Author response:
The following is the authors’ response to the original reviews.
We greatly appreciate the efforts of the reviewers, which have provided insightful and helpful comments to improve the manuscript. The feedback touches upon a number of topics, focusing on clarification or justification of experimental techniques and on understanding the mechanism by which P. aeruginosa detects HOCl. All reviewers raised the issue of how HOCl activates fro expression, including whether free or protein-bound methionine, cysteine, or other HOCl byproducts induce this expression. For the upcoming revision, we plan to perform experiments that address this issue and will discuss potential mechanistic models in light of the new data. In addition, we plan to perform additional experiments to address a reviewer’s concerns regarding the dependence of the fro response on HOCl production by neutrophils. The revision will correct imprecise statements pointed out by reviewers, and address all remaining issues requiring clarification or further discussion, including the range of HOCl sensitivity, relationship between HOCl and flow sensitivity, and justification for testing the fro response to nitric acid.
We have completed a number of experiments and responded thoroughly to reviewer comments below. We thank the reviewers again for their details comments, which suggested additional experiments and interpretations that have resulted in significant additional insight into the potential mechanism of HOCl sensing and its relevance with neutrophils.
Reviewer #1 (Public review):
Summary:
Foik et al. report that hypochlorous acid, a reactive chlorine species generated during host defense, activates the transcription of the froABCD in P. aeruginosa. This gene cluster had previously been associated with a potential role during the flow of fluids and appears to be regulated by the sigma factor FroR and its antisigma factor FroI. In the present study, the authors show that froABCD is expressed both in neutrophils and macrophages, which they claim is likely a result of HOCl but not H2O2 production. Fro expression is also induced in a murine model of corneal infection, which is characterized by immune cell invasion. Expression of the fro system can be quenched by several antioxidants, such as methionine, cysteine, and others. FroR-deficient cells that lack froABCD expression during HOCl stress appear more sensitive to the oxidant.
Strengths:
The authors provide a number of data supporting their claim that transcription of the froABCD system is induced by reactive chlorine species. This was shown by RNAseq, qRT-PCR, and through microscopy using a transcriptional reporter fusion. Likewise, elevated expression of froABCD was shown in vitro and in vivo, excluding potential in vitro artifacts. The manuscript, while mostly descriptive, is easy to follow, and the data were presented clearly.
We greatly appreciate the efforts of the reviewer and thank them for their succinct summary of the manuscript.
Weaknesses:
(1) Lines 60-62: Some of the authors' conclusions are not supported by the data and thus appear unfounded. One example: "we determine that fro upregulation.....These data suggest a novel mechanism..." Their data do not show that MSR upregulation is a direct effect of FroABCD. Instead, it could be possible that the FroR sigma factor also controls the expression of msr genes, which would be independent of froABCD.
We thank the reviewer for pointing out this important distinction. We have clarified in lines 63-65 in the clean version of the revision that MSR upregulation depends on FroR rather than FroABCD.
(2) The authors show increased fro transcription both in neutrophils and macrophages; however, the two types of immune cells differ quite dramatically with respect to myeloperoxidase activation and HOCl production.
Neither has this been discussed nor considered here.
We agree that the distinction between the cell types is important and have added a brief description of the differences in respiratory bursts and ROS production between the two cell types and our justification for focusing on neutrophils in lines 102-103. We think it’s very interesting that Fro appears to be activated by macrophages, which are not associated with HOCl production on their own. We think that it would be interesting to identify what is inducing Fro in macrophages in future work.
(3) With respect to the activation of fro expression upon challenge with conditioned media from stimulated neutrophils, does the conditioned media contain detectable amounts of HOCl? Do chloramines, which are byproducts of HOCl oxidation with amines, also stimulate expression?
This is an excellent question that addresses which molecules Fro is responding to from neutrophils. We have performed additional experiments that confirm that PMA-stimulated neutrophils produce HOCl (Fig. S2) through the use of a commercial hypochlorite sensor assay, which claims high specificity for detecting HOCl. We further confirmed that this production is inhibited by pretreatment with the MPO-specific inhibitor 4ABAH. These data support the interpretation that the Fro response to stimulated neutrophils requires MPO activity, of which the major product is HOCl. We have described this in lines 117-127.
Our data does not exclude the possibility that other MPO products could activate Fro expression. We were unable to obtain a reliable source of the major secondary MPO product, taurine chloramine, for our experiments, unfortunately. We believe that understanding the potential for secondary products to activate the response is an important and interesting question that can be explored in a future study. We have added a discussion of this in lines 367-376.
(4) A better control to prove that this fro expression is indeed induced by HOCl in activated neutrophils would be to conduct the experiments in the presence of a myeloperoxidase inhibitor.
We thank the reviewer for raising this point. We have performed the suggested set of experiments and found that indeed, the pre-treatment of neutrophils with MPO inhibitor 4-ABAH prior to PMA stimulation suppresses the activation of fro (Fig. 2D and Figure 2-figure supplement 1-2). The results are discussed in lines 117-127.
(5) The work was conducted with two different P. aeruginosa strains (i.e. AL143 and PAO1F). None of the figure legends provides details on which strain was used. For instance, in line 111, the authors refer to Figure S1B for data that I thought were done with PAO1F, while in 154, data were presented in the context of the infection model, which was conducted with the other strain.
We thank the reviewer for pointing this issue out. We have ensured that strain names appear in all the revised figure legends. To clarify, only mouse experiments and a related RT-qPCR assay used strain PAO1F due to prior IACUC approval of this strain and its use in previous publications.
(6) It would be good if immune cell recruitment at 2hrs and 20hrs PI could be quantified.
We previously quantified neutrophil recruitment at the site of corneal abrasion at 24 hours using the same conditions and strains (Ratitong, B. et al., J. Immun, 2022). While we do not have immune cell recruitment data for the 2 hr and 20 hr time points, the previous data show significant neutrophil recruitment near the latter time point, which is consistent with the interpretation that fro expression is activated by stimulated neutrophils. We have discussed this in lines 212-216.
(7) The conclusions of Figure 4 are, in my opinion, weak (line 187-188; "It is possible that ....."). These antioxidants likely quench the low amounts of NaOCl directly. This would significantly reduce the NaOCl concentrations to a level that no longer activates expression of fro. There is no direct evidence provided that oxidized methionine induces fro expression. Do the authors postulate that this is free methionine, or could methionine and/or cysteine oxidation in FroR increase the binding affinity of the sigma factor to the promoter? Another possibility is that NaOCl deactivates the anti-sigma factor. None of these scenarios has been considered here.
We acknowledge that our model of HOCl sensing was unclear and thank the reviewer for their insight. This critique is echoed by reviewer #2 in comment 3 as well. We recently found that the FroI anti-sigma factor has the highest concentration of methionine and cysteine residues of all known P. aeruginosa anti-sigma factors (Appendix 2—Table 1). Given that FroR and FroI form an extracytoplasmic function sigma – anti-sigma pair, which are associated with transducing extracellular signals to the cytoplasm, we have proposed an alternative model in which HOCl or secondary RCS molecules are detected through their oxidation of cysteine and methionine residues in FroI. This is discussed in lines 271-279 and lines 363-367.
(8) Line 184: The reaction constants of HOCl with Cys and Met are similar.
We thank the reviewer for pointing out this important clarification. We have revised the sentence to accurately reflect this in lines 243-244.
(9) Treatment with 16 uM NaOCl caused a growth arrest of ~15 hrs in the WT (Figure 5A), whereas no growth at all was recorded with 7.5 uM in Figure 3A.
We thank the reviewer for catching this. We have determined that the concentration of NaOCl in the reagent used for this particular experiment was lower than expected, thus requiring a higher concentration to achieve growth inhibition. We have repeated the experiment with new reagent and find that the results (now Figure 6A) are similar to the previous experiment but at a lower concentration of 4 micromolar, consistent with the concentration found to be sub-inhibitory in Figure 3A.
(10) The concentration range of NaOCl causing fro expression is extremely narrow, while oxidative burst rapidly generates HOCl at much higher concentrations. This should be discussed in more detail.
We appreciate the reviewer’s comment, which is related to reviewer #2’s comment #9. We have clarified the reported production rates of HOCl, which far surpass the bacterial MIC. After greater consideration, we believe secondary HOCl products including taurine chloramine could have a more significant role in vivo. While this molecule is less potent than HOCl, it is longer-lived, retains bactericidal activity, and retains the ability to oxidize methionine. We have discussed this in lines 377-405.
Reviewer #1 (Recommendations for the authors):
(1) Some statements in the text don't match the data shown in the Figures. For instance:
(a) Figure 2B shows ~65-fold fro expression, but the text states: "...increased expression of fro expression by 30fold..."(line 88).
The YFP/mCherry value of PMA-stimulated is 67.4 in the Figure (now Figure 2C). The fold-change is computed relative to unstimulated conditioned medium (third column, which has a value of 2.2), which is a 30-fold change. We have added a citation in the main text to the Source Data, which provides these raw values, to help clarify the computation for readers, and added in the legend that the value for unstimulated is greater than 1.
(b) Figure 3B shows ~35-fold fro expression at 1 uM NaOCl, but the text states: "...NaOCl increased fro expression by up to 74-fold..."(line 110).
We have clarified that the increase is relative to untreated, added that untreated value is below 1 in the caption, and provided a citation in the main text to the Source Data, which contains the raw values. The change is measured relative to untreated, for which the YFP/mCherry value is 0.48. The value at 1 uM is 35.7, giving a 74-fold change.
(2) Line 229: While the ∆froR strain was sensitive to HOCl, the strain was tolerant". Please revise.
We have corrected this typo (now lines 328- 329). We meant to convey that growth was not entirely inhibited in the froR strain.
Reviewer #2 (Public review):
Summary:
Foik et al. studied the regulation of the fro operon in response to HOCl, an oxidant derived from immune cells, especially neutrophils. They use a transcriptional fusion of YFP to the froA promoter in an mCherry-expressing P. aeruginosa strain to determine fro-induction under the microscope. They use this system to study fro expression in medium, in the presence of neutrophils and macrophages, neutrophil-conditioned medium, and several chemical stimuli, including NaCl, HOCl, hydrogen peroxide, nitric acid, hydrochloric acid, and sodium hydroxide. They also use a corneal infection model to demonstrate that froA is upregulated in P. aeruginosa 20 h post-infection and perform transcriptional analyses in WT and a froR mutant in response to HOCl.
Strengths:
Their data clearly shows that HOCl is a strong inducer of the fro Operon. The addition of HOClquenching chemicals together with HOCl abrogates the response. They also show that a froR mutant is more susceptible to HOCl than WT. Their transcriptomic data reveal genes under control of the FroR/FroI sigma factor/anti sigma factor system.
Weaknesses:
Although the presented evidence is mostly solid, some of their findings need to be evaluated more carefully; explaining the rationale behind some of the experiments might enhance the article, and some of the models proposed by the authors seem far-fetched, as outlined below:
We greatly appreciate the reviewer’s efforts and thank them for highlighting strengths and areas for improvement.
(1) In line 76 the authors claim "Relative to P. aeruginosa that were incubated in host cell-free media, P. aeruginosa in close proximity to human neutrophils or that were engulfed in mouse macrophages appeared to increase fro expression (Fig. 1C)". Counting bacterial cells in Figure 1C shows that 1 in 17 bacteria (5.8%) induce the froA-promotor in media in the absence of immune cells, while 4 in 72 bacteria (only 5.5%) do the same in the presence of neutrophils. Contrary to the authors' claims, it appears that P. aeruginosa actually decreases fro-expression in close proximity to neutrophils. There is a slight increase in fro-expression in bacteria co-incubated with macrophages (3 in 21, or 14.3%). A more rigorous statistical analysis might substantiate the authors' claim, but, as is, the claim "neutrophils increase fro expression" is untenable.
We believe the images alone do not give an adequate representation of the data and have quantified a larger portion of the data, which has been added as Figure 1D. The quantification supports the original claim that fro expression is increased during co-incubation with macrophages and neutrophils. Since there was not sufficient statistical sampling to distinguish engulfed P. aeruginosa from free ones, this part of the claim has been removed from the text (updated in lines 80-84).
(2) The authors should explain the rationale behind some of the chemicals used. Why did they use nitric acid? Especially at these high concentrations, a strong acid such as nitric acid might have a significant influence on the medium pH. I understand that the medium is phosphate-buffered, but 25 mM nitric acid in an unbuffered medium would shift the pH well below 2. Similar considerations apply to hydrochloric acid and sodium hydroxide.
We thank the reviewers for pointing out the need for this clarification. We have updated Figure 3D with a lower concentration of NaOH at 1 uM, which is the same concentration as NaOCl that activates fro expression. Due to the high buffering capacity of our medium, a high concentration of 6 mM NaOH was needed to induce a discernible change in pH and this high concentration of NaOH had no obvious effect on growth. Neither 1 uM nor 6 mM NaOH produced a change in fro expression, consistent with our previous findings that the effect is not due to sodium ions or higher pH. These updated findings are described in lines 181-190.
Since the effect of chloride is already controlled for using NaCl and the concentration of HCl used was not sufficient to cause a significant change in pH in the buffered medium, we have removed the HCl group from the data.
We have clarified that nitric acid was used because it is a strong oxidizer that is not found in neutrophils and that concentrations used were near the minimal inhibitory concentrations (lines 145-147 and lines 191-198). We acknowledge that the growth inhibition from HNO3 could be due to pH or oxidation. However, since no change in fro expression was observed at concentrations approaching the inhibitory concentration, we did not address the potential effects of low pH from nitric acid on fro expression.
(3) In line 187, the authors state that "It is possible that oxidized methionine increases fro expression" and they suggest a model to that effect in Figure 5D. It is unclear why the authors singled out methionine sulfoxide, since a number of other things get oxidized by HOCl. In line 184, the authors state, in the same vein, that "HOCl oxidizes methionine residues 100-fold more rapidly than other cellular components". The authors should state which other cellular compounds they are referring to. Certainly not cysteine and other thiols, which react equally fast and are highly abundant in the cell: P. aeruginosa contains 340 µM GSH, 140 µM CoA-SH (https://doi.org/10.1074/jbc.RA119.009934) plus free cysteine and cysteines in proteins (based on codon usage, 1.34% of amino acids in proteins are cysteine, while methionine is only slightly more present at 2.10%, although a number of starting methionines are removed from mature proteins).
We acknowledge that our HOCl sensing model had been vague and unclear and thank the reviewer for their insight. This critique is echoed by reviewer #1 in comment 7 as well.
Our initial suggestion that methionine sulfoxide was sensed was motivated by the observation that methionine sulfoxide reductases are upregulated by HOCl. However, we have revised this based on feedback from reviewers and further consideration of chlorine redox chemistry. Interestingly, we found that the FroI anti-sigma factor has the highest concentration of methionine and cysteine residues of all the P. aeruginosa anti-sigma factors (Appendix 2—table 1). Given that FroR and FroI form an extracytoplasmic function sigma – anti-sigma pair, which are associated with transducing extracellular signals to the cytoplasm, we propose a model in which HOCl or secondary RCS molecules are detected by their oxidation of cysteine and methionine residues in FroI. This is discussed in lines 271-278 and lines 362-366.
(4) Overall (and this is probably not addressable with the authors' data), some very interesting questions remain unanswered: what is the molecular mechanism of fro-induction? How is the FroR/FroI system modulated by HOCl? Does the system sense free or protein-bound methionine-sulfoxide? Are certain methionine residues in these proteins directly oxidized by HOCl? Many "HOCl-sensing" proteins are also modified at cysteine residues or amino groups; could those play a role? And lastly: what is the connection between shear/fluid flow and HOCl, or are these totally separate mechanisms of fro-induction?
We thank the reviewer for raising these excellent mechanistic questions. Issues relating to HOCl sensing are addressed in the preceding comment.
Regarding the connection to shear sensing, Padron et al., 2023 found that the detection of flow in P. aeruginosa can be attributed to chemical transport, in particular to H2O2 that was present in growth media. Based on the same principle, we expect Fro to be upregulated in flow at much lower concentrations than those observed in stationary fluids. The activation of Fro and the effects of HOCl would thus be expected to be flow-sensitive. We have commented on this important factor in the discussion in lines 406-417.
Reviewer #2 (Recommendations for the authors):
(1) To address 1, the authors could evaluate the microscopic images in the same manner in which they evaluated the other microscopic images, as, for example, presented in Figure 2B or Figure S1B.
See response to Weakness point (1).
(2) To address 2, please explain the choice of the chemicals (why nitric acid?), but also provide the pH of the media with those high concentrations of strong acids and bases, and interpret them in light of the permissible pH range for P. aeruginosa growth. More sensible controls might be a lower NaOH concentration in the range that would be reached through the amount of NaOH in the NaOCl stock at the highest NaOCl concentrations used. As for the acids, I don't see a reason to use these acids at these high concentrations. Please explain.
See response to Weakness point (2).
(3) To address 3: The authors could specify their methionine-sulfoxide model a bit more, so that testable hypotheses can be developed. If the authors think the FroR/FroI system senses free methionine sulfoxide, they or others could add methionine sulfoxide to the medium and check induction. If they think specific methionine residues in these proteins are oxidized, they could provide evolutionary evidence of conserved methionine residues. Or, based on a structure or structural prediction, they (or others) could mutate methionine residues, e.g., at the protein's surface or potential protein/protein-interaction sites and assess the effect on HOCl-based activation. Or they could consider other amino acids known to be highly reactive towards HOCl and mutate those in a future study.
See response to Weakness point (3).
Further comments:
(4) The headline of the figure legend of Figure S1 seems incomplete. Please mention the flow experiments shown in Figure S1A.
We have updated the title of this figure, which now appears in the eLife format as Figure 1 – figure supplement 1.
(5) What is the difference between the data presented in Figure 4C (bars "UTR" and "NaOCl") and the same bars in Figure S2A? Is this redundant or a re-plot?
In this revision, Figure 4C has become Figure 5B and Figure S2A is now Figure 5—figure supplement 1. Only the 1 uM NaOCl condition is replotted. We have described this in the legend for Figure 5—figure supplement 1.
(6) Line 228: hpd is more likely a gene of the aromatic amino acid catabolism.
We thank the reviewer for pointing this out. We have removed the ‘branched’ descriptor in this sentence, now in lines 325-326.
(7) Line 229: "While the ΔfroR strain was sensitive to HOCl, the strain was tolerant (Figure 5A)". Please clarify. Which strain was tolerant? WT?
We have corrected this typo (now line 328-329). We meant to convey that growth was not entirely inhibited in the froR strain.
(8) Line 247: "Activated neutrophils produce HOCl concentrations as high as 50 µM [24]." This "50 µM" number is often quoted; the citation trail typically leads to Weiss et al. 1982 (https://doi.org/10.1172/JCI110652). However, a more factually correct statement based on that paper would be "2 x 10^6 neutrophils, activated with 30 ng/mL PMA at 37C in 1 mL of Dulbecco's buffer can produce around 50 nmol HOCl per hour". In the particular reference 24, Dybpukt et al used a methodology similar to Weiss et al., and here around 50nmol were produced by the same number of cells in 30 min in response to 100 ng/mL PMA. Please clarify accordingly.
We thank the reviewer for bringing this to our attention. We have altered the language to indicate that the production rate is for this specific set of parameters. Related to this, Reviewer #1 Comment #10 requested a more detailed discussion of the significance of the Fro response, since it is at much lower HOCl concentration than produced by neutrophils. We have discussed this in lines 377-405.
(9) Line 275: "which is strain PA14 strain". Please clarify.
We have fixed this error and entered it into the Key Resource Table.
(10) Line 311: "Cultures containing densities below 10 P. aeruginosa per frame were concentrated using a syringe filter with 0.2 or 0.8 µm pore sizes (Millipore, Burlington, MA)." Isn't that a bit concerning when testing the induction of an operon that is supposedly activated by shear through fluid flow? Did the authors convince themselves that this procedure does not induce fro?
We do not expect the filtering procedure to cause changes in gene expression because cells are imaged immediately after filtering. Nonetheless, we performed additional experiments (Fig 3F and 2D) entirely without concentrating cells and found that YFP/mCherry levels were consistent with previous data in Figure 3. This rationale has been added to the Methods section under the “Fluorescence and Phase Contrast Microscopy” section.
