As an example, the work of Terashima-
Hasegawa et al. (ref 21) and how this relates to the current study seems to have been glossed over 
in the discussion. In the results section, subheadings and further details regarding the rationale for 
each experiment, and how these details fit into the context of a broader model are warranted. 

 

6. If space permits, the authors should provide a model for how everything fits together, including a 
discussion of the unknowns (see above), could be provided as an additional figure. 

 

7. Significance lines and asterisks are not always clear in figures. Some lines do not have their own 
asterisks. For example, for Figure 4B NLRC5, is the difference between short and long polyP **, NS, 
or something else. 

 

8. Why are there lines on the WB in Figure 3H? Were the control, LPS and LPS+polyP samples run on 
the same gel, for each antibody used? It would be inaccurate to compare treatment groups if 
samples were not analyzed on the same blots for each antibody. (I think it would be fine to do the 
total and phosho antibodies on separate blots, however). 

 

9. The authors could provide additional references to support their assertion that polyP chains made 
in human cells are ‘short’. Indeed, the Kornberg lab has previously seen longer chains in mammalian 
tissues (PMID:7890711). 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

Production of polyphosphate polymers is a ubiquitous trait of bacteria. In this study, Roewe J and co-
workers describe a role for long polyphosphates in immune suppression by bacteria. They show that 
long polyphosphates either produced by E. coli or exogenously co-administered into mice impede 
LPS-mediated inflammation and bacterial clearance. Overall the manuscript is clearly written and 
well controlled. This is a good study and well should be published in Nature Communications swiftly. 
I only have a few suggestions for improvement. 

 

Main points 

1) The authors have mainly focussed on TLR4-IFN-I signaling. However it seems that the inhibitory 
effect of polyphosphates is global. Do polyphosphate polymers also block other innate immune 
receptor signaling pathways relevant for anti-bacterial immunity for example TLR2 and TLR9 
mediated responses. 

2) One caveat of the study is the lack of defined mechanisms. Whereas the observed effects are 
likely due to overlapping mechanisms as discussed by the authors, can the authors to test whether 
polyphosphates polymers acts by targeting fundamental cellular processes important for receptor 
signaling, for example phagocytosis/endocytosis. 

3) Upon adding on to cells, are polyphosphates internalized? Or do they interact with and block 
immune agonists?