Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Bacteriophage protein Dap1 regulates evasion of antiphage immunity and Pseudomonas aeruginosa virulence impacting phage therapy in mice

Abstract

Bacteriophages have evolved diverse strategies to overcome host defence mechanisms and to redirect host metabolism to ensure successful propagation. Here we identify a phage protein named Dap1 from Pseudomonas aeruginosa phage PaoP5 that both modulates bacterial host behaviour and contributes to phage fitness. We show that expression of Dap1 in P. aeruginosa reduces bacterial motility and promotes biofilm formation through interference with DipA, a c-di-GMP phosphodiesterase, which causes an increase in c-di-GMP levels that trigger phenotypic changes. Results also show that deletion of dap1 in PaoP5 significantly reduces genome packaging. In this case, Dap1 directly binds to phage HNH endonuclease, prohibiting host Lon-mediated HNH degradation and promoting phage genome packaging. Moreover, PaoP5Δdap1 fails to rescue P. aeruginosa-infected mice, implying the significance of dap1 in phage therapy. Overall, these results highlight remarkable dual functionality in a phage protein, enabling the modulation of host behaviours and ensuring phage fitness.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Phage protein Dap1 regulates P. aeruginosa virulence by interacting with c-di-GMP phosphodiesterase DipA.
Fig. 2: PaoP5Δdap1 forms small plaques and produces fewer progenies.
Fig. 3: PaoP5Δdap1 is less efficient in genome packaging due to the decreased abundance of HNH endonuclease.
Fig. 4: Lon protease decreases the abundance of HNH endonuclease and inhibits phage genome packaging of PaoP5Δdap1.
Fig. 5: Dap1 binds to HNH endonuclease to prevent Lon-mediated HNH degradation.
Fig. 6: Dap1 is essential for the fitness of phage and the efficacy of phage therapy.

Similar content being viewed by others

Data availability

The analysed data and raw RNA-seq readings of PAO1 expressing dap1 were uploaded to the NCBI GEO (PRJNA1020646). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX68 partner repository with the dataset identifier PXD046148.

The databases used in this study include P. aeruginosa PAO1 genome (NC_002516.2), P. aeruginosa PA14 genome (NC_008463.1), P. aeruginosa phage PaoP5 genome (NC_029083.1), P. aeruginosa phage PaP_se genome (OL441337.1), P. aeruginosa phage JG004 (NC_019450.1) and P. aeruginosa phage PaP8 genome(OL754588.1). Source data are provided with this paper.

References

  1. Smith, W. P. J., Wucher, B. R., Nadell, C. D. & Foster, K. R. Bacterial defences: mechanisms, evolution and antimicrobial resistance. Nat. Rev. Microbiol. 21, 519–534 (2023).

    Article  CAS  PubMed  Google Scholar 

  2. Bernheim, A. & Sorek, R. The pan-immune system of bacteria: antiviral defence as a community resource. Nat. Rev. Microbiol. 18, 113–119 (2020).

    Article  CAS  PubMed  Google Scholar 

  3. Hsueh, B. Y. et al. Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria. Nat. Microbiol. 7, 1210–1220 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Tock, M. R. & Dryden, D. T. The biology of restriction and anti-restriction. Curr. Opin. Microbiol. 8, 466–472 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Samson, J. E., Magadán, A. H., Sabri, M. & Moineau, S. Revenge of the phages: defeating bacterial defences. Nat. Rev. Microbiol. 11, 675–687 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Huiting, E. et al. Bacteriophages inhibit and evade cGAS-like immune function in bacteria. Cell 186, 864–876.e21 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Davidson, A. R. et al. Anti-CRISPRs: protein inhibitors of CRISPR-Cas systems. Annu. Rev. Biochem. 89, 309–332 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rifat, D., Wright, N. T., Varney, K. M., Weber, D. J. & Black, L. W. Restriction endonuclease inhibitor IPI* of bacteriophage T4: a novel structure for a dedicated target. J. Mol. Biol. 375, 720–734 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Tesson, F. et al. Systematic and quantitative view of the antiviral arsenal of prokaryotes. Nat. Commun. 13, 2561 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Georjon, H. & Bernheim, A. The highly diverse antiphage defence systems of bacteria. Nat. Rev. Microbiol. 21, 686–700 (2023).

    Article  CAS  PubMed  Google Scholar 

  11. Boyle, T. A. & Hatoum-Aslan, A. Recurring and emerging themes in prokaryotic innate immunity. Curr. Opin. Microbiol. 73, 102324 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Goldfarb, T. et al. BREX is a novel phage resistance system widespread in microbial genomes. EMBO J. 34, 169–183 (2015).

    Article  CAS  PubMed  Google Scholar 

  13. Shen, B. W. et al. Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor. Nucleic Acids Res. 51, 3513–3528 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Liu, X. et al. Target RNA activates the protease activity of Craspase to confer antiviral defense. Mol. Cell 82, 4503–4518.e8 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Rouillon, C. et al. Antiviral signalling by a cyclic nucleotide activated CRISPR protease. Nature 614, 168–174 (2023).

    Article  CAS  PubMed  Google Scholar 

  16. Johnson, A. G. et al. Bacterial gasdermins reveal an ancient mechanism of cell death. Science 375, 221–225 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Roucourt, B. & Lavigne, R. The role of interactions between phage and bacterial proteins within the infected cell: a diverse and puzzling interactome. Environ. Microbiol. 11, 2789–2805 (2009).

    Article  CAS  PubMed  Google Scholar 

  18. Wan, X., Hendrix, H., Skurnik, M. & Lavigne, R. Phage-based target discovery and its exploitation towards novel antibacterial molecules. Curr. Opin. Microbiol. 68, 1–7 (2021).

    CAS  Google Scholar 

  19. Zhang, P. et al. Bacteriophage protein Gp46 is a cross-species inhibitor of nucleoid-associated HU proteins. Proc. Natl Acad. Sci. USA 119, e2116278119 (2022).

  20. Liu, J. et al. Antimicrobial drug discovery through bacteriophage genomics. Nat. Biotechnol. 22, 185–191 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Dion, M. B., Oechslin, F. & Moineau, S. Phage diversity, genomics and phylogeny. Nat. Rev. Microbiol. 18, 125–138 (2020).

    Article  CAS  PubMed  Google Scholar 

  22. Miller, E. S. et al. Bacteriophage T4 genome. Microbiol. Mol. Biol. Rev. 67, 86–156 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yuan, S., Shi, J., Jiang, J. & Ma, Y. Genome-scale top-down strategy to generate viable genome-reduced phages. Nucleic Acids Res. 50, 13183–13197 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Repoila, F., Tétart, F., Bouet, J. Y. & Krisch, H. M. Genomic polymorphism in the T-even bacteriophages. EMBO J. 13, 4181–4192 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Shen, M. et al. Characterization and comparative genomic analyses of Pseudomonas aeruginosa phage PaoP5: new members assigned to PAK_P1-like viruses. Sci. Rep. 6, 34067 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hendrix, H. et al. Metabolic reprogramming of Pseudomonas aeruginosa by phage-based quorum sensing modulation. Cell Rep. 38, 110372 (2022).

    Article  CAS  PubMed  Google Scholar 

  27. Tsao, Y. F. et al. Phage morons play an important role in Pseudomonas aeruginosa phenotypes. J. Bacteriol. https://doi.org/10.1128/jb.00189-18 (2018).

  28. Shah, M. et al. A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa. Mol. Cell 81, 571–583.e6 (2021).

    Article  CAS  PubMed  Google Scholar 

  29. Chen, G. et al. Structural basis for diguanylate cyclase activation by its binding partner in Pseudomonas aeruginosa. eLife 10, e67289 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kuchma, S. L. et al. BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. J. Bacteriol. 189, 8165–8178 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Xin, L. et al. Regulation of flagellar motor switching by c-di-GMP phosphodiesterases in Pseudomonas aeruginosa. J. Biol. Chem. 294, 13789–13799 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kong, W. et al. ChIP-seq reveals the global regulator AlgR mediating cyclic di-GMP synthesis in Pseudomonas aeruginosa. Nucleic Acids Res. 43, 8268–8282 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chen, L. et al. Characterization and genomic analysis of ValSw3-3, a new Siphoviridae bacteriophage infecting Vibrio alginolyticus. J. Virol. 94, e00066-20 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kala, S. et al. HNH proteins are a widespread component of phage DNA packaging machines. Proc. Natl Acad. Sci. USA 111, 6022–6027 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Leffers, G. G. Jr. & Gottesman, S. Lambda Xis degradation in vivo by Lon and FtsH. J. Bacteriol. 180, 1573–1577 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yang, N. et al. The Crc protein participates in down-regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa. Mol. Microbiol. 96, 526–547 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Herbst, K. et al. Intrinsic thermal sensing controls proteolysis of Yersinia virulence regulator RovA. PLoS Pathog. 5, e1000435 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Skorupski, K., Tomaschewski, J., Rüger, W. & Simon, L. D. A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease. J. Bacteriol. 170, 3016–3024 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mulvenna, N. et al. Xenogeneic modulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein. J. Biol. Chem. 294, 17501–17511 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dhungana, G., Nepal, R., Regmi, M. & Malla, R. Pharmacokinetics and pharmacodynamics of a novel virulent Klebsiella phage Kp_Pokalde_002 in a mouse model. Front. Cell. Infect. Microbiol. 11, 684704 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Girmenia, C. et al. Incidence, risk factors and outcome of pre-engraftment Gram-negative bacteremia after allogeneic and autologous hematopoietic stem cell transplantation: an Italian prospective multicenter survey. Clin. Infect. Dis. 65, 1884–1896 (2017).

    Article  PubMed  Google Scholar 

  42. Klastersky, J. et al. Bacteraemia in febrile neutropenic cancer patients. Int. J. Antimicrob. 30, S51–S59 (2007).

    Article  CAS  Google Scholar 

  43. Zhang, K. et al. Bacteriophage protein PEIP is a potent Bacillus subtilis enolase inhibitor. Cell Rep. 40, 111026 (2022).

    Article  CAS  PubMed  Google Scholar 

  44. Mühlen, S. & Dersch, P. Anti-virulence strategies to target bacterial infections. Curr. Top. Microbiol. Immunol. 398, 147–183 (2016).

    PubMed  Google Scholar 

  45. Rasko, D. A. & Sperandio, V. Anti-virulence strategies to combat bacteria-mediated disease. Nat. Rev. Drug Discov. 9, 117–128 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. De Smet, J. et al. Bacteriophage-mediated interference of the c-di-GMP signalling pathway in Pseudomonas aeruginosa. Microb. Biotechnol. 14, 967–978 (2021).

    Article  PubMed  Google Scholar 

  47. Ping, D. et al. Hitchhiking, collapse, and contingency in phage infections of migrating bacterial populations. ISME J. 14, 2007–2018 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Hengge, R. Trigger phosphodiesterases as a novel class of c-di-GMP effector proteins. Phil. Trans. R. Soc. Lond. B 371, 20150498 (2016).

    Article  Google Scholar 

  49. Rostøl, J. T. & Marraffini, L. (Ph)ighting phages: how bacteria resist their parasites. Cell Host Microbe 25, 184–194 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Uyttebroek, S. et al. Safety and efficacy of phage therapy in difficult-to-treat infections: a systematic review. Lancet Infect. Dis. 22, e208–e220 (2022).

    Article  CAS  PubMed  Google Scholar 

  51. Bao, J. et al. Non-active antibiotic and bacteriophage synergism to successfully treat recurrent urinary tract infection caused by extensively drug-resistant Klebsiella pneumoniae. Emerg. Microbes Infect. 9, 771–774 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. El Ghali, A. et al. Ciprofloxacin in combination with bacteriophage cocktails against multi-drug resistant Pseudomonas aeruginosa in ex vivo simulated endocardial vegetation models. Antimicrob. Agents Chemother. 67, e0072823 (2023).

    Article  PubMed  Google Scholar 

  53. Holger, D. J. et al. Phage–antibiotic combinations against multidrug-resistant Pseudomonas aeruginosa in in vitro static and dynamic biofilm models. Antimicrob. Agents Chemother. 67, e0057823 (2023).

    Article  PubMed  Google Scholar 

  54. Li, L. et al. First-in-human application of double-stranded RNA bacteriophage in the treatment of pulmonary Pseudomonas aeruginosa infection. Microb. Biotechnol. 16, 862–867 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Yang, Y. et al. Development of a bacteriophage cocktail to constrain the emergence of phage-resistant Pseudomonas aeruginosa. Front. Microbiol. 11, 327 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Yang, Y. et al. Characterization of the first double-stranded RNA bacteriophage infecting Pseudomonas aeruginosa. Sci. Rep. 6, 38795 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Cui, G. et al. PmiR senses 2-methylisocitrate levels to regulate bacterial virulence in Pseudomonas aeruginosa. Sci. Adv. 8, eadd4220 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Chua, S. L. et al. Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms. Nat. Commun. 7, 10750 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Shen, M. et al. A linear plasmid-like prophage of Actinomyces odontolyticus promotes biofilm assembly. Appl. Environ. Microbiol. 84, e01263-18 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Zhong, Q. et al. Transcriptomic analysis reveals the dependency of Pseudomonas aeruginosa genes for double-stranded RNA bacteriophage phiYY infection cycle. iScience 23, 101437 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Jones, C. J. et al. ChIP-seq and RNA-seq reveal an AmrZ-mediated mechanism for cyclic di-GMP synthesis and biofilm development by Pseudomonas aeruginosa. PLoS Pathog. 10, e1003984 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Hickman, J. W. & Harwood, C. S. Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol. Microbiol. 69, 376–389 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Yang, L. et al. Temperature-dependent carrier state mediated by H-NS promotes the long-term coexistence of Y. pestis and a phage in soil. PLoS Pathog. 19, e1011470 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Cen, L. et al. Exploitation of a bacterium-encoded lytic transglycosylase by a human oral lytic phage to facilitate infection. J. Virol. 96, e0106322 (2022).

    Article  PubMed  Google Scholar 

  66. Cai, X. et al. Cultivation of a lytic double-stranded RNA bacteriophage infecting Microvirgula aerodenitrificans reveals a mutualistic parasitic lifestyle. J. Virol. 95, e0039921 (2021).

    Article  PubMed  Google Scholar 

  67. Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Chen, T. et al. iProX in 2021: connecting proteomics data sharing with big data. Nucleic Acids Res. 50, D1522–d1527 (2022).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Z. Li from Northwest University for performing protein degradation assays. This study was supported by grants from the National Key Research and Development Program of China (2021YFA0911200 to S.L, 2022YFC2304400 to H.L. and 2022YFC2304401 to H.L.), the National Natural Science Foundation of China (32170188 to H.L and 32270175 to J.W.), the Shenzhen Science and Technology Program (20231120104808001) and the Key University Laboratory of Metabolism and Health of Guangdong, SUST. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Author information

Authors and Affiliations

Authors

Contributions

H.L. and S. Le conceptualized the project. L.W., J.W., F.T., Q.Y. and Z.Z. developed the methodology. H.L. and S. Le performed validation. S. Le, L.W., J.W., F.T., Q.Y., J.Z., Z.Z., J.L. and Q.Z. conducted investigations. H.L., S. Lu and S. Le curated data. X.H., H.L. and S. Le. reviewed and edited the manuscript.

Corresponding author

Correspondence to Haihua Liang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Microbiology thanks Dana Holger, Joshua Ramsay and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The host range of PaoP5 and the growth curve of the constructed strains that were determined in LB medium in the presence of 0.05 mM IPTG.

(a) PaoP5 efficiently lyses 54.1% of the 233 clinically isolated P. aeruginosa strains. (b) The biological activities of 60 hypothetical orfs from PaoP5 were investigated by expressing them individually from pME6032, a multicopy plasmid that could express the target gene when induced by IPTG. The growth of each constructed strain was monitored in LB medium supplemented with 0.05 mM IPTG in the 96-well plates. The OD600 was measured for each well using a Microplate Spectrophotometer every 2 h for 18 h. Expression of orf002, orf157, orf160, orf163, orf164, orf165, or orf176 in P. aeruginosa attenuated bacterial growth but orf003 (dap1), orf006, orf131, orf147, and orf166 did not. No significant difference was detected between the growth curve of the PAO1 and PAO1/p-dap1 according to the Two-way Repeated Measures ANOVA analysis (P = 0.115). Error bars indicate the means ± SD of three independent experiments.

Source data

Extended Data Fig. 2 RNA-seq analysis between PAO1 and PAO1/p-dap1.

(a) The GO enrichment of the differentially expressed genes in PAO1/EV and PAO1/p-dap1 strains, which is classified according to molecular function (MF), biological process (BP), and cellular component (CC), and the top 10 enriched GO was shown. P values were assessed by two-sided Fisher’s exact test and were adjusted for multiple hypothesis testing using the Benjamini–Hochberg correction; genes with p-values under a threshold of 0.05 were considered statistically significant. (b) The DEGs are classified based on the KEGG analysis, and the top 30 enriched pathways, including Quorum Sensing and biofilm formation, are displayed. (c) qRT-qPCR analysis of the indicated genes in PAO1/EV and PAO1/p-dap1 strains. Data represent mean ± s.d. (n = 3). Statistical significance was determined using a two-sided Student’s t-test.

Source data

Extended Data Fig. 3 Impact of dap1 on biofilm formation and the bacterial two-hybrid results of Dap1 with 42 proteins.

(a) The biofilm formation of PAO1/EV and PAO1/p-dap1 under LB conditions supplemented with different concentrations of IPTG was displayed with crystal violet staining and quantified with optical density measurement. Data represent mean ± s.d. (n = 3) and statistical significance was determined using a two-sided Student´s t-test. (b) Bacterial two-hybrid assay determined the interaction between Dap1 with 42 c-di-GMP metabolic enzymes. The pUT18C is fused to Dap1 protein and the pKT25 is fused to the target proteins. Interactions were visualized by a drop test on LB agar plates supplemented with X-gal. The blue colonies showing the interaction between Dap1 and the indicated proteins. Strains containing the pKT25 and pUT18 vectors were used as a negative control (C-), and the interaction between pUT18C-zip and pKT25-zip was used as a positive control (C+).

Source data

Extended Data Fig. 4 Interactions of Dap1 with other proteins.

(a) The bacterial two-hybrid assay indicated that Dap1 might interact with RocR and ProE. Interactions were visualized by a drop test on LB agar plates supplemented with X-gal, and quantified by measuring the β-galactosidase activity indicated in Miller Units. (b-c) Co-IP assays showing no interaction between Dap1 with either RocR (b) or ProE (c) Overnight cultures of E. coli harboring rocR-Flag (or proE-Flag) with either eGFP or dap1-eGFP were lysed. Cell-lysis supernatants were incubated with anti-Flag beads, and then proteins were detected by western blot with eGFP or Flag antibody. Each experiment was repeated three times independently with similar results. (d) The biofilm formation of the indicated strains was displayed with crystal violet staining and quantified with optical density measurement. Data in a and d represent mean ± s.d. (n = 3) and statistical significance was determined using a one-way ANOVA Dunnett’s multiple comparison test.

Source data

Extended Data Fig. 5 Expression of dap1 in PAO1 increased the transcript and protein level of CdrA.

(a) Strains harboring the cdrA-lux and p-dap1 plasmids were cultivated in LB medium supplemented with 0.05 mM IPTG. The promoter activity of cdrA was detected at the indicated time. Error bars indicated the mean ± s.d. of three biological replicates. (b) CdrA protein levels were measured in PAO1/EV and PAO1/p-dap1 strains. Bacteria were cultured to an OD600 of 0.6 and 1.5. Equivalent samples were loaded onto SDS-PAGE and detected with an anti-Flag antibody. α-RNA polymerase (RNAP) was used as a loading control.

Source data

Extended Data Fig. 6 DipA is an early expressed gene and deletion of dipA had no effect on phage plaque formation.

(a) RT-qPCR analysis of dap1 and orf57 at 1 min, 10 min, 15 min and 30 min after phage infection. The highest level of dap1 transcript was detected 10 mins after phage infection, while the mRNA of the structural protein Orf57 was highly expressed 30 min after phage infection. (b) The same number of phage PaoP5Δdap1 was mixed with PAO1 or ΔdipA, and used double-layer agar plates to observe the plaque number and size. Data in a-b represent mean ± s.d. (n = 3) and statistical significance was determined using a one-way ANOVA Dunnett’s multiple comparison test.

Source data

Extended Data Fig. 7 The differentially expressed proteins detected by proteomics analysis.

(a) The differentially expressed bacterial proteins (DEPs) of PaoP5 or PaoP5Δdap1 infected PAO1. Only 17 bacterial proteins were significantly changed during PaoP5 and PaoP5Δdap1 infection, including seven upregulated proteins and ten downregulated proteins. (b) Showing the KEGG enrichments of these DEPs between PaoP5 or PaoP5Δdap1. (c) The DEPs of PaoP5Δdap1 infected PAO1 and Δlon. P values were assessed by two-sided Fisher’s exact test and were adjusted for multiple hypothesis testing using the Benjamini–Hochberg correction, proteins with p-values under a threshold of 0.05 were considered as statistically significant. (d) The DEPs between PaoP5Δdap1 infected PAO1 and Δlon were classified according to the KEGG enrichment, and the top 20 KEGG catalogs, including two-component system, ABC transporters, and biofilm formation are displayed.

Source data

Extended Data Fig. 8 Representative transmission electron micrographs of phages.

Representative transmission electron micrographs of negatively stained phages produced in PAO1, PAO1/p-dap1, Δlon, and Δlon/p-lon. The empty capsids are black, and phages with white heads are packaged with the genomes. Each experiment was repeated three times independently with similar results.

Source data

Extended Data Fig. 9 The phage and bacterial counts in the liver of the mice.

Phage titer (a) and the bacterial CFU (b) detected in the liver of mice that were intraperitoneal injected with PAO1 and phage PaoP5Δdap1 or PaoP5 at an MOI of 10. The maximum titer of PaoP5 was detected at 8 h post phage injection (~1.75 × 108 PFU/gm). The phage titer gradually decreased after 24 h, and was cleared within 96 h. Meanwhile, the bacterial CFU in the liver decreased gradually and was cleared within 96 h. For the PaoP5Δdap1- treated, PAO1-infected mice, the phage titer continuously decreased to an undetectable level at 72 h, and the maximum titer of PaoP5Δdap1 in the liver (~5 × 106 PFU/gm) was much less than that of PaoP5-treated mice. a-b, Error bars indicated the mean ± s.d. (n = 4 mice). (c) Classification of the bacteria detected in the liver of the dead mice that were treated with PaoP5Δdap1. 4 mice that were dead 48 h after phage PaoP5Δdap1 therapy were selected, and 50 colonies were isolated from the liver of each mouse and 200 colonies were assessed for phage resistance. 82 colonies were phage sensitive, and 118 colonies were phage resistant to both PaoP5 and PaoP5Δdap1. 30 phage-resistant strains were sent for 16 s rDNA sequencing. Overall, 41% of these strains were phage-sensitive P. aeruginosa, 39% of the strains were phage-resistant P. aeruginosa, while the other phage-resistant strains are gut commensal bacteria, including Enterobacter hormaechei, Proteus vulgaris, Escherichia coli, Enterobacter ludwigii, Enterobacter kobei and Enterobacter roggenkampii. (d) PaoP5 forms large plaques on some clinical strains while PaoP5Δdap1 forms small plaques, but both phages could not infect PA14.

Source data

Extended Data Fig. 10 Model of the mechanisms by which Dap1 regulates bacterial virulence and evades the Lon-mediated anti-phage defense.

(a) Phage protein Dap1 interacting with DipA leads to an increase in the cellular c-di-GMP levels, which inhibits bacterial motility, promotes biofilm formation, and significantly attenuates the virulence of P. aeruginosa. (b) When PaoP5 infects PAO1, phage protein Dap1 binds to HNH endonuclease to evade Lon-mediated HNH degradation; thus, more progenies are packaged with genomic DNA. However, the HNH endonuclease is efficiently degraded by Lon in the absence of Dap1, which facilitates PaoP5Δdap1 to produce fewer progenies, and most capsids are empty.PA14.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Peer Review File

Supplementary Data 1–4

Supplementary Data 1. DEGs between PAO1 vs PAO1/p-dap1. Data 2. Proteomics analysis data of phage proteins. Data 3. Proteomics analysis data of bacterial proteins. Data 4. Antibodies used in this study.

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Unprocessed TEM micrographs.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Unprocessed TEM micrographs.

Source Data Fig. 4

Statistical and LC–MS source data.

Source Data Fig. 5

Unprocessed western blots and gels.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Unprocessed TEM micrographs.

Source Data Extended Data Fig. 9

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Le, S., Wei, L., Wang, J. et al. Bacteriophage protein Dap1 regulates evasion of antiphage immunity and Pseudomonas aeruginosa virulence impacting phage therapy in mice. Nat Microbiol 9, 1828–1841 (2024). https://doi.org/10.1038/s41564-024-01719-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-024-01719-5

Search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology