Targeted disruption of phage liquid crystalline droplets abolishes antibiotic tolerance of bacterial biofilms

  1. Abul K. Tarafder
  2. Miles Graham
  3. Luke K. Davis
  4. Shawna Pratt
  5. Jan Böhning
  6. Pavithra Manivannan
  7. Zhexin Wang
  8. Camila M. Clemente
  9. Raymond J Owens
  10. George A. O’Toole
  11. Philip Pearce
  12. Tanmay A.M. Bharat

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Abstract

All bacterial biofilms contain an extracellular matrix rich in filamentous molecules 1 that self-associate 2–4 , conferring emergent properties to bacteria 5 , including antibiotic tolerance 6 . Pseudomonas aeruginosa is a human pathogen that forms biofilms in diverse infectious settings 7,8 , where the upregulation of a filamentous bacteriophage Pf4, has been shown to be a key virulence factor that protects bacteria from antibiotics 9,10 . Here, we modelled biophysical characteristics of biofilm-linked liquid crystalline droplets formed by Pf4, which predicted that sub-stoichiometric phage binders had the ability to disrupt liquid crystals by changing the surface properties of the phage. We tested this prediction by developing nanobodies targeting the outer surface of the Pf4 phage, which disrupted in vitro reconstituted droplets, promoted antibiotic diffusion into bacteria, disrupted P. aeruginosa biofilm formation under a variety of conditions, and abolished antibiotic tolerance of biofilms. The inhibition strategy illustrated in this study could be extended to biofilms of other pathogenic bacteria, where filamentous molecules are pervasive in the extracellular matrix. Furthermore, our findings exemplify how targeting a biophysical mechanism, rather than a defined biochemical target, is a promising avenue for intervention, with the potential of applying this concept to other disease-related contexts.

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  1. Review Commons

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    Reply to the reviewers

    We have submitted a revision plan to Review Commons to address the criticisms of the reviewers. We will post the revised manuscript after completing the experiments.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    In the well-written manuscript by Tarafder et al., the authors follow up on their previous investigations of the filamentous bacteriophage Pf4, which self-assembles into a crystalline droplet surrounding Pseudomonas aeruginosa cells within a biofilm. Using theoretical coarse-grained molecular dynamics (MD) simulations, they predict that binding a small molecule or protein to the surface of bacteriophage Pf4 should disrupt the attraction-in this case depletion attraction-between individual phage particles. To test this hypothesis, nanobodies were raised against Pf4, and two promising candidates, Nb43 and Nb-D11, were identified. These nanobodies were characterized using biochemical assays, and binding of Nb43 to CoaB, the major coat protein, was visualized using cryo-EM. Using fluorescence microscopy and cryo-ET, the authors convincingly demonstrate that nanobodies can disrupt Pf4 crystalline droplet formation. Strikingly, nanobody-mediated disruption of Pf4 droplets also increases antibiotic susceptibility of P. aeruginosa both in vitro and in biofilm settings.

    Major comments

    1. Theoretical modelling: The MD simulations, as currently presented, do not add conceptual depth to the study. The idea that blocking an interaction site between phages (whether through active-site interference, obstruction of a protein-protein interface, or simple steric hindrance) would prevent alignment is straightforward and does not necessarily require MD simulations to justify. As such, this section feels superfluous and is currently the weakest point in an otherwise strong manuscript. Unless the simulations can meaningfully address at least some of the questions listed below, the authors should consider removing this part:

    The MD simulation is very simplistic, and filamentous phages are clearly not hard rods, as seen in the cryo-EM images. Would a certain degree of Pf4 flexibility allow to stabilize droplets even in the presence of low concentrations of Pf4 binders?

    How do the MD simulations explain that already pre-formed crystalline droplets can be penetrated and disassembled by small Pf4 binders?

    The authors state that Pf4 binders must be large relative to the depletant particles. Can this be demonstrated experimentally? Is there a sweet spot, as large molecules potentially cannot penetrate preformed droplets?

    1. Nanobody penetration into crystalline droplets (Extended Data Fig. 6a-d) vs. antibiotic penetration (Fig. 4) The authors show that Nb43 penetrates Pf4 droplets even at concentrations that do not disrupt droplet stability. How do the authors explain that a relatively large nanobody penetrates the crystalline droplet, whereas a much smaller antibiotic does not diffuse trough the droplet?

    In the experiments shown in Figure 4, the authors assess antibiotic activity against P. aeruginosa in the presence of Pf4 crystalline droplets. If I understand correctly, the additionally added Pf4 droplets do not physically encompass the bacteria, yet they still reduce antibiotic tolerance. If so, this appears to contradict the conclusion that Pf4 droplets act primarily as a diffusion barrier (as stated in the section title). Instead, this would suggest that Pf4 may reduce antibiotic potency through another mechanism (e.g., direct binding or sequestration). Would it be possible to test the addition of Pf4 alone, without the biopolymer alginate, to determine whether Pf4 itself is sufficient to increase antibiotic tolerance?

    Minor comments:

    • Title: The title is overstated. Please consider changing it to something similar to: "Targeted disruption of phage liquid crystalline droplets abolishes antibiotic tolerance in Pseudomonas aeruginosa biofilms."
    • Introduction sentence: "...where filamentous phage particles align along their axis in the presence of biopolymer,..." Please introduce what biopolymers are and specify which types are relevant here.
    • Amorphous Pf4 aggregates after Nb43 treatment (Fig 3b,e): The authors should discuss the nature of these aggregates. It appears that smaller spindles are both broken up and impeded in their formation after Nb43 treatment, whereas larger aggregates seem to persist.
    • Fig. 3c and 3f: Please describe how liquid crystalline structures were defined in the fluorescence images. Were thresholds for size, intensity, or morphology applied?
    • Use of P. aeruginosa ΔPAO728: For clarity, please explain why the strain lacking the Pf4 integrase is included in the in-vitro assays.

    Discussion:

    Neisseria meningitidis and Vibrio cholerae use filamentous phages to increase virulence. Do these phages also form liquid crystalline droplets? If not, how do the authors envision that the nanobody strategy described here could be applied to prevent infection? In general, the findings are hard to generalize to other biofilms matrices, which are highly heterogenous.

    Significance

    Bacterial biofilms and their associated antibiotic tolerance represent a major clinical burden, and new strategies to overcome these defenses are urgently needed. The strategy presented here-targeting and disrupting the protective extracellular matrix formed by liquid crystalline Pf4 phage droplets-is an exciting and innovative approach with clear translational potential for combating P. aeruginosa biofilms. The study is experimentally rigorous, well written, and carefully analyzed, and it represents a logical and impactful next step following the group's previous work. This manuscript will have significant impact on the field of P. aeruginosa biofilm research by providing a mechanistically grounded method to disrupt the protective biofilm architecture. However, it is important to note that the extracellular matrix architecture of biofilms formed by other bacterial species differs substantially, and thus the current findings cannot be directly generalized beyond P. aeruginosa without further investigation.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Tarafder et al. describes an interdisciplinary approach, combining biophysical modeling and microbiology, to target antibiotic tolerance in P. aeruginosa biofilms. A key conceptual contribution is the strategy of inhibiting a biophysical mechanism instead of a biochemical interaction. The study is logically organized, advancing from a theoretical model to the design of effective nanobody inhibitors, which are then validated across a series of experimental systems, from in vitro assays to complex static and flow-cell biofilms. The data robustly support the authors' conclusions, suggesting a potentially valuable approach for managing biofilm-based infection. Overall, this is a very interesting and robust study. The conclusions are well-supported by the evidence provided, and the manuscript is well-written, with figures that effectively illustrate the key results.

    Major comments:

    1. The fundamental characteristics of Nb43 and Nb-D11 (e.g., affinity, stability) should be provided. To solidify the central claim, the direct interaction between CoaB and Nb43 should be confirmed using an orthogonal biochemical method. urthermore, it is important to test whether Nb43 binds to the CoaB proteins from Pf1/Pf5/Pf6 to assess its specificity and broad application in other PA hosts such as MPAO1 and PA14
    2. In the static biofilm assay (Fig. 5a-b), the use of crystal violet staining only reports total biomass. To clarify the mechanism of action, experiments should distinguish whether Nb43 primarily prevents biofilm attachment/formation or actively eradicates an established biofilm. This is particularly relevant for the pre-incubation condition.
    3. The discussion should address the limitations of this therapeutic approach. A key concern is the potential for Pf4 reinfection and subsequent relapse of chronic infection, which is a major challenge in the field. Additionally, the manuscript would be strengthened by a more critical and direct comparison of this Nb-based strategy against existing anti-virulence or anti-biofilm alternatives, highlighting its potential advantages and drawbacks.

    Minor comments

    1. The prevention of Pf activation in P. aeruginosa biofilms is an important aspect that should be addressed in the Introduction and Discussion.
    2. In the Methods section for the biophysical model, the choice of specific parameters (e.g., phage length a=80 nm, depletant diameter σ=2.4 nm) is justified by referencing the system being modeled. However, a brief sentence explicitly stating that these values were chosen based on the known dimensions of Pf4 and alginate would be helpful for readers that are not familiar with the system.

    Significance

    This study provides a mechanistic insight into the advance and offers a complementary approach to treating biofilm-related infections, which remains an unexplored area in the field. The reported findings are likely to be of interest and significance to microbiologists and clinicians concerned with biofilm infections.

    My own expertise lies in the genetic and biochemical aspects of prophage induction and biofilm formation. Therefore, the details of nanobodies and their potential side effects fall outside the scope of my evaluation.

  4. Version published to 10.1101/2025.07.28.667140 on bioRxiv