Capsular polysaccharide restrains type VI secretion in Acinetobacter baumannii

  1. Nicolas Flaugnatti
  2. Loriane Bader
  3. Mary Croisier-Coeytaux
  4. Melanie Blokesch

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Abstract

The type VI secretion system (T6SS) is a sophisticated, contact-dependent nanomachine involved in interbacterial competition. To function effectively, the T6SS must penetrate the membranes of both attacker and target bacteria. Structures associated with the cell envelope, like polysaccharides chains, can therefore introduce spatial separation and steric hindrance, potentially affecting the efficacy of the T6SS. In this study, we examined how the capsular polysaccharide (CPS) of Acinetobacter baumannii affects T6SS’s antibacterial function. Our findings show that the CPS confers resistance against T6SS-mediated assaults from rival bacteria. Notably, under typical growth conditions, the presence of the surface-bound capsule also reduces the efficacy of the bacterium’s own T6SS. This T6SS impairment is further enhanced when CPS is overproduced due to genetic modifications or antibiotic treatment. Furthermore, we demonstrate that the bacterium adjusts the level of the T6SS inner tube protein Hcp according to its secretion capacity, by initiating a degradation process involving the ClpXP protease. Collectively, our findings contribute to a better understanding of the dynamic relationship between T6SS and CPS and how they respond swiftly to environmental challenges.

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  1. Version published to 10.7554/elife.101032 on eLife
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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    #1) Summary: The transport of effector proteins across membranes from the producing bacterium into a target cell is at the core of bacterial secretion systems. How an additional layer in form of a capsule affects effector export and the susceptibility towards effector import is not fully understood. Here, Flaugnatti and colleagues combined bacterial genetics with phenotypic assays and electron microscopy to demonstrate a dual role of a bacterial capsule in preventing T6SS-mediated effector export and promoting protection from effector import by another bacterium's T6SS. The wide variety of methods used, complementation of the mutants, and validation of the findings across strains strengthen the author's conclusions. Although the main conclusions seem straight forward, the authors unravel the unexpected complexity underlying these phenotypes with strong mechanistic work. In brief, a capsule-deficient mutant (∆itra) is shown to assemble its T6SS similar to the WT, yet secretes more Hcp than the WT and is better in T6SS-mediated killing of other bacteria. A capsule-overproducing mutant (∆bfmS) shows both, a partial deficiency in T6SS assembly and an additional reduction in exported Hcp, and is worse in T6SS-mediated killing than the WT. A mutant with a capsule similar to WT and deficient in cell sensing (∆tslA) forms the least T6SS apparatuses and is yet better in T6SS-mediated killing than the overcapsulated mutant. Together, these data show an effect of the capsule on (i) T6SS apparatus assembly, (ii) effector export, (iii) effector import, and (iv) the need for clearance of accumulating non-secreted Hcp by ClpXP. The work on a clinical isolate of Acinetobacter tumefaciens and the data on an impaired T6SS activity on other cells by antibiotic-induced capsulation is a strong demonstration of the work's clinical relevance in addition to the findings' conceptual novelty.

    •        In my view, the manuscript is outstanding with very high quality of experimental data, very well written text and very clear presentation of the data in figures. A few minor comments and suggestions below that I think would strengthen the manuscript.*

    __ Authors’ reply #1: __We thank the reviewer for their enthusiasm.

    Major comment:

    #2) OPTIONAL: Fig. 4c/l. 320: Having an indirect effect of an antibiotic on T6SS activity by antibiotic-induced capsule formation is very intriguing and contributes to the clinical relevance of the overall findings. When I saw the data in Fig. 4c, the graph instantaneously reminded me of the panel in Fig. 2a, where a similar phenotype is observed by changing the predator:prey ratio in the absence of any antibiotic. The authors themselves comment on the possibility of antibiotic-induced, reduced predator growth (and thereby a change in predator:prey ratio) as a one factor impacting the phenotype here. I am wondering if this data could be strengthened or better disentangled to test more precisely if it is the antibiotic induced capsule formation per se that affects T6SS-mediated killing by A. baumanii in the presence of antibiotics. Would it help to take the bfmS mutant along as a control for direct comparison to see if antibiotic-induced capsule formation of the WT to similar levels of the mutant results in the same killing phenotype? Would it help to test for T6SS-mediated killing in the presence and absence of antibiotics at multiple predator:prey ratios? Could the effect of the antibiotic on A. baumanii growth be measured and considered when choosing the ratio at which the bacteria are mixed?

    __ Authors’ reply #2: __The point raised by the reviewer is very important. As we have stated in the manuscript, the capsule-induced production using antibiotics impacts the growth of A. baumannii and could therefore change the predator-prey ratio, potentially affecting the observed phenotype. However, the antibiotic is expected to equally impact the non-encapsulated ΔitrA strain, yet this strain maintains very strong T6SS killing activity in the presence of chloramphenicol. Thus, we do not believe the predator-prey ratio is causing the observed effect. To address this point more directly, we nonetheless propose to: i) repeat the experiments with different predator-prey ratios (1:1, 2:1, and 5:1), and ii) include a bfmS mutant as a control.

    Minor comments:

    #3) Figure 1D, l. 155, I might have missed this, do the authors happen to have the numbers of E. cloacae as well? This would strengthen the claim on A. baumannii survival because of E. cloacae is being killed.

    __ Authors’ reply #3: __The reviewer is correct; we did not include the survival of E. cloacae in the initial manuscript due to technical reasons (counter-selection of E. cloacae). However, we propose to repeat the experiment using an E. cloacae strain carrying a plasmid conferring kanamycin resistance. This will allow us to counter-select E. cloacae after contact with the A. baumannii predator to determine if E. cloacae is killed by A. baumannii in a T6SS-dependent manner.

    __#4) __Figure 2, I suggest to write out the species name of the prey in the box with the ratio. With E. cloacae being referred to in the previous figure and starting with similar letters than E. coli, I wasn't sure at first sight what E. c. refers to.

    __ Authors’ reply #4: __We appreciate the comment and will revise the figure as suggested.

    __#5) __use of the term "T6SS activity" throughout the manuscript (e.g. l. 182, l. 187). I leave this up to the authors. To me, it seems like an umbrella term for the initial observation and I see that such a term can be very handy for the writing. I just would like to mention that the use of the term was not always intuitive to me and sometimes even a bit misleading. For example, l. 182 refers to "increased T6SS activity". As a reader, I only know about 'T6SS activity on other cells' or 'a T6SS-mediated effect on other cells' at this point. T6SS apparatus assembly/firing activity is tested for specifically later and it turns out to differ between mutants. By the time the term is used in the discussion, it captures multiple nuanced phenotypes described by then. The more precise definition of the term in l. 200 helped to capture what exactly is meant by the authors.

    __ Authors’ reply #5: __We propose rephrasing the sentences to include the term "T6SS-secretion activity" when referring to Hcp secretion assays and "T6SS-mediated killing activity" when referring to killing experiments.

    __#6) __l. 198-199 "Collectively, our findings indicate that CPS does not hinder the secretion process of the T6SS or the consequent elimination of competing cells". I might be missing something, I cannot entirely follow this sentence. Didn't the authors just show that the CPS does hinder T6SS-mediated elimination of competing cells in panel 2A and less secreted Hcp in the encapsulated WT compared to the non-encapsulated mutant in panel 2B?

    __ Authors’ reply #6:__ We thank the reviewer for this comment. We realize that the sentence wasn’t well phrased, resulting in confusion. What we meant was that the T6SS is functional regarding its T6SS-mediated killing and secretion in the WT strain, while we also showed that the non-capsulated strain kills and secretes more T6SS material in the supernatant. Thus, there seems to be a balance between capsule production and T6SS activity in the WT. We will revise the sentence to better reflect this meaning.

    #7)* l. 224, typo, "in"*

    __ Authors’ reply #7:__ We will correct this typo. Thank you.

    __#8) __Two connected comments: l. 338, Just a thought, I am wondering about the title of the section. After reading it a second time, I think it is technically correct. When reading it first, I was a bit confused when getting to the data because apparatus assmebly is impaired in the capsule-overproducing strain and although "preserved", doesn't the data indicate that there is less T6SS assembly in the bfmS mutant and that this might be because of less cell sensing and isn't this a main point that there is a difference in apparatus assembly in the capsule overproducing strain compared to WT (other than no difference in apparatus assembly in the strain without capsule)? To me it seems not fully acknowledged as a finding in the interpretation of the data that less cells of the bfmS mutant have a T6SS apparatus. Isn't that interesting? A title along the lines of "Capsule-overproducing strain has preserved sensory function and assembles less T6SS apparatuses" would have been more intuitive for me. l. 352, In case I didn't miss a reference to this data earlier in the manuscript, I am wondering if it would be worth mentioning the finding on the reduced apparatus assembly of the bfmS mutant earlier, together with Figure 3 already. At least a sentence that mentions already that there is more coming later. When I got to this line in the manuscript and read the findings on the apparatus assembly, I first needed to go back to figure 3 and look at the data there again in light of this finding. It is mentioned here on the side but I think very important for the interpretation of the phenotypic data of the bfmS mutant shown earlier, isn't it? The tslA mutant is used beautifully here.

    __ Authors’ reply #8:__ We thank the reviewer for the suggestion and the kind comment about the beautiful usage of the tslA mutant. We will modify the title of the corresponding paragraph as suggested to make it more intuitive.

            Regarding the comment about mentioning the T6SS apparatus assembly defect in the *bfmS* mutant earlier, we respectfully disagree. While we agree that this point is important and can partially explain the difference in killing activity, we believe that showing it together with the *tslA* mutant (Figure 5) makes more sense and is easier for the reader to understand.

    __#9) __Discussion: optional comment. On the one hand, I like the concise discussion. On the other hand, I see more potential here for bringing it all together (potentially at the expense of shortening some of the introduction). I think the subtleties of the findings are complex. For example, I could envision a graphical summary with a working model on all the effects of a capsule on the T6SS and its potential clinical relevance making the study accessible to even more readers.

    __ Authors’ reply #9: __In the revised manuscript, we will include a graphical summary/model.

    Significance

    __#10) __General assessment: I consider the story very strong in terms of novelty, experimental approaches used, quality of the data, quality of the writing and figures of the manuscript. In my view, the aspects that could be improved are optional/minor and concern only one figure and some phrasing.

    •        Advance: I see major advance in the findings (i, mechanistic) on the mechanism of how the capsule interferes with T6SS, (ii, fundamental) on the discovery of ClpXP degrading Hcp, and (iii, clinical) on the meaning of antibiotic treatment for the T6SS of this clinically relevant and often multi-drug resistant bacterial species, which strongly complements existing work on the T6SS and antibiotics in A. baumanii (e.g. of the Feldman group). As the authors write themselves, the starting points of the study of a capsule protecting from a T6SS and the effect of a T6SS on other cells being negatively impacted by a capsule were known, although not studied in one species and not understood mechanistically.*

    •        Audience: I see the result of interest to a broad audience in the fields of bacteria-bacteria interactions, Acinetobacter baumanii, type VI secretion, antimicrobial resistance, bacterial capsules.*

    __ Authors’ reply #10: __We once again thank the reviewer and highly appreciate their positive and constructive feedback on our work. We hope the reviewer will be satisfied with the revised version of our manuscript.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    #11) In the manuscript by Flaugnatti et al., the authors provide clear evidence of the interplay between capsule outer coat production and the Type VI secretion system (T6SS) in Acinetobacter baumannii. The authors demonstrate that the presence of the capsule or the activity of the T6SS enhances survival against attacking bacteria. However, they also show that in their model bacterium, the (over)production of the capsule likely hinders T6SS dynamics, thereby reducing overall killing efficiency. Additionally, they reveal that the amount of the T6SS component Hcp is regulated in cells that can no longer assemble and/or secrete via the T6SS, presumably by the ClpXP protease. Overall, the experiments are well designed, and most conclusions are supported by the data and appropriate controls. I have however some suggestions that could further strengthen the manuscript prior to publication.

    __ Authors’ reply #11: __We are grateful for the reviewer’s enthusiasm and will implement their comments and suggestions in the revised version of the manuscript.

    Major comments:

    __#12) __Line 164. The authors use E. coli as prey to test the T6SS activity of A. baumannii. Why not directly use the E. cloacae strain (with or without T6SS) for this purpose? This would provide direct evidence that A. baumannii uses its T6SS to kill E. cloacae, thus confirming the authors conclusions in this section.

    __ Authors’ reply #12: __We thank the reviewer for this comment. We used E. coli to assess the functionality of the T6SS in different strains of A. baumannii, as it is commonly done in the T6SS field. However, as suggested by reviewer 1 (see comment #3) and in response to this query, we will also provide survival data of E. cloacae in the revised manuscript using a plasmid-carrying E. cloacae derivative that allows direct selection.

    __#13) __In Figure 2, the authors show that a non-capsulated strain kills more effectively and secretes more than a WT, but has a similar number of T6SS. They suggest in their conclusion that "the observed increase in T6SS activity in the non-capsulated strain suggests a compensatory mechanism for the absence of the protective capsule layer." This conclusion implies the presence of an "active" regulatory mechanism that would increase the number of successful T6SS firing events, which has not been demonstrated. Could it not simply be that the capsule blocks some shots that cannot penetrate and are therefore ineffective? This hypothesis is mentioned in lines 204-208. The authors should clarify the conclusion of this section. Given the challenge this may pose in A. baumannii, I suggest that the authors quantify the assembly/firing dynamics of the T6SS under WT and ΔitrA conditions. This would help distinguish between the two hypotheses explaining better firing in non-capsulated cells: i.e., if the number of assembled T6SS is the same in both strains (Fig 2C & 2D), do non-capsulated cells assemble/fire faster, indicating an adaptation in regulation, or do we observe the same dynamics, suggesting a simple physical barrier blocking the passage of certain T6SS firing events?

    __ Authors’ reply #13:__ We realize that the sentence, and more specifically the word "compensatory," might have been misleading and thank the reviewer for bringing this to our attention. What we meant to convey is that there is a balance between capsule production and T6SS activity; if disturbed, the balance shifts in one direction or the other. Specifically, there is more protection through the production of a thicker capsule (e.g., in the ∆bfmSmutant or under sub-MIC conditions of antibiotics, regulated by the Bfm system, as mentioned in the text) or more T6SS activity when less capsule is present (e.g., in the ΔitrA mutant, which we propose is caused by the lack of the steric hindrance). We will rephrase this sentence in the revised manuscript to better convey this message.

            Regarding the quantification of T6SS dynamic assembly/firing events between the capsulated (WT) and non-capsulated (ΔitrA) strains, we do not think this is required for this study, as the amount of secreted Hcp reflects the overall activity of the system. Importantly, we also do not have the technical means to quantify assembly/firing rates under Biosafety 2 conditions, as this requires specialized microscopes with very fast acquisition options (see, for instance, Basler, Pilhofer *et al.*, 2012, *Nature*). Indeed, very few labs in the T6SS field have been able to measure such rates.

    __#14) __Line 428-429. It is mentioned that the deletion of lon does not have a notable effect. However, I observe that the absence of Lon alone causes a more rapid degradation of Hcp in the cells compared to the WT strain (Fig 7B). How do the authors explain that the absence of this protease (whether under conditions of Hcp accumulation or not) increases the degradation of this protein in the cell? This explanation should be included in the manuscript.

    __ Authors’ reply #14: __That’s a fair point. We didn’t address this point further, as the deletion of lon didn’t resolve the issue of why Hcp is degraded. In fact, the opposite seems to be the case, as there is less Hcp in the ∆lon strain compared to the WT. While this observation is not directly relevant to the question of why Hcp is degraded late during growth in secretion-impaired strains, we will properly mention it in the revised manuscript.

            Please also note that a strong growth defect of a Δ*lon*Δ*clpXP* double mutant impaired further investigation in this direction.

    Minor comments:

    #15) Throughout the manuscript, the authors use the term "predator" to refer to A. baumannii. Predation is a specific phenomenon that involves killing for nourishment. To my knowledge, the T6SS has never been shown to be a predation weapon but rather a weapon for interbacterial competition, which is a different concept. If this has not been demonstrated in A. baumannii, the authors should replace the term "predator" with "attacker" (or an equivalent term) to clarify the context.

    __ Authors’ reply #15: __We thank the reviewer for this comment. The term “predator,” as highlighted by the reviewer, typically implies killing for nourishment/cellular products. In the context of T6SS, it facilitates the killing of competitors, releasing DNA into the environment that can subsequently be acquired through natural competence for transformation, as observed in species like Vibrio cholerae (our work by Borgeaud et al., 2015, Science) or other Acinetobacter species such as Acinetobacter baylyi (Ringel et al., 2017, Cell Rep.; Cooper et al., 2017, eLife). The acquisition of DNA reflects the killing for cellular products of the prey. As most A. baumannii strains are also naturally competent, this justifies the usage of the predator and prey nomenclature.

            Apart from this fact, it seems to be a matter of nomenclature, with many papers in the field using one term or the other. Yet, ultimately, this doesn’t change any of the scientific findings. Therefore, to satisfy the reviewer, we will change “predator” to “attacker” throughout the revised manuscript.

    __#16) __Line 274. Since the authors stated that in the Wzc mutant, the capsule is "predominantly found in the supernatant and only loosely attached to the cell," this result is not unexpected. This finding is also consistent with the previous results from Fig. 3A & B, which show sensitivity to complement-mediated killing and the weak amount of (ab)normal CPS produced in that strain, further confirmed by Fig. 3E.

    __ Authors’ reply #16__: We fully agree with the reviewer’s suggestion and will remove the statement.

    __#17) __Line 299. The authors speculate that "... T6SS may deploy through gaps akin to arrow-slits in the capsule's mesh...". However, this is very unlikely since a WT strain kills (Fig. 3C) and secretes (Fig. 2B & 3D) less effectively than the itrA mutant, suggesting that the T6SS is not assembled in the "right places" devoid of CPS; otherwise, we would expect similar T6SS activity. Based on the results in Fig. 2 (and my earlier comment), this implies that A. baumannii assembles its T6SS randomly, and in the presence of the capsule, its shots would need to be in the right place to penetrate the envelope and reach the target. Could the authors comment on this point and provide a model figure to better visualize the interplay between the capsule and T6SS under the three major conditions: WT, non-capsulated, and capsule overproduction?

    __ Authors’ reply #17: __We thank the reviewer and agree with their comment. We discussed the hypothesis of T6SS deployment through gaps, drawing a parallel to what was proposed for biofilm and T6SS in V. cholerae(Toska et al., 2018, PNAS). However, as mentioned earlier, we believe that the effect of the capsule on T6SS activity is primarily due to steric hindrance, which increases the distance between the T6SS apparatus and the prey cell. To clarify our findings further, we will include a model summarizing our results, as requested by reviewer 1 (see comment #9).

    __ #18)__ In Fig. 5A, the microscopy panels should be adjusted to the same dynamic range as the WT (which represents a true signal), which does not appear to be the case for the tlsA mutant panel for instance. The image gives the impression of a large amount of free TssB-msfGFP in the cytoplasm. However, this effect is due to the dynamic range being adjusted to display a signal. This observation is consistent with the fact that the amount of TssB-msfGFP protein is identical across all strains (Fig. S2F).

    __ Authors’ reply #18: __We will adjust the images to the range of the WT in the revised manuscript, as suggested. However, regardless of how these images are presented, the enumeration of T6SS structures will remain unchanged, which was the sole point of this experiment.

    __#19) __Unless I am mistaken, the authors do not comment on the fact that in a ΔbfmS strain, the number of T6SS is halved compared to a WT or ΔitrA strain. If capsule overproduction only partially limits the TslA-dependant T6SS assembly, how can this result be explained? Is it related to the degradation of Hcp in this background, which ultimately limits the formation of T6SS? If so, it would be interesting to mention this connection in the section "Prolonged secretion inhibition triggers Hcp degradation”

    __ Authors’ reply #19: __We did mention that the T6SS assembly of the ΔbfmS mutant is reduced compared to the WT (or ΔitrA), likely due to the defect in sensing the prey (lines 369-374 and 468-472 of the initial manuscript). However, we will revise the sentence to improve clarity in the revised version of the manuscript.

    * *

    Significance

    __#20) __This work is highly intriguing as it not only delves into the specific mechanisms involved but also connects fundamental elements in bacterial competition, i.e., the necessity for self-protection and aggression for survival. The manuscript offers valuable insights into cellular dynamics at a microscale level and prompts new inquiries into the regulation of these systems on a population scale. The work is well-done and the writing is also clear. I am convinced that this work represents another significant step towards understanding bacterial mechanisms and will undoubtedly spark considerable interest in the field.

    __ Authors’ reply #20: __We sincerely thank reviewer #2 for their constructive inputs, which will improve our manuscript.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    __#21) __The manuscript by Flaugnatti et al investigates the relationship between functions of the T6SS in A. baumannii and production of capsular polysaccharide. The manuscript argues that (1) capsule protects A. baumannii against T6SS-mediated attack by other bacteria, (2) capsule also interferes with the bacterium's own T6SS activity, and (3) the T6SS inner tube protein Hcp is regulated by degradation by ClpXP. The main critiques regard the first two conclusions, which seem to be based solely on use of a mutant that has a confounding effect as described below; and to strengthen the third claim by further exploring the results of overexpressing Hcp and by determining whether there is a fitness benefit for Hcp regulation.

    __ Authors’ reply #21: __We thank reviewer #3 for their relevant input. We will conduct additional experiments based on their comments, and these will be incorporated into the revised manuscript.

    __Main items:____ __

    __#22) __Throughout the paper, an itrA deletion mutant is used as the capsule-deficient strain and conclusions are drawn about role of capsule based on this mutant. However, itrA deletion also eliminates the protein O-glycosylation pathway (Lees-miller et al 2013), a potential confounder. Analysis of mutants specifically deficient in the high-molecular weight capsule but not protein glycosylation, and/or mutants in the protein o-glycosylation enzyme, should be incorporated into the study to enhance the ability to make conclusions about the role of the capsule.

    __ Authors’ reply #22: __Fair point. We thank the reviewer for this important suggestion. To distinguish between the O-glycosylation pathway and capsule production, we will generate a ∆pglL strain (specific to O-glycosylation), as suggested, and will repeat the key experiments (similar to Fig. 2A and 2B). We are almost done with the engineering of this mutant strain and therefore don’t expect any major delays.

    __#23) __Evidence could be provided to support the idea raised in lines 482-483 that T6SS component accumulation is toxic ("degradation [of T6SS components] could serve as a strategy to alleviate proteotoxic stress..."). For example, growth curves of ∆clpXP strains with and without hcp could be analyzed, to determine how degrading Hcp is helping the bacteria.

    __ Authors’ reply #23: __We will perform growth curves of ΔclpXP strains with and without hcp, as suggested by the reviewer. However, we are uncertain whether we will be able to observe differences between these strains, as the conditions under which such degradation is significant may be challenging to replicate under standard laboratory conditions.

    __#24) __The possible ClpXP recognition sequence identified at the C terminus of Hcp is interesting-does overexpression of an Hcp variant lacking/altered in this motif alter its protein levels compared to WT Hcp?

    __ Authors’ reply #24: __We thank the reviewer for this suggestion. We are in the process of performing the suggested experiment and will include the data in the manuscript.

    __Minor items:____ __

    __#25) __*A better explanation could be provided for why overexpressing hcp in WT but not in ∆hcp leads to increased Hcp protein levels. There is a statement about Hcp being regulated post transcriptionally, possibly by degradation (lines 422-423), but would that not also result in regulation in the WT strain? *

    __ Authors’ reply #25: __The reviewer is absolutely correct here. Despite careful genetic engineering, we believe that the hcp mutant used may have a polar effect, causing Hcp accumulation only in the ∆hcp + p-hcp strain but not in the WT + p-hcp strain, which remains capable of secretion. The ∆hcp strain therefore mimics the secretion-impaired tssB mutant. We will clarify this in the revised manuscript.

    __#26) __*An untreated control is needed in Fig. 4B. *

    __ Authors’ reply #26: __The untreated samples were shown in all previous figures. However, we understand the reviewer's point and will repeat the experiment with the untreated control included in the same experiment.

    #27) *line 179: please clarify "reflecting better invading bacteria" *

    __ Authors’ reply #27: __We appreciate the reviewer mentioning this oversight. We meant to compare this to a situation where a bacterium invades an already existing community, resulting in a predator-prey ratio below 1. We will clarify this further in the revised manuscript.

    #28) *line 351: consider rewording the statement that ∆tslA results in decreased in T6SS assembly and activity using the tssB-msfGFP microscopy assay; it is not clear that activity is measured in this assay. *

    __ Authors’ reply #28: __The reviewer is correct. We will revise the sentence accordingly to better reflect the T6SS assembly.

    __#29) __*lines 260-265: This experiment could use clarifying, but it would seem that it requires analysis of the secreted capsule levels in the tssB mutant to show it does not produce extracellular capsule to the same extent that ∆bfmS does. *

    __ Authors’ reply #29: __We thank the reviewer for the suggestion and will include these experimental data in the revised manuscript.

    __#30) __*Fig. 6C and 7A labelling could be improved to avoid potential confusion that the bar graphs are quantifying the western blot. E.g., could add a corresponding vertical label to the Western data, or consider changing "relative expression of hcp" to something reflecting analysis of transcript levels. *

    __ Authors’ reply #30: __We will improve this figure by splitting the qPCR and Western blot data into independent panels. This will eliminate any confusion.

    __#31) __lines 416-417 and Fig. 7A: states that "hcp mRNA levels increased significantly", but more careful wording could be used because the WT's transcript change is not significant after overexpression (though it is significant in ∆hcp).

    __ Authors’ reply #31: __Point well taken. We will improve the sentence (and Figure) to make its meaning unambiguous.

    __#32) __lines 479-480 states that in secretion-impaired strains accumulation of Hcp is mitigated by ClpXP; while this was shown for ∆tssB, was this also the case for ∆bfmS?

    __ Authors’ reply #32: __This is indeed an interesting suggestion. We are in the process of generating the double mutant ∆bfmS∆clpXP and will include the experimental results in the revised manuscript.

    Significance

    __#33) __*The strengths of the study are the focus on a clinically significant pathogen, the potential novel roles for the important capsule virulence factor of A. baumannii, and the identification of novel points of control of the T6SS. The analyses of T6SS function are thorough and carefully performed. *

    __ Authors’ reply #33: __We thank the reviewer for their comments, which we believe will significantly strengthen our work, particularly regarding the capsule aspect.

  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Flaugnatti et al investigates the relationship between functions of the T6SS in A. baumannii and production of capsular polysaccharide. The manuscript argues that (1) capsule protects A. baumannii against T6SS-mediated attack by other bacteria, (2) capsule also interferes with the bacterium's own T6SS activity, and (3) the T6SS inner tube protein Hcp is regulated by degradation by ClpXP. The main critiques regard the first two conclusions, which seem to be based solely on use of a mutant that has a confounding effect as described below; and to strengthen the third claim by further exploring the results of overexpressing Hcp and by determining whether there is a fitness benefit for Hcp regulation.

    Main items:

    • Throughout the paper, an itrA deletion mutant is used as the capsule-deficient strain and conclusions are drawn about role of capsule based on this mutant. However, itrA deletion also eliminates the protein O-glycosylation pathway (Lees-miller et al 2013), a potential confounder. Analysis of mutants specifically deficient in the high-molecular weight capsule but not protein glycosylation, and/or mutants in the protein o-glycosylation enzyme, should be incorporated into the study to enhance the ability to make conclusions about the role of the capsule.
    • Evidence could be provided to support the idea raised in lines 482-483 that T6SS component accumulation is toxic ("degradation [of T6SS components] could serve as a strategy to alleviate proteotoxic stress..."). For example, growth curves of ∆clpXP strains with and without hcp could be analyzed, to determine how degrading Hcp is helping the bacteria.
    • The possible ClpXP recognition sequence identified at the C terminus of Hcp is interesting--does overexpression of an Hcp variant lacking/altered in this motif alter its protein levels compared to WT Hcp?

    Minor items:

    • A better explanation could be provided for why overexpressing hcp in WT but not in ∆hcp leads to increased Hcp protein levels. There is a statement about Hcp being regulated post transcriptionally, possibly by degradation (lines 422-423), but would that not also result in regulation in the WT strain?
    • An untreated control is needed in Fig. 4B.
    • line 179: please clarify "reflecting better invading bacteria"
    • line 351: consider rewording the statement that ∆tslA results in decreased in T6SS assembly and activity using the tssB-msfGFP microscopy assay; it is not clear that activity is measured in this assay.
    • lines 260-265: This experiment could use clarifying, but it would seem that it requires analysis of the secreted capsule levels in the tssB mutant to show it does not produce extracellular capsule to the same extent that ∆bfmS does.
    • Fig. 6C and 7A labelling could be improved to avoid potential confusion that the bar graphs are quantifying the western blot. E.g., could add a corresponding vertical label to the Western data, or consider changing "relative expression of hcp" to something reflecting analysis of transcript levels.
    • lines 416-417 and Fig. 7A: states that "hcp mRNA levels increased significantly", but more careful wording could be used because the WT's transcript change is not significant after overexpression (though it is significant in ∆hcp)
    • lines 479-480 states that in secretion-impaired strains accumulation of Hcp is mitigated by ClpXP; while this was shown for ∆tssB, was this also the case for ∆bfmS?

    Significance

    The strengths of the study are the focus on a clinically significant pathogen, the potential novel roles for the important capsule virulence factor of A. baumannii, and the identification of novel points of control of the T6SS. The analyses of T6SS function are thorough and carefully performed.

  4. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript by Flaugnatti et al., the authors provide clear evidence of the interplay between capsule outer coat production and the Type VI secretion system (T6SS) in Acinetobacter baumannii. The authors demonstrate that the presence of the capsule or the activity of the T6SS enhances survival against attacking bacteria. However, they also show that in their model bacterium, the (over)production of the capsule likely hinders T6SS dynamics, thereby reducing overall killing efficiency. Additionally, they reveal that the amount of the T6SS component Hcp is regulated in cells that can no longer assemble and/or secrete via the T6SS, presumably by the ClpXP protease. Overall, the experiments are well designed, and most conclusions are supported by the data and appropriate controls. I have however some suggestions that could further strengthen the manuscript prior to publication.

    Major comments:

    Line 164. The authors use E. coli as prey to test the T6SS activity of A. baumannii. Why not directly use the E. cloacae strain (with or without T6SS) for this purpose? This would provide direct evidence that A. baumannii uses its T6SS to kill E. cloacae, thus confirming the authors conclusions in this section.. In Figure 2, the authors show that a non-capsulated strain kills more effectively and secretes more than a WT, but has a similar number of T6SS. They suggest in their conclusion that "the observed increase in T6SS activity in the non-capsulated strain suggests a compensatory mechanism for the absence of the protective capsule layer." This conclusion implies the presence of an "active" regulatory mechanism that would increase the number of successful T6SS firing events, which has not been demonstrated. Could it not simply be that the capsule blocks some shots that cannot penetrate and are therefore ineffective? This hypothesis is mentioned in lines 204-208. The authors should clarify the conclusion of this section. Given the challenge this may pose in A. baumannii, I suggest that the authors quantify the assembly/firing dynamics of the T6SS under WT and ΔitrA conditions. This would help distinguish between the two hypotheses explaining better firing in non-capsulated cells: i.e., if the number of assembled T6SS is the same in both strains (Fig 2C & 2D), do non-capsulated cells assemble/fire faster, indicating an adaptation in regulation, or do we observe the same dynamics, suggesting a simple physical barrier blocking the passage of certain T6SS firing events? Line 428-429. It is mentioned that the deletion of lon does not have a notable effect. However, I observe that the absence of Lon alone causes a more rapid degradation of Hcp in the cells compared to the WT strain (Fig 7B). How do the authors explain that the absence of this protease (whether under conditions of Hcp accumulation or not) increases the degradation of this protein in the cell? This explanation should be included in the manuscript.

    Minor comments:

    • a) Throughout the manuscript, the authors use the term "predator" to refer to A. baumannii. Predation is a specific phenomenon that involves killing for nourishment. To my knowledge, the T6SS has never been shown to be a predation weapon but rather a weapon for interbacterial competition, which is a different concept. If this has not been demonstrated in A. baumannii, the authors should replace the term "predator" with "attacker" (or an equivalent term) to clarify the context.
    • b) Line 274. Since the authors stated that in the Wzc mutant, the capsule is "predominantly found in the supernatant and only loosely attached to the cell," this result is not unexpected. This finding is also consistent with the previous results from Fig. 3A & B, which show sensitivity to complement-mediated killing and the weak amount of (ab)normal CPS produced in that strain, further confirmed by Fig. 3E.
    • c) Line 299. The authors speculate that "... T6SS may deploy through gaps akin to arrow-slits in the capsule's mesh...". However, this is very unlikely since a WT strain kills (Fig. 3C) and secretes (Fig. 2B & 3D) less effectively than the itrA mutant, suggesting that the T6SS is not assembled in the "right places" devoid of CPS; otherwise, we would expect similar T6SS activity. Based on the results in Fig. 2 (and my earlier comment), this implies that A. baumannii assembles its T6SS randomly, and in the presence of the capsule, its shots would need to be in the right place to penetrate the envelope and reach the target. Could the authors comment on this point and provide a model figure to better visualize the interplay between the capsule and T6SS under the three major conditions: WT, non-capsulated, and capsule overproduction?
    • d) In Fig. 5A, the microscopy panels should be adjusted to the same dynamic range as the WT (which represents a true signal), which does not appear to be the case for the tlsA mutant panel for instance. The image gives the impression of a large amount of free TssB-msfGFP in the cytoplasm. However, this effect is due to the dynamic range being adjusted to display a signal. This observation is consistent with the fact that the amount of TssB-msfGFP protein is identical across all strains (Fig. S2F).
    • e) Unless I am mistaken, the authors do not comment on the fact that in a ΔbfmS strain, the number of T6SS is halved compared to a WT or ΔitrA strain. If capsule overproduction only partially limits the TslA-dependant T6SS assembly, how can this result be explained? Is it related to the degradation of Hcp in this background, which ultimately limits the formation of T6SS? If so, it would be interesting to mention this connection in the section "Prolonged secretion inhibition triggers Hcp degradation."

    Referee Cross-Commenting

    Overall, I agree with the concerns raised by reviewers 1 and 3. This (already) very good manuscript will undoubtedly benefit from these comments.

    Significance

    This work is highly intriguing as it not only delves into the specific mechanisms involved but also connects fundamental elements in bacterial competition, i.e., the necessity for self-protection and aggression for survival. The manuscript offers valuable insights into cellular dynamics at a microscale level and prompts new inquiries into the regulation of these systems on a population scale. The work is well-done and the writing is also clear.

    I am convinced that this work represents another significant step towards understanding bacterial mechanisms and will undoubtedly spark considerable interest in the field.

    Expertise: T6SS, fluorescence microscopy, predation, interbacterial competition

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The transport of effector proteins across membranes from the producing bacterium into a target cell is at the core of bacterial secretion systems. How an additional layer in form of a capsule affects effector export and the susceptibility towards effector import is not fully understood. Here, Flaugnatti and colleagues combined bacterial genetics with phenotypic assays and electron microscopy to demonstrate a dual role of a bacterial capsule in preventing T6SS-mediated effector export and promoting protection from effector import by another bacterium's T6SS. The wide variety of methods used, complementation of the mutants, and validation of the findings across strains strengthen the author's conclusions.

    Although the main conclusions seem straight forward, the authors unravel the unexpected complexity underlying these phenotypes with strong mechanistic work. In brief, a capsule-deficient mutant (∆itra) is shown to assemble its T6SS similar to the WT, yet secretes more Hcp than the WT and is better in T6SS-mediated killing of other bacteria. A capsule-overproducing mutant (∆bfmS) shows both, a partial deficiency in T6SS assembly and an additional reduction in exported Hcp, and is worse in T6SS-mediated killing than the WT. A mutant with a capsule similar to WT and deficient in cell sensing (∆tslA) forms the least T6SS apparatuses and is yet better in T6SS-mediated killing than the overcapsulated mutant. Together, these data show an effect of the capsule on (i) T6SS apparatus assembly, (ii) effector export, (iii) effector import, and (iv) the need for clearance of accumulating non-secreted Hcp by ClpXP.

    The work on a clinical isolate of Acinetobacter tumefaciens and the data on an impaired T6SS activity on other cells by antibiotic-induced capsulation is a strong demonstration of the work's clinical relevance in addition to the findings' conceptual novelty.

    In my view, the manuscript is outstanding with very high quality of experimental data, very well written text and very clear presentation of the data in figures. A few minor comments and suggestions below that I think would strengthen the manuscript.

    Major comment:

    OPTIONAL: Fig. 4c/l. 320: Having an indirect effect of an antibiotic on T6SS activity by antibiotic-induced capsule formation is very intriguing and contributes to the clinical relevance of the overall findings. When I saw the data in Fig. 4c, the graph instantaneously reminded me of the panel in Fig. 2a, where a similar phenotype is observed by changing the predator:prey ratio in the absence of any antibiotic. The authors themselves comment on the possibility of antibiotic-induced, reduced predator growth (and thereby a change in predator:prey ratio) as a one factor impacting the phenotype here. I am wondering if this data could be strengthened or better disentangled to test more precisely if it is the antibiotic induced capsule formation per se that affects T6SS-mediated killing by A. baumanii in the presence of antibiotics. Would it help to take the bfmS mutant along as a control for direct comparison to see if antibiotic-induced capsule formation of the WT to similar levels of the mutant results in the same killing phenotype? Would it help to test for T6SS-mediated killing in the presence and absence of antibiotics at multiple predator:prey ratios? Could the effect of the antibiotic on A. baumanii growth be measured and considered when choosing the ratio at which the bacteria are mixed?

    Minor comments:

    • Figure 1D, l. 155ff, I might have missed this, do the authors happen to have the numbers of E. cloacae as well? This would strengthen the claim on A. baumanii survival because of E. cloacae is being killed.
    • Figure 2, I suggest to write out the species name of the prey in the box with the ratio. With E. cloacae being referred to in the previous figure and starting with similar letters than E. coli, I wasn't sure at first sight what E. c. refers to.
    • use of the term "T6SS activity" throughout the manuscript (e.g. l. 182, l. 187). I leave this up to the authors. To me, it seems like an umbrella term for the initial observation and I see that such a term can be very handy for the writing. I just would like to mention that the use of the term was not always intuitive to me and sometimes even a bit misleading. For example, l. 182 refers to "increased T6SS activity". As a reader, I only know about 'T6SS activity on other cells' or 'a T6SS-mediated effect on other cells' at this point. T6SS apparatus assembly/firing activity is tested for specifically later and it turns out to differ between mutants. By the time the term is used in the discussion, it captures multiple nuanced phenotypes described by then. The more precise definition of the term in l. 200 helped to capture what exactly is meant by the authors.
    • l. 198f "Collectively, our findings indicate that CPS does not hinder the secretion process of 199 the T6SS or the consequent elimination of competing cells". I might be missing something, I cannot entirely follow this sentence. Didn't the authors just show that the CPS does hinder T6SS-mediated elimination of competing cells in panel 2A and less secreted Hcp in the encapsulated WT compared to the non-encapsulated mutant in panel 2B?
    • l. 224, typo, "in"
    • Two connected comments: l. 338, Just a thought, I am wondering about the title of the section. After reading it a second time, I think it is technically correct. When reading it first, I was a bit confused when getting to the data because apparatus assmebly is impaired in the capsule-overproducing strain and although "preserved", doesn't the data indicate that there is less T6SS assembly in the bfmS mutant and that this might be because of less cell sensing and isn't this a main point that there is a difference in apparatus assembly in the capsule overproducing strain compared to WT (other than no difference in apparatus assembly in the strain without capsule)? To me it seems not fully acknowledged as a finding in the interpretation of the data that less cells of the bfmS mutant have a T6SS apparatus. Isn't that interesting? A title along the lines of "Capsule-overproducing strain has preserved sensory function and assembles less T6SS apparatuses" would have been more intuitive for me. l. 352, In case I didn't miss a reference to this data earlier in the manuscript, I am wondering if it would be worth mentioning the finding on the reduced apparatus assembly of the bfmS mutant earlier, together with Figure 3 already. At least a sentence that mentions already that there is more coming later. When I got to this line in the manuscript and read the findings on the apparatus assembly, I first needed to go back to figure 3 and look at the data there again in light of this finding. It is mentioned here on the side but I think very important for the interpretation of the phenotypic data of the bfmS mutant shown earlier, isn't it? The tslA mutant is used beautifully here.
    • Discussion: optional comment. On the one hand, I like the concise discussion. On the other hand, I see more potential here for bringing it all together (potentially at the expense of shortening some of the introduction). I think the subtleties of the findings are complex. For example, I could envision a graphical summary with a working model on all the effects of a capsule on the T6SS and its potential clinical relevance making the study accessible to even more readers.

    Significance

    General assessment

    I consider the story very strong in terms of novelty, experimental approaches used, quality of the data, quality of the writing and figures of the manuscript. In my view, the aspects that could be improved are optional/minor and concern only one figure and some phrasing.

    Advance

    I see major advance in the findings (i, mechanistic) on the mechanism of how the capsule interferes with T6SS, (ii, fundamental) on the discovery of ClpXP degrading Hcp, and (iii, clinical) on the meaning of antibiotic treatment for the T6SS of this clinically relevant and often multi-drug resistant bacterial species, which strongly complements existing work on the T6SS and antibiotics in A. baumanii (e.g. of the Feldman group). As the authors write themselves, the starting points of the study of a capsule protecting from a T6SS and the effect of a T6SS on other cells being negatively impacted by a capsule were known, although not studied in one species and not understood mechanistically.

    Audience

    I see the result of interest to a broad audience in the fields of bacteria-bacteria interactions, Acinetobacter baumanii, type VI secretion, antimicrobial resistance, bacterial capsules

  6. Version published to 10.1101/2024.04.23.590769v1 on bioRxiv