Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism
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Evaluation Summary:
Using a genome wide screen, the authors identify novel pathways that protect Pseudomonas aeruginosa against Type-6 secretion-mediated by other bacterial species. Importantly these pathways each protect against specific effectors. As an example, the authors further show that one factor Arc3B protects against phospholipase activity by antagonizing the production of lysophospholipids induced by phospholipase-type effectors. These findings uncover potentially conserved and unsuspected bacterial-defense pathways.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that Pseudomonas aeruginosa possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters ( arc1-3 ). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.
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Author Response:
Reviewer #1 (Public Review):
In this manuscript, the authors explore mechanisms in Pseudomonas aeruginosa involved in defending cells from T6SS-dependent attacks by other bacteria. Using a genome-wide Tnseq approach, the authors identify three gene clusters involved in this defensive response. They also report that these gene clusters are activated by the GacS/GacA/Rsm pathway. The authors also convincingly show that each of the three gene clusters encode proteins involved in the defence against specific toxins. Finally, one of the defence proteins is analyzed in more detail and found to prevent the accumulation of lysophospholipids generated by the Tle3 phospholipase toxin.
I did not identify any weaknesses except that the manuscript is incredibly densely written, making it difficult to read.
We are grateful to …
Author Response:
Reviewer #1 (Public Review):
In this manuscript, the authors explore mechanisms in Pseudomonas aeruginosa involved in defending cells from T6SS-dependent attacks by other bacteria. Using a genome-wide Tnseq approach, the authors identify three gene clusters involved in this defensive response. They also report that these gene clusters are activated by the GacS/GacA/Rsm pathway. The authors also convincingly show that each of the three gene clusters encode proteins involved in the defence against specific toxins. Finally, one of the defence proteins is analyzed in more detail and found to prevent the accumulation of lysophospholipids generated by the Tle3 phospholipase toxin.
I did not identify any weaknesses except that the manuscript is incredibly densely written, making it difficult to read.
We are grateful to the reviewer for their positive assessment of our study. We regret any difficulty that our writing style may have caused and we thank the reviewer for providing this comment. We have incorporated revisions throughout the manuscript to improve readability.
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Evaluation Summary:
Using a genome wide screen, the authors identify novel pathways that protect Pseudomonas aeruginosa against Type-6 secretion-mediated by other bacterial species. Importantly these pathways each protect against specific effectors. As an example, the authors further show that one factor Arc3B protects against phospholipase activity by antagonizing the production of lysophospholipids induced by phospholipase-type effectors. These findings uncover potentially conserved and unsuspected bacterial-defense pathways.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, the authors explore mechanisms in Pseudomonas aeruginosa involved in defending cells from T6SS-dependent attacks by other bacteria. Using a genome-wide Tnseq approach, the authors identify three gene clusters involved in this defensive response. They also report that these gene clusters are activated by the GacS/GacA/Rsm pathway. The authors also convincingly show that each of the three gene clusters encode proteins involved in the defence against specific toxins. Finally, one of the defence proteins is analyzed in more detail and found to prevent the accumulation of lysophospholipids generated by the Tle3 phospholipase toxin.
I did not identify any weaknesses except that the manuscript is incredibly densely written, making it difficult to read.
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Reviewer #2 (Public Review):
In this work, Ting and colleagues performed genome-wide screens to identify genes affecting P. aeruginosa (Pa) ability to cope with B. thailandensis (B.thai) type 6 secretion (T6S) antagonistic action. The group had previously shown that during Pa-B.thai competition the Pa Gac/Rsm regulatory cascade is activated in response of kin cell lysis, leading to an increase of the H1-T6S synthesis as part of the PARA (P. aeruginosa response to antagonism) regulon. Here, three additional gene clusters (named arc1-3) regulated by the Gac/Rsm cascade are identified and shown to be important to counteract the damage caused by B.thai T6S. Interestingly, arc2 and arc3 genes provide a fitness advantage towards specific B.thai T6S effectors: ColA and Tle3, respectively. Since the arc1-arc3 genes are encoding for hypothetical …
Reviewer #2 (Public Review):
In this work, Ting and colleagues performed genome-wide screens to identify genes affecting P. aeruginosa (Pa) ability to cope with B. thailandensis (B.thai) type 6 secretion (T6S) antagonistic action. The group had previously shown that during Pa-B.thai competition the Pa Gac/Rsm regulatory cascade is activated in response of kin cell lysis, leading to an increase of the H1-T6S synthesis as part of the PARA (P. aeruginosa response to antagonism) regulon. Here, three additional gene clusters (named arc1-3) regulated by the Gac/Rsm cascade are identified and shown to be important to counteract the damage caused by B.thai T6S. Interestingly, arc2 and arc3 genes provide a fitness advantage towards specific B.thai T6S effectors: ColA and Tle3, respectively. Since the arc1-arc3 genes are encoding for hypothetical proteins which function is unknown, the authors then focused on the characterization of the molecular action of Arc3A and Arc3B, showing its absence leads to an accumulation of lysophopholipids that is dependent on Tle3 activity (and independently from Aas recycling).
Overall, the study is remarkable, exciting and well-conceived, as it reveals new molecular pathways affecting bacterial susceptibility to T6S effectors. The novelty resides on the fact that the arc1-3 "protective genes" are not classical toxin immunity genes, but are widespread genes probably involved in housekeeping or damage repair cellular functions, like the Arc3 genes and phospholipids homeostasis.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5468049.
In this manuscript, Ting et al. describe three gene clusters - named antagonism resistance clusters (arc1-3) - that are upregulated by the Gac/Rsm danger sensing system and proposed to work as an innate immunity system against antibacterial attacks. This work builds on their previous studies (LeRoux et al., 2015 eLife; LeRoux et al., 2015 J Mol Biol) in which the authors have identified a danger sensing mechanism that bacteria use to sense lysis of neighboring kin cells and activate an offensive T6SS weapon - which was also dependent on the Gac/Rsm regulators.
The arc clusters appear to provide antagonism against specific toxic T6SS effectors, with arc3 cluster being most important to …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5468049.
In this manuscript, Ting et al. describe three gene clusters - named antagonism resistance clusters (arc1-3) - that are upregulated by the Gac/Rsm danger sensing system and proposed to work as an innate immunity system against antibacterial attacks. This work builds on their previous studies (LeRoux et al., 2015 eLife; LeRoux et al., 2015 J Mol Biol) in which the authors have identified a danger sensing mechanism that bacteria use to sense lysis of neighboring kin cells and activate an offensive T6SS weapon - which was also dependent on the Gac/Rsm regulators.
The arc clusters appear to provide antagonism against specific toxic T6SS effectors, with arc3 cluster being most important to antagonize the toxic effect of the lipase Tle3.
During interbacterial competition assays between Pseudomonas aeruginosa and Burkholderia thailandensis, arc3 protects P. aeruginosa from detergent-like lysophospholipid toxic products generated by the action of Tle3.
Overall, the article is very interesting and touches on a hot topic in the field: the identification of mechanisms that protect bacteria from T6SS attacks independent of specific immunity proteins.
I have only one major comment:
Is the activation/expression of arc3 cluster regulated by an increase of lysophospholipids in the bacterial membrane? The expression of arc3 genes could be measured in bacteria exposed to extracellular phospholipase from another source or in bacteria expressing the Tle3 toxin from a plasmid. As the authors are claiming arc1-3 genes are innate immune systems, it would be interesting to analyze whether these clusters could be activated without interbacterial contact (downstream of Gac/Rsm) and only by the intracellular detection of the specific danger signal (lysophospholipids). Could they also try adding pure lysophospholipid to mimic the signaling without any bacterial contact?
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