The protective function of an immunity protein against the cis -toxic effects of a Xanthomonas Type IV Secretion System Effector

  1. Gabriel U. Oka
  2. Diorge P. Souza
  3. Germán G. Sgro
  4. Cristiane R. Guzzo
  5. German Dunger
  6. Chuck S. Farah

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Abstract

Many bacterial species use specialized secretion systems to translocate proteinaceous toxic effectors into target bacterial cells. In most cases, effectors are encoded in bicistronic operons with their cognate immunity proteins. The current model is that immunity proteins could, in principle, provide protection in two different ways: i) by avoiding self-intoxication (suicide or cis -intoxication) or ii) by inhibiting intoxication due to “friendly-fire” translocation from neighboring sister cells (fratricide or trans -intoxication). Here, we set out to distinguish between these two protection mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS), where killing is due to the action of a cocktail of secreted effectors (X-Tfes) that are inhibited by their cognate immunity proteins (X-Tfis). We use a set of X. citri mutants lacking multiple X-Tfe/X-Tfi pairs to show that X-Tfis are not absolutely required to protect against trans -intoxication. Our investigation then focused on the in vivo function of the lysozyme-like effector X-Tfe XAC2609 and its cognate immunity protein X-Tfi XAC2610 . We observe the accumulation of damage in the X. citri cell envelope and inhibition of biofilm formation due to the action of X-Tfe XAC2609 in the absence of X-Tfi XAC2610 . We show that X-Tfe XAC2609 toxicity is independent of an active X-T4SS and that X-Tfi XAC2610 protects the cell colony against X-Tfe XAC2609 -induced cis -intoxication via autolysis. In vitro assays employing X-Tfi XAC2610 mutants were used to test and validate an AlphaFold2-derived model of the X-Tfe XAC2609 -X-Tfi XAC2610 complex which presents topological similarities with the distantly related Tse1/Tsi1 complex from P. aeruginosa and the the i-type lysozyme from Meretrix lusoria (MI-iLys) in complex with PliI-Ah from Aeromonas hydrophila . While immunity proteins in other systems have been shown to protect against attacks by sister cells ( trans -intoxication), this is the first description of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis -intoxication.

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    General Statements

    We are happy to resubmit our manuscript “The protective function of an immunity protein against the cis-toxic effects of a Xanothomonas Type IV Secretion System Effector” by Gabriel Oka et al. This paper shows that the cohort of immunity proteins associated with the cocktail of toxic effectors secreted by the Xanthomonas citri T4SS are not required to protect against toxins injected by neighboring cells but rather provide protection against endogenous toxins of the cell in which they were produced. To our knowledge, this the first description of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis-intoxication, a point we emphasize in the revised introduction.

    We thank the reviewers for their thorough revision of the manuscript. Two of the three reviewers clearly expressed the opinion that the manuscript would be of general interest and should be published. We have carried out a number of new experiments and data analyses to respond to most of the suggestions of all three reviewers and believe that the manuscript is significantly improved as a result.

    Reviewer #1 (Evidence, reproducibility and clarity):

    The manuscript by Oka et al. shows that one effector of X. citri is likely translocated into the periplasm where it cleaves PG unless inhibited by its cognate immunity protein. Interestingly, this effector is required for killing of target cells like E. coli in T4SS-dependent manner but it does not seem to be delivered into X. citri cells by T4SS. Authors show using various assays that cells lacking the immunity protein have various phenotypes including lysis and defect in biofilm formation, however, despite "cis-intoxication" the ability to kill other bacteria or infect plants remains unaffected. The manuscript is well written and in general all the experiments have proper controls and thus the conclusions seem solid. The results described here are novel and interesting as they are unexpected.

    Major issues that should be addressed:

    - Test various deletion variants of the toxin to identify which part of the protein is responsible for its translocation into the periplasm. This may help to identify the possible mechanism of translocation of the toxin into the periplasm. Alternatively, the authors may attempt to select for non-toxic point mutants of the toxin. This could be done by a random PCR mutagenesis of the toxin and a selection of the surviving mutants in the absence of the immunity protein.

    Thank you for your insightful experimental suggestions using PCR mutagenesis to investigate the molecular mechanisms of the alternative translocation of X-Tfes to the periplasm. However, I regret to inform you that the first five authors of this manuscript are no longer a member of my lab. Therefore, please consider accepting the results shown in Figure 5A where we observe that the N-terminal domain of X-TfeXAC2609 that lacks the XVIPCD domain still abolishes biofilm formation in the absence of X-TfiXAC2610. Also, note that the E48A point mutation in the active site of the GH19 domain that abolishes the in vitro activity of X-TfeXAC2609 (Souza et al. 2015), also abolishes X-TFEXAC2609 toxicity in vivo in the absence of X-TfiXAC2610(Figure 5). Furthermore, in the predicted structure of the X-TfiXAC2610(54-267)-X-TfeXAC2609(1-194) complex (Figure 6), A tyrosine side chain in the conserved loop in X-TfiXAC2610 interacts directly with Glu48 in the X-TfeXAC2609 active site. One possibility for further investigation is the remaining region of X-TfeXAC2609(195-306) as a putative translocation domain. Sequence analysis of this region indicates that it encodes a canonical peptidoglycan binding domain. Another possibility is the existing intrinsic leakage of cytoplasmic proteins to the periplasm. As we understand it, the leakage of cytoplasmic proteins to the periplasm is not a well-documented phenomenon, although there is some evidence that suggests it may occur (PMID: 28808000, PMC3016450 references cited in the revised manuscript). This poorly characterized T4SS-independent pathway of translocation as indicated in Figure 1B (pathway 2)****.

    - Test if localization of the immunity protein to the cytoplasm blocks its activity. An immunity protein mutant that lacks its secretion signal should not protect against cis-intoxication.

    To address the question, we conducted new assays on colony opacity, as shown in Figure S2C. The X. citri ∆X-TfiXAC2610 strain was transformed with a plasmid expressing a cytoplasmic version of X-TfiXAC2610 that lacks the signal peptide and lipobox (X-TfiXAC2610(His-22-267)). Figure S2C shows that this cytosolic version of X-TfiXAC2610 protects X. citri from the toxic effects of X-TfeXAC2609 in the ∆X-TfiXAC2610 background. This suggests that X-TfiXAC2610(His-22-267) may directly interact with X-TfeXAC2609 in the cytoplasm, leading to the inhibition of X-TfeXAC2609 hydrolase activity and/or inhibition of its translocation into the periplasm. This is now mentioned in the results section of the revised manuscript

    While many experiments support the conclusion that the toxin is responsible for "cis-intoxication, the test of "trans-intoxication" should be done again but with the same setup as was used for testing of killing of E. coli. The CPRG based assay is far more sensitive than counting survival by plating to count CFUs. This test should be done at a relatively high initial OD so that there is an immediate contact between the "killer" and the "prey" bacteria (lacking immunity/effector). If needed, LacZ should be over-expressed in X. citri to make use of the CPRG based assay. In addition, such assay could be used also for "cis-intoxication" to supplement the potentially hard to quantify biofilm experiments shown in Fig. 4 (e.g. test all the T4SS mutants for "cis-intoxication").

    We are confident that the X-Tfis do not play a role in protecting against T4SS-mediated trans-intoxication since we continue to observe X-TfeXAC2609-dependent intoxication even in the absence of a functional XT4SS (see experiments using strains lacking X-T4SS subunits in Figures 2, S2, 3, 4 and 5. This is not to say that trans-intoxication does not occur. In fact, it does, and there is an independent mechanism that protects against it. We will provide details of the mechanism that protects against trans-intoxication in a forthcoming manuscript. In the present manuscript, we are addressing the phenomenon of cis-intoxication. To support our conclusion that the immunity proteins are not involved in the prevention of trans-intoxication does not occur in X. citri, we have included one additional supplementary video: Movie S7 shows that wild-type Xanthomonas citri does not kill and X. citri Δ8Δ2609-GFP. The absence of killing events in these experiments indicates that the X-T4SS-associated X-Tfi immunity proteins are not required for protection against X-T4SS-mediated sibling attack.

    In addition, such assay could be used also for "cis-intoxication" to supplement the potentially hard to quantify biofilm experiments shown in Fig. 4 (e.g. test all the T4SS mutants for "cis-intoxication").

    - Fig. 2A needs a positive control. For example, test killing of E. coli under the same conditions.

    Figure 2A of the revised manuscript now shows a CPRG assay competition assay that clearly demonstrates X-T4SS-dependent killing of E.coli MG by X. citri. We have now included the results of CFU experiments of X. citri vs E. coli competitions in a new Supplementary Figure (Figure S1) that are consistent with the CPRG assays. We note that our group has published similar results in the past (Souza et al. 2015; Oliveira et al. 2016; Oka et al. 2022). CFU measurements of X. citri vs E. coli competition assays are performed under slightly different conditions from the X. citri vs X. citri assays shown in Fig 2B. This is because E. coli grows at a significantly faster rate than X. citri so the initial cell ratios in these experiments have to be modified.

    - Authors should look at the paper by Ho et al. PNAS 2017, which describes trafficking of VgrG of V. cholerae into the periplasm of E. coli without an obvious secretion signal. The effector of X. citri may behave similarly.

    We thank the reviewer for this observation and now mention the paper by Ho et al. in the Discussion of the revised manuscript. Using a number of different algorithms (TatP, SignalP 6.0) we do not find any evidence of putative signal sequences. In the Discussion, we also mention the manuscript by Dong et al., 2013 that showed that the immunity protein TsiV3 that neutralizes VgrG3 is critical to prevent trans-intoxication.

    - Provide some form of quantification of the phenotypes (cell rounding and cell death) observed using live-cell imaging.

    As suggested by the reviewer, we performed a quantitative analysis of the propidium iodide (PI) permeability by calculating the percentage of PI permeable cells observed in movies S1-S5. This data is now presented in Figure 3 and Table S4 of the revised manuscript.

    - Provide quantification of biofilm related phenotypes as well as of the citrus canker development assay

    As suggested by the reviewer, we have carried out experiments to quantify the amount of biofilm using a crystal violet assay (absorbance at 570 nm). The results are presented in Figure S5 of the revised manuscript.

    Reviewer #1 (Significance):

    The study provides an interesting insight into immunity proteins against anti-bacterial toxins. It points to a need to protect against "cis-intoxication". This is novel and interesting to a wide audience of microbiologists interested in bacterial competition as this could be true also for other toxins.

    We thank the reviewer for his/her positive recommendation.

    It would be however important to identify how is the toxin translocating to the periplasm of the producing bacterium. Some insight into the mechanism would vastly improve the study. My expertise is in understanding bacterial interactions and competition but I lack a direct experience with assays specific for X. citri.

    We agree that an understanding of the mechanism of translocation into the periplasm would be interesting but is beyond the scope of the present manuscript. However, we do point out that this has been observed previously by other groups in the fourth paragraph of the Discussion of the revised manuscript: “... In the case of X-TfeXAC2609, the toxin somehow makes its way into the cell periplasm where, in the absence of X-TfiXAC2610, it degrades the peptidoglycan layer. Analysis of the X-TfeXAC2609 sequence by the SignalP 6.0 (Teufel et al., 2022) and other algorithms failed to detect any putative N-terminal signal peptide. Although the mechanism responsible for X-TfeXAC2609 transfer into the periplasm is at the moment unknown, we have shown that it is independent of a functional X-T4SS and of the XVIPCD secretion signal. Other bacterial proteins have been shown to transfer into the periplasm without any obvious secretion signal, for example VgrG3 from Vibrio cholerae (Ho et al. 2017) and recombinant forms of HdeA and chymotrypsin inhibitor 2 (Banes and Pielak, 2011).”

    Reviewer #2 (Evidence, reproducibility and clarity):

    This manuscript explores the role of an immunity protein of the Xanthomonas type IV secretion system (X-T4SS). In contrast to most T4SSs that conjugate plasmids or transfer effectors into host cells, this system is able to kill other bacteria similar to the role of T6SSs. Here, the authors tested whether the immunity protein XAC2610 functions to prevent cis-intoxication (by self) and/or trans-intoxication (by sister cells). They provide data that the XAC2610 immunity protein functions to protect cis intoxication, but not trans-intoxication, by the T4SS effector XAC2609 (which functions as a peptidoglycan hydrolase). Based on AlphaFold modeling, they went on to identify a residue in XAC2610 that is critical for inhibiting the activity of the XAC2609 toxin. Overall the data is fairly solid and generally support the conclusions the authors made.

    Major comments:

    One of the major conclusions of the manuscript is that XAC2610 does not prevent trans-intoxication and the data in the manuscript support this conclusion. However, I wonder if this is an oversimplification. Notably, the authors observed that wild type Xantho was unable to kill a target cell lacking 8 different toxin/immunity systems (Fig. 1A). One could conclude that none of these immunity proteins function in preventing trans-intoxication ... or ... perhaps it appears that none perform this role because wild-type Xantho never attacks its siblings? For example, it is conceivable that Xantho uses a general mechanism, perhaps somewhat similar to phage exclusion or plasmid incompatibility, to prevent sibling attack? To me this seems more likely than none of the eight immunity proteins play a role in preventing trans-intoxication. Moreover, the phenotype observed for the ∆2610 mutant in preventing cis-intoxication is somewhat subtle, likely because the toxin and the immunity protein are topologically restricted to the cytoplasm and the periplasm, respectively. This would make sense if this were not the primary role for 2610.

    Ideally the authors will be able to test this theory by demonstrating that a wild-type Xantho strain can attack (but likely not kill) its siblings. Alternatively, could the authors test if related, but not identical, Xantho strains that express 2609/2610 are able to kill their ∆2610 mutant, i.e. do "cousins" attack each other? Not sure about the semantics but this could be described as preventing trans-intoxication. If they are unable to do either experiment, that is ok but they should at least describe this concept in their discussion (assuming they agree).

    We thank the reviewer for his insightful comments. Indeed, this manuscript is focussed solely on the role of X-Tfi immunity proteins which we show to be principally involved in avoiding cis-intoxication (self-intoxication). The question of trans-intoxication will be left to an upcoming manuscript by our group. In fact we have identified a key factor (not an X-Tfi) that is responsible for inhibiting trans-intoxication. As suggested by the reviewer, we have now added the following text to the end of the third paragraph of the Discussion: “Nevertheless, the fact that wild-type X. citri is unable to kill strains lacking immunity proteins is intriguing. That cells in some way avoid trans-intoxication is revealed by the fact that X. citri wild-type cells carrying an X-T4SS and full cohort of X-Tfes do not kill the X. citri Δ8Δ2609-GFP, the X. citri ∆X-TfeXAC2609∆X-TfiXAC2610, or any other X-T4SS-deficient strain tested points to a still-to-be-characterized mechanism of protection against trans-intoxication (fratricide) that will be addressed in future studies by our group.”

    Minor comments:

    1. Figure 1 is a bit confusing in terms of the layout. It would be beneficial if the authors separated parts A and B by a few spaces.

    As suggested, we have modified the layout of Figure 1 to more clearly distinguish between the two mechanisms tested.

    1. Figure 2A should start off by showing that the Xantho T4SS can kill other bacteria (e.g. Fig S4A). This would set up the paper better.

    As suggested, old Figure S4A has now been transferred to Figure 2A in the revised manuscript.

    1. Fig. 2A should include p values.

    As suggested, p values have been provided in the legend of Figure 2B (old Figure 2A).

    1. Fig. 2B is really hard to see and should be removed from the manuscript (although I do appreciate the novelty of the technique using Marilyn Monroe).

    We have transferred the old Fig. 2B to the Supplementary Material (Fig S2) of the revised manuscript. We agree that the effect is subtle, but we want to maintain the figure since the transparency of the ΔXAC2610 X. citri colonies over time were the first observations that led us to investigate this phenomenon. Additionally, to reduce potential human bias and to enhance the objectivity of the assay, we employed a Convolutional Neural Network (CNN) to analyze all the colonies presented in Fig S2. This method provides a confidence tendency index for opacity and transparency variations. A detailed description of this new methodology is in the "Materials and Methods" section (Convolutional Neural Network (CNN) analysis).

    1. Instead all of the data in Fig. 2B should be shown in a new version of Fig. 2C. Fig. 2C should include additional controls including:
    2. A wild type strain containing 2609 and 2610 mutants
    3. A complete virB operon deletion in combination with 2609 and 2610 mutants
    4. ∆8 strain
    5. 2609 lacking its T4SS signal sequence
    6. 2609 targeted to the periplasm with a sec signal sequence
    7. etc.

    We sincerely value the comprehensive suggestions for improving what was previously presented as Fig. 2D. (Current version of Figure 2B is the Fig S2 as mentioned in the previous observation). We encounter a practical challenge here: the primary authors responsible for these experiments, especially the first five, have since departed from our lab. This situation limits our immediate capacity to execute the extensive set of experiments you've proposed.

    Recognizing the significance of the controls you've outlined for a quantitative analysis of the colony phenotypes (Fig. 2C (current version)) we have instead supplemented our study with a rigorous quantitative analysis of the microscopy assays referenced in Movies S1-S5, Figure 3, Table S4. These analyses further emphasize our observations concerning colony transparency (Fig S2).

    1. Figure 2C. The VirB7 western band looks like in the 2610 complemented strain.

    Thank you for pointing out the discrepancy in our previous manuscript at line 366, which pertains to the description of the mutants in old Fig. 2. The double mutant, ΔX-TfiXAC2610ΔvirB7 strain, was actually complemented with X-TfiXAC2610 (as stated in the current version (Fig S2B), and not with VirB7. Additionally, we have corrected the legend of the figure (line 684 previous version) from (∆X-TfiXAC2610∆VirB7c) to (∆X-TfiXAC2610c∆VirB7). We apologize for the mix-up in our earlier description and are grateful for your meticulous review and feedback in this matter. Furthermore, we agree that, in this particular experiment, the VirB7 band seems weaker but it is clearly visible in the 2610 complemented strain.

    1. Figure 3C should include a comparison of exponential vs. stationary phase cells. In addition, the results for the ∆2610 mutant and the ∆2610 ∆B7 double mutant appear to be different(?). P values should be provided. If it is statistically significant, then this should be explained in the manuscript. It was not clear how the % damaged cells were calculated? # of cells? Stats?

    The statistical analysis that the reviewer suggested has been provided in the new version of the Figure 4C and its legend. In addition, we have also included a supplementary Table S5 that presents the total number of cells analyzed in these experiments.

    1. The majority of Figure 4 should be replaced by assaying the effect of a virB operon deletion rather than showing the individual mutants.

    We believe that retaining old Figure 4 (****Figure 5 of the revised manuscript) is important. By showcasing results from this specific set of single mutants, we are able to rule out the possibility that X-TfeXAC2609 translocation into the periplasm is mediated by a distinct X-T4SS subunit or subcomplex. We've expanded on this rationale at the start of the paragraph to provide a more comprehensive justification for our approach.

    1. Discussion:
    2. The last one to two paragraphs of the results belong in the Discussion.
    3. A more detailed description of cis-intoxication would be useful.

    As suggested, the last two paragraphs the Results section of the original manuscript have now been moved to the end of the Discussion.

    As suggested by the reviewer, third paragraph of the Discussion describes cis-intoxication in more detail.

    Reviewer #2 (Significance):

    This work provides a conceptual advance in understanding the protective function of a T4SS immunity protein, X-Tfe XAC2610, against the cis-toxic effects of the T4SS effector, X-Tfi XAC2610. It will likely be of interest to scientists interested in T4SSs & T6SSs and interbacterial competition. Overall this is a thought-provoking manuscript and should be published in a respectable journal.

    We sincerely thank Reviewer #2 for the thoughtful appraisal and positive feedback regarding our work. We are gratified to hear that the reviewer recognizes the conceptual advance our research brings to the understanding of T4SS immunity proteins and are encouraged by the acknowledgment that this manuscript will be of interest to our peers. We truly appreciate the endorsement for publication in a reputable journal.

    Reviewer #3 (Evidence, reproducibility and clarity):

    In this study, the authors suggest that TfeXAC2609-TfiXAC2610 represent a novel deviation from the established paradigm in contact-dependent interbacterial secretion systems. X. citri strains lacking the predicted immunity protein, TfiXAC2610, do not suffer a competitive disadvantage when grown in T4SS-inducing conditions against a wild-type strain. Furthermore, cells lacking the immunity develop aberrant morphology and auto-lyse. The mechanism for self-intoxication by Tfe****XAC2609 is independent of a functional T4SS, and intoxication is exacerbated when the toxin's T4SS-signal sequence is removed.

    Major Points

    1. The authors of the study do not provide sufficient evidence that TfeXAC2609 contributes to T4SS mediated killing. Does the toxin behave in a synergistic way, rather than mediate killing independently? Does removing the toxin and immunity change the competitive advantage of X. citri?

    We have shown in a previous publication that X-TfeXAC2609 does contribute to X-T4SS mediated killing (Oka et al, 2022). In that published paper we show that even in the absence of seven other toxin/antitoxin pairs, X-T4SS mediated transfer of only one effector (X-TfeXAC2609 or X-TfeXAC3634) can kill E. coli cells.

    Removing only X-TfeXAC2609 and X-TfiXAC2610 does not significantly reduce the ability of X. citri cells to kill E. coli (Fig. 2A of the revised manuscript). This is expected since this double mutant still retains seven other toxin/immunity pairs.

    Suggested Experiments: Competing, against E. coli, both WT X. citri and X. citri ΔXAC2609 ΔXAC2610, and determining whether there is a change in relative competitive advantage, or expressing TfeXAC2609 in a heterologous system and marking any observed toxic phenotype.

    The results of the experiment suggested by the reviewer have now been included in part A of the revised version of Figure 2. The effect of deleting only one toxin such as X-TfeXAC2609 results in no detectable difference in killing efficiency, most likely due to the presence of the eight other X-Tfes, three of which have been shown (XAC3634) or are predicted (XAC0466 and XAC1918) to have pedptidoglycan hydrolase activity (Oka et al, 2022, Souza, 2015, Sgro et al, 2019).

    1. Authors should directly answer where the toxin is active and localized in the cell.

    Suggested Experiments: Western blot subcellular fractionation (cytoplasm, periplasm, etc) to determine the localization of each protein.

    In response to the query about the toxin's activity and localization within the cell, we acknowledge the importance of such experiments to shed light on these aspects. However, I would like to highlight that the five first authors of this work are no longer affiliated with our lab. Consequently, we are facing constraints in terms of manpower and expertise to undertake comprehensive experiments such as the suggested subcellular fractionation.

    Also, our earlier work demonstrated the importance of the XVIPCD for secretion via X-T4SS (Souza, 2015) and in vivo activity of X-TfeXAC2609 (Oka et al., 2022). Moreover, using heterologous proteins expressed in E. coli (Souza, 2015) and our current observation that the absence of X-TfiXAC2610 induces spheroplast formation (Fig 4A-B, Movie S6) strongly suggest that the peptidoglycan glycohydrolase activity of the N-terminal domain of X-TfeXAC2609 acts in the periplasm.

    1. There is no evidence that TfeXAC2609 plays any role in inter-bacterial killing besides that is predicted from its genetic arrangement and in vitro assays from a previous publication.

    Suggested Experiments: Again, with the available antibodies, detecting whether TfeXAC2609 is being secreted, either in competition settings against X. citri or E. coli; given that there is no killing observed in Fig. 2B, it may also be a suitable control for this experiment.

    We have published in vivo evidence in the past:

    Souza et al, 2015 showed that X-TfeXAC2609 is secreted when in contact with E. coli cells.

    Oka et al, 2022 showed that an X. citri strain expressing X-TfeXAC2609/X-TfiXAC2610 but lacking seven other toxin/antitoxin pairs can still kill E. coli.

    1. The structural and co-evolutionary analysis seems to miss an essential point - that the lack of fratricide protection is not due to a novel protein-protein interaction.

    We do not understand this comment. As we point out in the manuscript, X-TfiXAC2610 does not protect against fratricide (trans-intoxication) but instead does protect against suicide (cis-intoxication). This protection requires a X-TfeXAC2609-X-TfiXAC2610 protein-protein interaction supported by the structural and co-evolutionary analysis as well as the experimental data using the X-TfiXAC2610 Y170A mutant (Fig. 6D of the revised manuscript). Moreover, we believe that the structural and sequence analysis significant expand the knowledge of the broader family of immunity proteins to which X-TfiXAC2610 belongs (Fig. S10 and Fig. S11 of the revised manuscript).

    1. The role of the immunity in biofilm formation is confusing. Cells lacking the immunity die within 96 hours (the auto-lysis phenotype). Given that the immunity is required for viability in this time frame, wouldn't it also be required for viability after five days?

    Suggested Amendments: Remove or de-emphasize.

    In the manuscript we use several different techniques to show that cells lacking the X-TfiXAC2610 immunity protein are less viable than the wild-type strain under certain conditions (growth on LB agar plates, biofilm formation) but perhaps not under others (ie in direct short-term competition experiments against E. coli and in long-term (2 week) in planta citrus canker assays). This is consistent with the fact that ultrastructural analysis by transmission electron microscopy shows that when grown in liquid media, only around 2% of X. citri cells lacking X-TfiXAC2610 present significant damage to their cell envelope (only 0.1% of wild-type cells show damage).

    1. Why does cell permeability increase with the loss of the T4SS signal sequence? Without there being greater evidence to support that an alternative secretion system is secreting or transporting the toxin into the periplasm, which may compete with the T4SS, additional hypotheses should be experimentally probed.

    The reviewer is comparing the propidium iodide permeability results observed for the ΔX-TfiXAC2610 mutant (carrying an empty pBRA plasmid) that expresses full-length X-TfeXAC2609 from its chromossomal gene with the ΔX-TfeXAC2609/ΔX-TfiXAC2610 double mutant carrying the pBRA-X-TfeXAC2609NT plasmid that expresses the X-TfeXAC2609 protein lacking the T4SS signal sequence from a very strong inducible promoter. Therefore, it can be expected that the levels of the truncated effector could be significantly greater than that of the full-length effector, leading to more damage.

    Note that, in the absence of X-TfiXAC2610, cell permeability increases only if X-TfeXAC2609 is present, with or without its XVIPCD T4SS signal sequence. This is consistent with a cis-intoxication mechanism which is independent of the X-T4SS-mediated transfer of the toxin from one cell to another. As we mention in the revised manuscript, and as pointed out by reviewer 1, Ho et al have also observed that when a lysozyme-containing domain of the T6SS effector VgrG3 is expressed in E. coli or in Vibrio cholerae, it can be detected in the periplasm in spite of the lack of a detectable signal sequence and in the absence of a functional T6SS. Ho et al attributed this observation to a “cryptic” secretion mechanism.

    1. Unclear if the the loss of cell envelope integrity is a direct effect of TfeXAC2609 activity and not an artifact of cell death. The microscopy also does not show a consistent change in morphology amongst intoxicated cells as there are healthy cells adjacent to lysed cells. This needs to be investigated in much more mechanistic detail.

    We observed that the X-TfeXAC2609 toxicity is dependent on its lysozyme domain since a point mutation in the active site residue (E48A) abolishes the toxicity-related phenotype in the biofilm assay (****Figure 5****).

    1. The role for immunity proteins in cis-intoxication is not novel as proposed by the authors. For example, see PMID:22511866 and PMID:26456113 where the authors used an inducible degradation system to show that in a T6SS null strain, cis-intoxication occurs when immunity is depleted.

    We thank the reviewer for pointing out these observations which are now mentioned and cited in the Introduction and in the Discussion of the revised manuscript.

    Minor Revisions

    1. Inconsistent use of the term "self-killing"; either refers to the killing of kin cells, or self (interchangeably used to refer to trans and cis killing).

    The term “self-killing” no longer appears in the manuscript.

    1. Terms trans-intoxication and cis-intoxication are convoluted and not constructive to the points being communicated. Self-killing vs kin-killing seem more intuitive and clearer. We prefer to maintain the use of the terms cis-intoxication and trans-intoxication which we defined in the Introduction, at the beginning of the Results section and in the Discussion as well as in Figure 1.
    2. Readability would be improved by the removal of double negatives.

    We have tried to avoid these whenever possible.

    1. Bacterial competition assay in methods only refers to the E. coli competition, not the one between the different genotypes of X. citri****.

    Both methods were described in the same paragraph in the original manuscript. For clarity, this has now been divided into two sections in the revised manuscript: “X. citri vs E. coli competition assays” and “X. citri vs X. citri competition assays”.

    1. Strain naming scheme presented on pg. 16 doesn't conform to traditional, and clearer, nomenclature typically used.

    We have checked the manuscript to make sure that strain naming was consistent throughout the manuscript.

    1. On Pg 25, there is a typo "X-TfiXAC2609" as opposed to X-TfeXAC2609

    Thank you for the observation. This has now been corrected.

    1. Line 619 - "or several other immunity proteins in competition assays"... where was this data shown? No immediate connection to any figures from this paper nor are there any references.

    This is shown in Figure 2B and in Movie S7 which is now cited directly in the revised manuscript.

    Reviewer #3 (Significance):

    Overall it is difficult to take paradigm-conflicting conclusions at face-value when they are not presented alongside concrete experimental evidence. Without directly showing that the toxin localizes to the periplasm, the explanation that "the toxin somehow makes its way into the cell periplasm [independent of the T4SS] where it degrades the peptidoglycan layer" hinders the other conclusions presented by the authors. Consequently, my enthusiasm for this work is minimal.

    We deeply appreciate the insightful feedback from Reviewer #3, particularly regarding the concerns about paradigm-conflicting conclusions. We are steadfast in our commitment to ensuring that our findings are both rigorous and scientifically relevant.

    Evidence for Toxin Localization: We understand the criticality of concrete experimental evidence for toxin localization to the periplasm. While our data suggest an yet to be discovered translocation pathway of X-TfeXAC2609 from the cytoplasm to the periplasm, we recognize the importance of providing direct evidence. We are actively working on methodologies to understand this phenomenon. However, we do not believe that answering this question is absolutely necessary to understand the main conclusions of the present manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    In this study, the authors suggest that TfeXAC2609-TfiXAC2610 represent a novel deviation from the established paradigm in contact-dependent interbacterial secretion systems. X. citri strains lacking the predicted immunity protein, TfiXAC2610, do not suffer a competitive disadvantage when grown in T4SS-inducing conditions against a wild-type strain. Furthermore, cells lacking the immunity develop aberrant morphology and auto-lyse. The mechanism for self-intoxication by TfeXAC2609 is independent of a functional T4SS, and intoxication is exacerbated when the toxin's T4SS-signal sequence is removed.

    Major Points

    1. The authors of the study do not provide sufficient evidence that TfeXAC2609 contributes to T4SS mediated killing. Does the toxin behave in a synergistic way, rather than mediate killing independently? Does removing the toxin and immunity change the competitive advantage of X. citri?
      Suggested Experiments: Competing, against E. coli, both WT X. citri and X. citri ΔXAC2609 ΔXAC2610, and determining whether there is a change in relative competitive advantage, or expressing TfeXAC2609 in a heterologous system and marking any observed toxic phenotype.
    2. Authors should directly answer where the toxin is active and localized in the cell.
      Suggested Experiments: Western blot subcellular fractionation (cytoplasm, periplasm, etc) to determine the localization of each protein.
    3. There is no evidence that TfeXAC2609 plays any role in inter-bacterial killing besides that is predicted from its genetic arrangement and in vitro assays from a previous publication.
      Suggested Experiments: Again, with the available antibodies, detecting whether TfeXAC2609 is being secreted, either in competition settings against X. citri or E. coli; given that there is no killing observed in Fig. 2B, it may also be a suitable control for this experiment.
    4. The structural and co-evolutionary analysis seems to miss an essential point - that the lack of fratricide protection is not due to a novel protein-protein interaction.
    5. The role of the immunity in biofilm formation is confusing. Cells lacking the immunity die within 96 hours (the auto-lysis phenotype). Given that the immunity is required for viability in this time frame, wouldn't it also be required for viability after five days?
      Suggested Amendments: Remove or de-emphasize.
    6. Why does cell permeability increase with the loss of the T4SS signal sequence? Without there being greater evidence to support that an alternative secretion system is secreting or transporting the toxin into the periplasm, which may compete with the T4SS, additional hypotheses should be experimentally probed.
    7. Unclear if the the loss of cell envelope integrity is a direct effect of TfeXAC2609 activity and not an artifact of cell death. The microscopy also does not show a consistent change in morphology amongst intoxicated cells as there are healthy cells adjacent to lysed cells. This needs to be investigated in much more mechanistic detail.
    8. The role for immunity proteins in cis-intoxication is not novel as proposed by the authors. For example, see PMID:22511866 and PMID:26456113 where the authors used an inducible degradation system to show that in a T6SS null strain, cis-intoxication occurs when immunity is depleted.

    Minor Revisions

    1. Inconsistent use of the term "self-killing"; either refers to the killing of kin cells, or self (interchangeably used to refer to trans and cis killing).
    2. Terms trans-intoxication and cis-intoxication are convoluted and not constructive to the points being communicated. Self-killing vs kin-killing seem more intuitive and clearer
    3. Readability would be improved by the removal of double negatives.
    4. Bacterial competition assay in methods only refers to the E. coli competition, not the one between the different genotypes of X. citri.
    5. Strain naming scheme presented on pg. 16 doesn't conform to traditional, and clearer, nomenclature typically used.
    6. On Pg 25, there is a typo "X-TfiXAC2609" as opposed to X-TfeXAC2609
    7. Line 619 - "or several other immunity proteins in competition assays"... where was this data shown? No immediate connection to any figures from this paper nor are there any references.

    Significance

    Overall it is difficult to take paradigm-conflicting conclusions at face-value when they are not presented alongside concrete experimental evidence. Without directly showing that the toxin localizes to the periplasm, the explanation that "the toxin somehow makes its way into the cell periplasm [independent of the T4SS] where it degrades the peptidoglycan layer" hinders the other conclusions presented by the authors. Consequently, my enthusiasm for this work is minimal.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript explores the role of an immunity protein of the Xanthomonas type IV secretion system (X-T4SS). In contrast to most T4SSs that conjugate plasmids or transfer effectors into host cells, this system is able to kill other bacteria similar to the role of T6SSs. Here, the authors tested whether the immunity protein XAC2610 functions to prevent cis-intoxication (by self) and/or trans-intoxication (by sister cells). They provide data that the XAC2610 immunity protein functions to protect cis intoxication, but not trans-intoxication, by the T4SS effector XAC2609 (which functions as a peptidoglycan hydrolase). Based on AlphaFold modeling, they went on to identify a residue in XAC2610 that is critical for inhibiting the activity of the XAC2609 toxin. Overall the data is fairly solid and generally support the conclusions the authors made.

    Major comments:

    One of the major conclusions of the manuscript is that XAC2610 does not prevent trans-intoxication and the data in the manuscript support this conclusion. However, I wonder if this is an oversimplification. Notably, the authors observed that wild type Xantho was unable to kill a target cell lacking 8 different toxin/immunity systems (Fig. 1A). One could conclude that none of these immunity proteins function in preventing trans-intoxication ... or ... perhaps it appears that none perform this role because wild-type Xantho never attacks its siblings? For example, it is conceivable that Xantho uses a general mechanism, perhaps somewhat similar to phage exclusion or plasmid incompatibility, to prevent sibling attack? To me this seems more likely than none of the eight immunity proteins play a role in preventing trans-intoxication. Moreover, the phenotype observed for the ∆2610 mutant in preventing cis-intoxication is somewhat subtle, likely because the toxin and the immunity protein are topologically restricted to the cytoplasm and the periplasm, respectively. This would make sense if this were not the primary role for 2610.

    Ideally the authors will be able to test this theory by demonstrating that a wild-type Xantho strain can attack (but likely not kill) its siblings. Alternatively, could the authors test if related, but not identical, Xantho strains that express 2609/2610 are able to kill their ∆2610 mutant, i.e. do "cousins" attack each other? Not sure about the semantics but this could be described as preventing trans-intoxication. If they are unable to do either experiment, that is ok but they should at least describe this concept in their discussion (assuming they agree).

    Since the cis-intoxication phenotype of the ∆2610 mutant is subtle, it would strengthen the authors' conclusions on cis-intoxication if they artificially targeted XAC2609 to the periplasm with a sec signal sequence. If the authors are correct, this should be a lethal event in the absence of the 2610 immunity protein. This might be useful in terms of figuring out how the 2609 toxin normally gets into the periplasm, a major unanswered question in this manuscript.

    Minor comments:

    1. Figure 1 is a bit confusing in terms of the layout. It would be beneficial if the authors separated parts A and B by a few spaces.
    2. Figure 2A should start off by showing that the Xantho T4SS can kill other bacteria (e.g. Fig S4A). This would set up the paper better.
    3. Fig. 2A should include p values.
    4. Fig. 2B is really hard to see and should be removed from the manuscript (although I do appreciate the novelty of the technique using Marilyn Monroe).
    5. Instead all of the data in Fig. 2B should be shown in a new version of Fig. 2C. Fig. 2C should include additional controls including:
    • a. A wild type strain containing 2609 and 2610 mutants
    • b. A complete virB operon deletion in combination with 2609 and 2610 mutants
    • c. ∆8 strain
    • d. 2609 lacking its T4SS signal sequence
    • e. 2609 targeted to the periplasm with a sec signal sequence
    • f. etc.
    1. Figure 2C. The VirB7 western band looks like in the 2610 complemented strain.
    2. Figure 3C should include a comparison of exponential vs. stationary phase cells. In addition, the results for the ∆2610 mutant and the ∆2610 ∆B7 double mutant appear to be different(?). P values should be provided. If it is statistically significant, then this should be explained in the manuscript. It was not clear how the % damaged cells were calculated? # of cells? Stats?
    3. The majority of Figure 4 should be replaced by assaying the effect of a virB operon deletion rather than showing the individual mutants.
    4. Discussion:
    • a. The last one to two paragraphs of the results belong in the Discussion.
    • b. A more detailed description of cis-intoxication would be useful.

    Significance

    This work provides a conceptual advance in understanding the protective function of a T4SS immunity protein, X-Tfe XAC2610, against the cis-toxic effects of the T4SS effector, X-Tfi XAC2610. It will likely be of interest to scientists interested in T4SSs & T6SSs and interbacterial competition. Overall this is a thought-provoking manuscript and should be published in a respectable journal.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Oka et al. shows that one effector of X. citri is likely translocated into the periplasm where it cleaves PG unless inhibited by its cognate immunity protein. Interestingly, this effector is required for killing of target cells like E. coli in T4SS-dependent manner but it does not seem to be delivered into X. citri cells by T4SS. Authors show using various assays that cells lacking the immunity protein have various phenotypes including lysis and defect in biofilm formation, however, despite "cis-intoxication" the ability to kill other bacteria or infect plants remains unaffected. The manuscript is well written and in general all the experiments have proper controls and thus the conclusions seem solid. The results described here are novel and interesting as they as unexpected.

    Major issues that should be addressed:

    • Test various deletion variants of the toxin to identify which part of the protein is responsible for its translocation into the periplasm. This may help to identify the possible mechanism of translocation of the toxin into the periplasm. Alternatively, the authors may attempt to select for non-toxic point mutants of the toxin. This could be done by a random PCR mutagenesis of the toxin and a selection of the surviving mutants in the absence of the immunity protein.
    • Test if localization of the immunity protein to the cytoplasm blocks its activity. An immunity protein mutant that lacks its secretion signal should not protect against cis-intoxication.
    • While many experiments support the conclusion that the toxin is responsible for "cis-intoxication, the test of "trans-intoxication" should be done again but with the same setup as was used for testing of killing of E. coli. The CPRG based assay is far more sensitive than counting survival by plating to count CFUs. This test should be done at a relatively high initial OD so that there is an immediate contact between the "killer" and the "prey" bacteria (lacking immunity/effector). If needed, LacZ should be over-expressed in X. citri to make use of the CPRG based assay. In addition, such assay could be used also for "cis-intoxication" to supplement the potentially hard to quantify biofilm experiments shown in Fig. 4 (e.g. test all the T4SS mutants for "cis-intoxication").
    • Fig. 2A needs a positive control. For example, test killing of E. coli under the same conditions.
    • Authors should look at the paper by Ho et al. PNAS 2017, which describes trafficking of VgrG of V. cholerae into the periplasm of E. coli without an obvious secretion signal. The effector of X. citri may behave similarly.
    • Provide some form of quantification of the phenotypes (cell rounding and cell death) observed using live-cell imaging.
    • Provide quantification of biofilm related phenotypes as well as of the citrus canker development assay.

    Significance

    The study provides an interesting insight into immunity proteins against anti-bacterial toxins. It points to a need to protect against "cis-intoxication". This is novel and interesting to a wide audience of microbiologists interested in bacterial competition as this could be true also for other toxins. It would be however important to identify how is the toxin translocating to the periplasm of the producing bacterium. Some insight into the mechanism would vastly improve the study. My expertise is in understanding bacterial interactions and competition but I lack a direct experience with assays specific for X. citri.

  5. Version published to 10.1101/2023.08.28.555164v1 on bioRxiv