A pyocin-like T6SS effector mediates bacterial competition in Yersinia pseudotuberculosis

ABSTRACT Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa, respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK_0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis. Next, we sought to determine its identity and characterize its toxicity. We found that YPK_0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis. We further identified YPK_0952 as a DNase dependent on Mg2+, Ni2+, Mn2+, and Co2+ bivalent metal ions, and the homologous immune protein YPK_0953 can inhibit its activity. In summary, YPK_0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK_0953 prevents self-attack in Y. pseudotuberculosis. IMPORTANCE Bacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK_0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis. YPK_0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation.


General comment.
There is a gap in knowledge in the characterization of Y. pseudotuberculosis T6SS effectors.This analysis may unveil new principles of general interest beyond Y. pseudotuberculosis.The presented work is sounded although the authors do not provide conclusive evidence demonstrating that the effector is indeed secreted in a T6SS-dependent manner, and that the effector exerts a killing effect when deliver via the T6SS in a contact-dependent manner.These two experiments are crucial to sustain their conclusions.In addition some other controls and clarifications are needed.This reviewer hopes the authors will find the comments useful to strengthen this manuscript.
Major comments.1.No statically analysis is done in the case of figures 2 and 4. 2. Authors have not demonstrated the secretion of the effector in a T6SS-3 dependent manner.This experiment is easily done using a tagged version of the protean (either in the chromosome e or in a plasmid) and assess the secretion to the supernatant by the wild-type and the T6SS-3 null (non-functional) mutant strain.In case, there is still secretion (or lower), authors should then assess the secretion by any of the other T6SS systems.3. A point the authors make is the contact-dependent delivery of the toxin.This is important for this type of pyocin-like toxins that can exert their antimicrobial effect without need of being injected.As such their results could be interpreted by t6SS secretion of the toxin, and its subsequent internalization from the supernatant instead of being injected into the cell.Some work should be done to provide evidence.This reviewer suggests these two experiments: (i) separate prey and predator by a membrane and assess killing; (ii) test the antimicrobial properties (and capacity to degrade DNA) of condition media from wild-type, t6SS-3 null strain, and the effector mutant.Authors may wish to consider assessing the contact-depedent translocation by microscopy.4. Pull-dpwn experiments.Could the authors explain the two bands observed in the anti-his blot? 5. Fig 4 .Panel A does not show any effect for the t6SS effector.Whereas the results with EDTA may support the role of divalent cations, panel B cannot be considered conclusive for the role of the specific cations (the buffer may be "contaminated").Besides there are differences in the outcome of the different cations, and, for example, it might be tempting to speculate that this is a Mn toxin.Further robust biochemistry experiments are warranted to clarify this point.Panel E; the signal of the vector alone (as well as other controls) is unusually high for a flow cytometry experiment.Authors need to provide as supplementary material the gating showing the particle size and the FITC channel.In addition, other technique is needed to substantiate these observations (for example immunoblotting).6.Fig 5A is bit confusing.On the one hand, this reviewer suggests to show the counts of ONLY the prey.In this context, wt versus wt there should not be any differences as this would be the max value.Potentially, this result should be similar to WT-clpV3.If here you have more bacteria then authors need to speculate/explain why this is the case because these strains should contain the immunity proteins.In panels A and B, it will be informative to show the control of prey incubated for the same time with no other bacteria but the same volume of PBS.6. Authors do not provide any rationale for the chosen site-directed mutants generated.Are these resides within the active site?any other reason?This should be specified in the text, and if needed provide some modeling information.7. Authors need to revise extensively the manuscript to increase the readability by avoiding superfluous expressions, avoid long sentences, limit the use of passive voice, and ensure there are no grammatical errors.There are too many to pinpoint but just as examples: lines 65-67, lines 71-75, line 77-79, lines 228-230, line 370, line 414 and many others.3.In vivo work.Authors need to show in the graph the limit of the detection of the plating.4. Authors need to justify why they had only infected female.Infecting only one sex is not a good practice.
Reviewer #2 (Comments for the Author): 1.The overall structure and logic of the article are clear, but the content is limited.I hope the author can further explore the mechanism of mediating bacterial competition.2.The writing of this manuscript is rough, for example, the description of certain experimental methods is too simple and not suitable for researchers to conduct experimental operations according to the current methods.3.As for mouse colonization determination, I suggest supplementing experimental conditions such as the number of mice in each group, the weight of colon and small intestine tissues, etc.

Response to reviewers
We are pleased to see the reviewers' enthusiasm toward our manuscript and appreciate their insightful suggestions that have helped strengthen the revised manuscript.Here, we provide a detailed response to each reviewer's comments and the corresponding changes made to the revised manuscript.Please note the following: Text in black font corresponds to the reviewers' comments, blue font text is our response to the reviewers' comments, and highlighted text represents the excerpts from the revised manuscript.

Response to Reviewer 1
Yang and coworkers aim to shed light into the mode of action of a putative T6SS effector encoded within one of the T6SS clusters of Y. pseudotuberculosis.They leveraged molecular microbiology and biochemistry methods to establish the mode of action.In vivo work challenging a gut infection model showed the contribution of this effector to virulence.

Re:
We greatly appreciate the reviewer's invaluable comments and suggestions on our work, which are extremely helpful for us to improve our manuscript.

General comment
There is a gap in knowledge in the characterization of Y. pseudotuberculosis T6SS effectors.This analysis may unveil new principles of general interest beyond Y.
pseudotuberculosis.The presented work is sounded although the authors do not provide conclusive evidence demonstrating that the effector is indeed secreted in a T6SS-dependent manner, and that the effector exerts a killing effect when deliver via the T6SS in a contact-dependent manner.These two experiments are crucial to sustain their conclusions.In addition some other controls and clarifications are needed.This reviewer hopes the authors will find the comments useful to strengthen this manuscript.
Re: Thanks for the positive comments and constructive suggestions!The dependence of YPK_0952 on T6SS-3 for secretion and its killing effect via T6SS in a contact-dependent manner are very important to our conclusion.
1) The reliance of YPK_0952 on T6SS-3 secretion has been confirmed by our group and published in the reference 23 of the manuscript, where the corresponding description in the manuscript, page 14, " The primary open reading frame (ORF), YPK_0952, is distinguished by a typical PAAR domain at its N-terminus and an S-type pyocin domain at its C-terminus.YPK_0952 predominantly functions as a T6SS effector secreted by T6SS-3 (23).", and the literature describes it as " To identify novel T6SS effectors, we searched the Yptb YPIII genome for genes containing the Proline-Alanine-Alanine-Rginine (PAAR) domain, a conserved effector-targeting domain that is linked or adjacent to numerous known T6SS effectors 7,21,22 .A gene locus encoding multiple hypothetical T6SS effector-immunity pairs was identified (YPK_0952-0958, Fig. 1a).Both the first and last open-reading frame (ORF) of this locus contain PAAR domains.The first ORF, YPK_0952, contains a typical PAAR domain at its N-terminus and an S-type pyocin domain at its C-terminus.When VSVG-tagged YPK_0952 was produced in YPIII, the secreted protein was readily detected in the supernatant.However, YPK_0952 secretion was abrogated in the Δ4clpV mutant, in which all four essential ATPase genes in the four sets of T6SSs were deleted, strongly suggesting that YPK_0952 is a T6SS effector.The secretion of YPK_0952 was dramatically diminished with deletion of clpV3, but not with deletion of clpV1, clpV2, or clpV4, further indicating that YPK_0952 is a T6SS effector mainly associated with T6SS-3 (Supplementary Fig. 1a).Similarly, we showed that YPK_0954, which does not contain a PAAR domain but is located downstream of YPK_0952, is also a T6SS-3 effector (Fig. 1b and Supplementary Fig. 1b)." 2) The intra-species contact competition experiment (Fig. 5A) shows that the wild-type Y.pseudotuberculosis has a distinct advantage in competition with ∆ypk_0952∆ypk_0953 (YPK_0952 and YPK_0953 deletion mutant) and ∆clpV3 (clpV3 deletion mutant).The inter-species contact competition experiment (Fig. 5B) demonstrates that both ∆ypk_0952 and ∆clpV3 have a significantly lower competitive advantage over E. coli DH5α compared to the wild-type Y.
pseudotuberculosis.It is evident from this that YPK_0952 exerts its lethal effect by T6SS in a contact-dependent manner.
Comment 1: No statically analysis is done in the case of figures 2 and 4.
Re: Thank you for raising this important issue.For the experiments involved in Fig. 2 and Fig. 4, we referred to the methods described in reference 23 from the manuscript.1) Fig. 2 presents the toxicity growth experiments of Y. pseudotuberculosis and E. coli, where the number of bacteria increases to varying degrees as the culture time extends.We conducted statistical analyses, the data in the manuscript represent mean ± SD, n = 3, with three biological replicates.
2) Fig. 4E showcases a flow cytometry assay, where each sample was tested for ten thousand cells, meeting the requirements for a TUNEL assay.The results obtained are accurate and reliable.
Comment 2: Authors have not demonstrated the secretion of the effector in a T6SS-3 dependent manner.This experiment is easily done using a tagged version of the protean (either in the chromosome e or in a plasmid) and assess the secretion to the supernatant by the wild-type and the T6SS-3 null (non-functional) mutant strain.In case, there is still secretion (or lower), authors should then assess the secretion by any of the other T6SS systems.
Re: Thanks for the kind reminder from the reviewer.Our group's earlier work has already demonstrated that YPK_0952 is secreted by T6SS-3, as mentioned on page 14 of the manuscript.The researchers examined the secretion of YPK_0952 in the wild-type and various T6SS mutants.It was discovered that the secretion of YPK_0952 was completely undetectable in mutants lacking all four sets of T6SS, and significantly reduced in the T6SS-3 mutant, indicating that YPK_0952 is secreted via T6SS-3.The description in manuscript reference 23 is as follows: "Supplementary This is important for this type of pyocin-like toxins that can exert their antimicrobial effect without need of being injected.As such their results could be interpreted by t6SS secretion of the toxin, and its subsequent internalization from the supernatant instead of being injected into the cell.Some work should be done to provide evidence.This reviewer suggests these two experiments: (i) separate prey and predator by a membrane and assess killing; (ii) test the antimicrobial properties (and capacity to degrade DNA) of condition media from wild-type, t6SS-3 null strain, and the effector mutant.Authors may wish to consider assessing the contact-depedent translocation by microscopy.

Re:
We thank the reviewer for the supportive comments on our work.The manuscript reports that the pyocin-like effector protein YPK_0952 is delivered into other cells by T6SS through contact-dependent delivery, exerting its bactericidal effect.In contrast, contact-independent delivery does not require injection and completes toxin transfer through cell surface receptors.Therefore, the experiment involving the killing of bacteria by internalization of supernatant containing the YPK_0952 toxin belongs to contact-independent delivery of toxins, and is not part of the contact-dependent competition mentioned in our text.the results with EDTA may support the role of divalent cations, panel B cannot be considered conclusive for the role of the specific cations (the buffer may be "contaminated").Besides there are differences in the outcome of the different cations, and, for example, it might be tempting to speculate that this is a Mn toxin.Further robust biochemistry experiments are warranted to clarify this point.Panel E; the signal of the vector alone (as well as other controls) is unusually high for a flow cytometry experiment.Authors need to provide as supplementary material the gating showing the particle size and the FITC channel.In addition, other technique is needed to substantiate these observations (for example immunoblotting).

Re:
We appreciate the valuable suggestions.1) As shown in Fig. 4A, His 6 -YPK_0952 is capable of degrading DNA, and the experimental results are the same as those with the addition of DNase I alone, thereby proving that His 6 -YPK_0952 is a DNase.As for your concerns regarding Fig. 4B, we have reformulated the reaction buffer (20 mM MES, 100 mM NaCl, pH 6.9) and conducted the experiment with only the corresponding metal ions added to each lane, ensuring that the buffer was not contaminated.At the same time, we set up a control by adding only λ DNA to the lane to demonstrate that the reaction buffer was not contaminated.Furthermore, our intention is to demonstrate that YPK_0952 is a DNase that relies on various divalent metal cations, rather than a specific type of divalent metal cation enzyme (such as, Mn 2+ ).The updated Fig. 4B on page 28 of the manuscript is as follows.3) E. coli BL21 (DE3) containing plasmids with pET28a, pET28a-ypk_0952, pET28a-ypk_0952-ypk_0953, pET28a-ypk_0952 F441A or pET28a-ypk_0952 Y450A were induced with 0.5 mM IPTG for 4 hours followed by western blotting.The results showed that all toxin were expressed except for the empty vector.Fig. 4E in the manuscript demonstrates that only the YPK_0952 was TUNEL positive.This indicates that only YPK_0952 has DNase activity that can fragment DNA within E. coli BL21 (DE3).
Determination of YPK_0952 expression levels in E. coli BL21 (DE3) harboring pET28a or its derivatives using an anti-His antibody.
Comment 6: Fig 5A is bit confusing.On the one hand, this reviewer suggests to show the counts of ONLY the prey.In this context, wt versus wt there should not be any differences as this would be the max value.Potentially, this result should be similar to WT-clpV3.If here you have more bacteria then authors need to speculate/explain why this is the case because these strains should contain the immunity proteins.In panels A and B, it will be informative to show the control of prey incubated for the same time with no other bacteria but the same volume of PBS.
Re: Sorry for the imprecise description in the former manuscript.

1)
We have made changes to Figure 5A, now only displaying the CFU of prey, on page 29.
2) Regarding the question of why the WT has more bacteria after competing with the ∆clpV3 mutant that contains the immune protein YPK_0953.We think that in T6SS-mediated contact-dependent competition, the ∆clpV3 mutant's inability to secrete effector proteins into predator strains prevents it from counterattacking.
Comment 7: Authors do not provide any rationale for the chosen site-directed mutants generated.Are these resides within the active site?any other reason?This should be specified in the text, and if needed provide some modeling information.
Re: Thank you for your suggestion!The manuscript mentions two site-directed mutants, YPK_0952 F441A and YPK_0952 Y450A , which are located within the toxic activity region.Fig. 1A demonstrates that F441 and Y450 are highly conserved sites in YPK_0952, and Fig. 2C and 2D show that the amino acid region from position 411 to 510 is its toxic region.By performing site-directed mutagenesis toxicity screening on multiple highly conserved sites within this toxic region, we found that mutations at F441 and Y450 result in the loss of YPK_0952 toxicity, thereby identifying these two sites as key toxic amino acids.We have added the site-directed mutagenesis toxicity screening experiment to the updated supplementary information, Supplementary Figure 2, on page 3. to compete for resources within complex microbial communities.Control of key nutrients is a central aspect of this survival struggle, for instance, iron is an essential element for cell function, and most bacteria can secrete siderophores to efficiently bind and capture iron ions (37).Beyond nutritional competition, bacteria employ various strategies to optimize their survival capabilities.For example, P. aeruginosa can utilize its motility to overtake other microorganisms such as Agrobacterium tumefaciens, demonstrating how movement can aid bacteria in avoiding or enhancing competitive interactions (38).Chemical warfare is also part of the competitive nature between bacteria, with the production of antibiotics being a common mode of attack (39).However, in the face of antibiotic assault, some bacteria can acquire resistance through genetic mutations and the spread of these genes.Biofilm formation represents another bacterial strategy to counteract antibiotic attacks.The polysaccharide components within biofilms can reduce the penetration of antibiotics, thereby protecting the resident bacteria (40).Additionally, the T6SS is an effective competitive weapon for bacteria that is capable of injecting toxins directly into target cells and possesses the ability to kill a variety of cell types, including bacterial and eukaryotic cells.In summary, bacteria maintain their niche within microbial communities through a variety of complex survival strategies, constantly adapting to and impacting their environment."Comment 2: The writing of this manuscript is rough, for example, the description of certain experimental methods is too simple and not suitable for researchers to conduct experimental operations according to the current methods.
Re: Thanks for the kind reminder from the reviewer.We have revised the experimental methods in the manuscript, providing detailed descriptions to ensure researchers can replicate the experiments using the current methods.
Comment 3: As for mouse colonization determination, I suggest supplementing experimental conditions such as the number of mice in each group, the weight of colon and small intestine tissues, etc.

Re:
We appreciate the valuable suggestion.In the updated manuscript, we have added a description related to the "Murine colonization assay", on page 13: "Female 6-week-old BALB/c mice were acclimated in the laboratory for 3 days.Mice were orally gavaged with 10 9 CFUs of the indicated Y. pseudotuberculosis strains and monitored for 24 h.At the end of the experiment, six mice from each group were euthanized, and colon tissues (from the WT group: 0.43 g, 0.29 g, 0.39 g, 0.34 g, 0.35 g, 0.36 g; from the Δypk_0952Δypk_0953 group: 0.32 g, 0.51 g, 0.39 g, 0.33 g, 0.33 g, 0.36 g) and small intestine tissues (from the WT group: 0.95 g, 1.07 g, 0.85 g, 0.9 g, 0.81 g, 0.76 g; from the Δypk_0952Δypk_0953 group: 0.84 g, 0.96 g, 1.14 g, 0.86 g, 1.49 g, 1.1 g) were weighed.The colon and small intestine tissues were then homogenized in 0.5 mL of PBS on ice, and samples of different dilutions were plated on selective YLB agar containing appropriate antibiotics for CFU counting (23).This was followed by subsequent absolute quantification of CFU by normalization of each sample to the initial pellet weight."Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
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Reviewer #1 (Comments for the Author): The authors provided a detailed response to the issues raised by the reviewers, and additional data is provided.However, two outstanding question remain: added.For figuire, they have the quantitative data available (fluorescenece intensity -median and SD-) and they do indicate that the experiments were done three indepdent tiomes.So there is no barrier to run a one WAY ANOVA analysis with multiple comparisons correction.
2. Authors still have not addressed experimentally the possibility that the toxin is secreted by the T6SS but it is NOT injected into the prey cells by the T6SS.This is one of the key points of the manuscript, The two experiments suggested previously (comment 3) can be easily done by the authors with the tools they have using very simple assays.

Minor comments. 1 .
Authors should consider including as supplementary material a figure showing the T6SS-3 locus and the positions of the genes under consideration in this study.2. Fig 5. Donor and recipient are not the most used terminology for these assays.Please use instead prey and predator.
Figure 1: YPK_0954 (Tce1) is a T6SS-3 secreted nuclease effector and YPK_0955 (Tci1) is its immunity protein.a-b, YPK_0952 and YPK_0954 are T6SS-3 effectors.Plasmids directing the expression of YPK_0952-VSVG (a) or YPK_0954-VSVG (b) were introduced into the indicated Y. pseudotuberculosis strains.Total cell pellet (Pellet) and secreted proteins in culture supernatant (Sup) were isolated and probed for the presence of the fusion protein.The cytosolic RNA polymerase (RNAP) or isocitrate dehydrogenase (ICDH) was similarly detected as a control."Comment 3: A point the authors make is the contact-dependent delivery of the toxin.

Comment 4 :
Pull-down experiments.Could the authors explain the two bands observed in the anti-his blot?Re: Thank you very much for your insightful comments and suggestions to improve our pull-down results!The observation of two bands in the anti-His blot may be due to impurities in the His 6 -YPK_0952 protein used in the GST pull-down experiments.We have repurified the His 6 -YPK_0952 protein and conducted the GST pull-down experiment again.The revised results have been updated in the manuscript on page 27.The updated Fig. 3A is as follows.

Figure 3 .
Figure 3. Direct binding between YPK_0952 and YPK_0953 was detected using GST pull-down and bacterial two-hybrid assays.(A)His 6 -YPK_0952 was incubated with GST-YPK_0953, GST, or an irrelevant recombinant protein GST-YPK_3549, and the protein complexes captured on glutathione beads were detected using western blotting.(B) Interactions were assessed using MacConkey maltose plates (upper) and the β-galactosidase assay

Figure 4 .
Figure 4. YPK_0952 exhibits DNase activity.(A) In vitro DNase activity assay showed the integrity of λ DNA (0.35 µg) co-incubated with YPK_0952 or DNase I in the reaction buffer with or without EDTA at 37°C for 30 min.Reaction products were analyzed using agarose gel electrophoresis.(B) YPK_0952 and λ DNA co-incubated with the reaction buffer containing different divalent metal ions.(C) YPK_0953 inhibited DNase activity of YPK_0952.The DNase activity of the YPK_0952 and YPK_0953 co-incubated were tested in the reaction buffer.(D) The DNase activity of the YPK_0952 F441A and YPK_0952 Y450A variants was tested along with YPK_0952 and DNase I in the reaction buffer.(E) YPK_0952-induced genomic DNA fragmentation was detected before (Left) and 4 h after (Right) IPTG induction in the TUNEL assay.DNA fragmentation was detected based on the monitoring of fluorescence intensity (indicated on the x-axis) using flow cytometry.The counts resulting from cell sorting are shown on the y-axis.2)We have added gating for the FITC channel and display granularity as supplementary material in the flow cytometry experiment, found in the supplementary information on page 5.The updated Supplementary Figure4in the supplementary information on page 5 is as follows.

Figure 5 .
Figure 5. YPK_0952 mediates contact-dependent T6SS killing.(A) Intra-species growth competition between the indicated Y. pseudotuberculosis predator and prey strains.Predator and prey strains (1:1) were mixed and then grown for 48 h on a solid support.The CFU of the prey strains was measured based on plate counts.Error bars represent the mean ± SD of prey CFU from three independent experiments, with two-tailed, unpaired Student's t-test.*P < 0.0332; **P < 0.0021; ***P < 0.0002, ****P < 0.0001.(B) Inter-species growth competition experiments between the indicated Y. pseudotuberculosis predator and E. coli DH5α prey strains.