Genome-directed study reveals the diversity of Salmonella T6SS effectors and identifies a novel family of lipid-targeting antibacterial toxins

  1. Gianlucca G. Nicastro
  2. Stephanie Sibinelli-Sousa
  3. Julia Takuno Hespanhol
  4. Thomas W. C. Santos
  5. Joseph P. Munoz
  6. Rosangela S. Santos
  7. Blanca M. Perez-Sepulveda
  8. Sayuri Miyamoto
  9. L. Aravind
  10. Robson F. de Souza
  11. Ethel Bayer-Santos

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Abstract

Bacterial warfare is a common and ancient phenomenon in nature, where bacterial species use strategies to inhibit the growth or kill competitors. This involves the production and deployment of antibacterial toxins that disrupt essential cellular processes in target cells. The continuous arms race in which bacteria acquire new toxin and immunity proteins to promote increased adaptation to their environment is responsible for the diversification of this toxin repertoire. Here, we deployed in-silico strategies to analyze 10,000 genomes and identify effectors secreted via the type VI secretion system of Salmonella . We identified 128 candidates, which are widespread in a vast array of Salmonella serovars and other bacterial species. Tox-Act1 is among the most frequent candidates and was selected for in-depth characterization. Tox-Act1 contains a permuted NlpC/P60 papain-like catalytic core characteristic of lipid-targeting members rather than the typical peptidases or amidases. Evolutionary analysis revealed the relationship of Tox-Act1 with acyltransferases. Biochemical assays with purified toxin and lipidomics of intoxicated cells showed that Tox-Act1 exhibits phospholipase activity, cleaving off acyl groups from phosphatidylglycerol and phosphatidylethanolamine. In addition, we demonstrate that Tox-Act1 is secreted in a T6SS-dependent manner and provide a competitive advantage during colonization of the gut of infected mice. This work broadens our understanding of toxin domains and provides the first direct characterization of a lipid-targeting NlpC/P60 domain in biological conflicts.

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    Reply to the reviewers

    'The authors do not wish to provide a response at this time.'

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    This manuscript presents a large-scale comparative genomics analysis of Salmonella genomes to identify and characterize the repertoire of Type VI Secretion System (T6SS) effectors. The authors combine bioinformatic predictions with experimental validation of one novel toxin domain (Tox-Act1), revealing a unique catalytic activity not previously reported in bacterial toxins. While the study is comprehensive and offers valuable insights into T6SS diversity, the insufficient description of computational methods and limited accessibility of underlying data reduce reproducibility and impact.

    Major comments

    1. The computational methods are inadequately described in the Materials and Methods section, and the authors did not provide the underlying datasets. These omissions make it impossible to reproduce the analysis or to apply the approach to other organisms.
    2. The criteria used to distinguish between T6SS effectors and non-effectors are unclear. The reliance on proximity to structural genes ("guilt-by-association") is insufficient and may have led to the omission of cargo effectors not proximal to these structural genes.
    3. No information is provided in the Materials and Methods section about the graph-based clustering strategy mentioned in the main text (Rows 109-111), including the Jaccard index and Louvain algorithm.
    4. The definition and identification of T6SS subtypes, including the use of the term "orphan," are not explained (Rows 111-112).
    5. The phylogenetic analysis of the newly identified domain Tox-Act1 lacks consistency and detail. For example, Rows 324-326 state: "To predict the function of Tox-Act1, we sought to understand its evolutionary relationship by constructing a phylogenetic tree using the sequences of Tox-Act1, TseH and additional permuted members, such as LRAT and YiiX." However, this contradicts Rows 342-344 and Figure 4A, which describe the phylogenetic tree as being built from permuted NlpC/P60 members, and indicate that a single query was used for PSI-BLAST, marked with a red star. It is unclear whether Tox-Act1, TseH, or another sequence was used as the initial PSI-BLAST query.
    6. The Tox-Act1 domain investigated is labeled as an acyltransferase, but the evidence presented supports only phospholipid-degrading activity. In my opinion, the naming should better reflect the activity demonstrated by the data.
    7. Table S1 should include representative protein accessions for each T6SS toxin domain. This is essential for evaluating the novelty of the identified domains and for enabling their use in future analyses. The repeated use of "This study" (96 times) as a reference, without further detail, is confusing and unhelpful. In my view, referencing the current study is appropriate only when the manuscript provides sufficient information on the corresponding domain.
    8. In general, the authors should place greater emphasis on ensuring that the proteins and genomes analyzed in this study can be reliably identified. Genomic accessions and locus tags should be traceable in public databases such as NCBI, and the supplemental information must correspond accurately to the main text. For example, I was unable to find information on FD01543424_00914, which was used as the query for the alignment of STox_15 (the name used in the supplemental information, while in the main text it is referred to as Tox-Act1; see related comment below).
    9. A supplementary table listing all Salmonella effectors and their domain annotations is missing. This is essential for transparency, reproducibility, and future use of the data.
    10. The GitHub repository contains a large volume of data and code but lacks detailed documentation and clear instructions, including example files. This greatly limits reproducibility and usability. The current organization of the repository makes it difficult to locate specific results; for example, Tox-Act1 is referred to as STox_15 in the GitHub files, but this is not mentioned in the manuscript. The authors should improve data organization and provide a README file for clarity.

    Minor comments

    1. The introduction should discuss previous work on Salmonella T6SS effectors, including Blondel et al. (2023) (ref 71 in the manuscript), Amaya et al. (2022), and Amaya et al. (2024).
    2. In Figure 1C, genomic examples should include strain names and locus tags.
    3. In Figure 1F, 'ND' should be replaced with 'Unknown' or 'Not Determined'.
    4. Figure 1E is overly complex and, in my opinion, does not add value, especially since the accompanying text is sufficient on its own. Moreover, the authors acknowledge that their initial analysis missed the similarity between Tox-Act1 and both DUF4105 and the TseH effector, which raises concerns about the accuracy and usefulness of this graph.
    5. Figure 3D lacks information about the number of replicates (n=?).
    6. Discrepancies in domain annotations:
      • Row 232: STox_47 is missing from Table S1.
      • Row 233: STox_18 is pore-forming and STox_53 is a nuclease (per Table S1), which contradicts the main text.
    7. Multiple grammatical and typographical errors exist throughout the text, including:
      • Row 41: "provide" should be "provides"
      • Rows 131, 222: "immunities" should be "immunity proteins"
      • Rows 170, 253, 288: "thee" should be "three"
      • Row 388: "corresponds" should be "correspond"
      • Row 389: "chomatogram" should be "chromatogram"
    8. Rows 257-259: The claim that PAAR and RHS domains assist in translocation across the bacterial inner membrane is presented as fact, but this is only a hypothesis and should be stated more cautiously.
    9. Figure 3A: The selection of representative genomic loci is unclear. For example, FD01843896 is shown in the figure, but cloning was performed using FD01848827, and the HHPred analysis was based on FD01543424. The rationale for using different sequences at each step should be clarified.
    10. Rows 296-299: The absence of a secretion assay in the study is notable. If this is due to the inability to activate the SPI-6 T6SS of Salmonella enterica serovar Typhimurium, as discussed in these lines, it should be explicitly mentioned in the text.
    11. Figure 4C (sequence logo) is not described in the Materials and Methods section.
    12. Row 467: The retrieval date of the gff files from the 10KSG database is missing.
    13. Rows 474-476: The domain models used for T6SS cluster prediction are not described.

    Significance

    This is a comprehensive study involving a large number of Salmonella genomes, potentially identifying many new T6SS effectors and toxic activities. One new domain analyzed in this work is experimentally investigated and shown to have a unique catalytic activity not previously observed in toxins. However, the bioinformatic methods are not described in sufficient detail, making it difficult to assess or reproduce the work. Protein accession numbers are missing, even for representative toxins, and locus tags are not traceable, making the identified effectors not readily accessible. There are many inaccuracies throughout the text and supplemental data. The Tox-Act1 domain investigated is labeled as an acyltransferase, but the evidence only supports phospholipid-degrading activity. While the study includes many graphs and histograms, they often obscure the main findings. Consequently, the audience is likely to be limited.

    Nevertheless, despite these concerns, I believe this is an important work that could be valuable to the broad community once a more thorough revision is undertaken, not only by addressing the specific comments raised, but also by rechecking the analyses, reorganizing the presentation, and ensuring that all data and annotations are clearly accessible and traceable.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The manuscript titled "Genome-directed study reveals the diversity of Salmonella T6SS effectors and identifies a novel family of lipid-targeting antibacterial toxins" presents a comprehensive in silico analysis of T6SS-associated effector and immunity genes across approximately 10,000 Salmonella genomes. In addition, the authors selected one of the newly identified effectors, Tox-Act1, for detailed biochemical characterization. To my knowledge, this study represents the most extensive genome-wide mining effort to date for T6SS-associated effectors and immunity proteins in Salmonella, employing a range of state-of-the-art computational prediction tools. The in vitro enzymatic characterization of Tox-Act1 further validates the in silico approach and adds a novel functional perspective to the dataset. Overall, the study provides a rich and comprehensive dataset. However, for readers without a strong bioinformatics background, the logic and workflow of the in silico prediction pipeline may be challenging to follow. Consequently, my comments focus primarily on the biochemical analysis of Tox-Act1, rather than the computational aspects of the study.

    Major comments:

    1. In Figure 3, the authors first demonstrated that Tox-Act1 and Imm-Act1 constitute a functional antibacterial toxin-immunity pair using a heterologous E. coli expression system. They then proceeded to an in vivo mouse colonization model, showing that prey cells lacking the tox-act1/imm-act1 locus exhibited reduced competitiveness when co-infected with a Salmonella strain carrying the endogenous tox-act1, compared to a ∆tssL mutant. As this is the first report identifying and characterizing Tox-Act1 function in Salmonella, the authors should provide additional experimental evidence addressing the following key points: (i) Whether Tox-Act1 is secreted by Salmonella in a T6SS-dependent manner; (ii) Whether target cells lacking imm-act1 (in either Salmonella or E. coli) can be intoxicated by Salmonella secreting Tox-Act1; (iii) Whether the observed competitive advantage in vitro conferred by Tox-Act1 is dependent on its phospholipase activity. Given that Salmonella T6SS can be activated by hns deletion, such experiments should be feasible and are crucial for the functional validation of any newly identified T6SS effector. Addressing these points would substantially strengthen the mechanistic basis of the study and reinforce the biological importance and relevance of Tox-Act1.
    2. In Figure 4, the authors present the evolutionary relationship between Tox-Act1 and the previously identified T6SS effector TseH from Vibrio, and they propose that these two effectors may share similar enzymatic activities and overlapping cellular targets. Given the ongoing debate and unresolved questions regarding the biochemical function of TseH, the authors should leverage their established in vitro phospholipase assay to test whether TseH exhibits phospholipase activity similar to that of Tox-Act1. Demonstrating such activity would not only substantiate the proposed functional conservation but also provide critical biochemical insight into a long-standing question in the T6SS field.
    3. In Figures 5C and 5D, the authors performed lipidomic analyses on E. coli cells heterologously expressing Tox-Act1 and reported that specific phospholipid species are altered in a manner dependent on Tox-Act1's phospholipase activity. However, the data presented in Figure 5D only include changes in the abundance of PG, FFA, LPG, and LPE. To provide a comprehensive overview of the lipidomic alterations, the authors should present the full dataset of all identified phospholipid species. This is essential to evaluate the extent and specificity of lipid remodeling induced by Tox-Act1. It is currently unclear whether the observed reduction in PG is the only statistically significant change or if additional lipid species were similarly affected but not shown. Furthermore, the authors claim that Tox-Act1 functions as a phospholipase A1. However, in Figures 5A and 5B, the signal corresponding to intact phospholipids remains relatively high, raising concerns about the apparent weak enzymatic activity in this assay. This observation contrasts with previously characterized phospholipase toxins in the antibacterial toxin field, such as Tle1 from Burkholderia, which exhibit robust activity under in vitro conditions. To substantiate the enzymatic potency of Tox-Act1 and clarify this discrepancy, the authors should include a side-by-side comparison using the same in vitro assay with a well-established phospholipase toxin (e.g., Tle1) as a positive control. This would allow for a direct evaluation of the relative enzymatic strength of Tox-Act1 and support the interpretation of its lipid-targeting function.

    Minor Comments:

    1. Line 32: Please specify "Type VI Secretion System (T6SS)" when first introducing the term in the abstract, to ensure clarity for a broad readership.
    2. There are inconsistencies between the numerical values reported in the main text and those shown in the figures. For instance, the manuscript repeatedly states that approximately 10,000 Salmonella genomes were analyzed in the in silico search, whereas Figure 1 indicates a total of 10,419 genomes. Similarly, Line 108 mentions 42,560 genomic sites, yet Figure 1 displays a count of 49,080. Please ensure that all numerical data are consistent across the manuscript and figures to avoid confusion or misinterpretation.
    3. The definition of "Orphan clusters" is not provided. Please specify the criteria used to define these clusters and clarify the rationale for grouping them separately from the other clusters (i1-i4) shown in Figure 1A. It would be helpful to explicitly state how they differ from the canonical clusters.
    4. Lines 114-119: The sentence structure in this section is overly long and difficult to follow. Please revise this portion for clarity and conciseness to ensure that the intended message is clearly conveyed.
    5. The color coding in Figure 1C is incomplete; only a few categories are indicated in the legend. Please revise the legend to include all color codes used in the figure for accurate interpretation.
    6. Lines 278-280: The authors state that "cells lysed without losing their rod shape, which suggests that the peptidoglycan was not affected... indicating that this is not the target of Tox-Act1." Please provide appropriate references or supporting evidence for this interpretation. Clarification is needed to explain the morphological criteria being used to infer peptidoglycan integrity.
    7. Please define "competitive index" in the legend of Figure 3D to ensure the metric is clearly understood by readers unfamiliar with the term.
    8. It is unclear to me why the author use (data not shown) in Line 315. Please provide evidence to support the claim in the paragraph.
    9. In Figure 4D, the authors compare the activity of wild-type and catalytic mutant Tox-Act1, but protein expression levels are not shown. Please include immunoblot or other relevant data to confirm equivalent expression of both constructs, to rule out differential expression as a confounding factor.

    Referee cross-commenting

    I agree with Reviewer #3 that the authors should provide more details on their search for better reproducibility.

    Significance

    This manuscript presents a large-scale in silico analysis of Salmonella T6SS effectors and immunity proteins, accompanied by the biochemical characterization of a novel phospholipase effector, Tox-Act1. The genome-wide dataset is comprehensive, representing the most extensive mining effort of its kind to date. The study is strengthened by in vitro validation of Tox-Act1 activity and its role in interbacterial competition. However, the manuscript would benefit from additional experimental data to confirm key mechanistic aspects, including T6SS-dependent secretion of Tox-Act1, its toxicity toward target cells lacking immunity, and the contribution of phospholipase activity to its antibacterial function. Comparative assays with established T6SS phospholipases (e.g., Tle1) are recommended to clarify enzymatic potency. Further, the authors should apply their phospholipase assay to test TseH activity and resolve long-standing questions in the field. Several areas also require clarification or correction, including inconsistencies in reported genome counts, incomplete figure legends, unclear terminology (e.g., "Orphan clusters"), and missing experimental controls (e.g., protein expression levels, full lipidomic dataset). Minor edits to improve clarity and consistency are also suggested. Overall, the study is significant and of high potential impact but requires additional experimental validation and revisions to improve clarity and completeness.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: In this study, authors used in silico approaches to analyse 10,000 bacterial genomes and identified 128 candidate effectors secreted via the T6SS of Salmonella. Among these, Tox-Act1 was selected for detailed characterisation. The authors demonstrated that Tox-Act1 harbours a permuted NlpC/P60 catalytic domain with phospholipase activity, targeting key membrane lipids. Furthermore, they confirmed that Tox-Act1 is secreted in a T6SS-dependent manner and enhances bacterial competitiveness during gut colonisation in mice, providing new insights into lipid-targeting toxin domains in interbacterial interactions. My concerns raised are all minor and should be readily addressable by the authors.

    Minor Concerns:

    Line 279-280: The statement that the peptidoglycan is not a target of Tox-Act1 is somewhat strong at this stage of the manuscript. The preservation of cell shape does not necessarily imply that the peptidoglycan remains unaltered at a subcellular level. Given that Tox-Act1 belongs to the NlpC/P60 family, members of which include known peptidases, the authors should moderate this assertion. Replacing "is not" with "is likely not" or using conditional phrasing would be more appropriate here.

    Lines 328-331: The conclusion that the Tox-Act1 clade is deployed in biological conflicts is not fully explained or substantiated. The authors are encouraged to provide a brief rationale to support this conclusion.

    Figure 4D: There appears to be a labelling inconsistency. The immunity protein is referred to as "Slmm15," which may relate to the original name of Tox-Act1 (i.e., STox_15), but the correct label should likely be "Imm-Act1."

    Line 401 and elsewhere: The correct spelling is "L-arabinose" with a capital "L". The manuscript should be checked for consistency in this regard.

    Throughout the text and figures: Bacterial species names are often incorrectly formatted, e.g., "S. Panama" (Line 226) should be written in scientific style as S. panama, with italics and the species name in lowercase. A systematic revision of species names is recommended to enhance rigour.

    Figure 3D: The X-axis labelling is somewhat confusing. The use of terms such as "attackers" and "prey" is misleading in this context, as the experiment tests the in vivo survival capacity of different Salmonella strains (WT or T6SS mutants mixed with toxin/immunity double mutants) in a mouse model, rather than a direct bacterial killing assay. Clarifying this would greatly improve readability.

    Significance

    Overall, this study is well-executed. The approach used to identify a previously uncharacterised diversity of T6SS effectors in Salmonella is robust and provides a valuable framework that could be extended to other systems involved in interbacterial competition. This renders the work relevant and of interest for publication. While the manuscript occasionally lacks clarity in explaining the rationale behind certain experimental choices, the narrative remains generally accessible.

    Field of expertise: Secretion systems, interbacterial competition, bacterial predation, live-cell imaging, protein network

  5. Version published to 10.1101/2024.09.27.615498 on bioRxiv