Oral Presentation 51st Lorne Proteins Conference 2026

A protein antibiotic inhibits the BAM complex to kill without cell entry (131174)

Fabian Munder 1 2 3 , Matthew Johnson 2 3 , Imogen Samuels 3 , Laura McCaughey 4 , Oleksii Zdorevskyi 5 , Chunxiao Wang 3 , Ashleigh Kropp 1 2 3 , Lauren Zavan 6 , Erin P. Price 7 8 , Derek S. Sarovich 7 8 , Swati Varshney 9 , Christopher McDevitt 6 , Hari Venugopal 10 , Vivek Sharma 5 11 , Matthew T. Doyle 12 13 14 , Francesca Short 1 15 , Debnath Ghosal 2 3 , James P. R. Connolly 16 , Gavin J. Knott 17 , Rhys Grinter 1 2 3
  1. Department of Microbiology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia
  2. Centre for Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia
  3. Department of Biochemistry & Pharmacology, Bio21, The University of Melbourne, Melbourne, VIC, Australia
  4. School of Infection and Immunity, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
  5. Department of Physics, University of Helsinki, Helsinki, Finland
  6. Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, VIC, Australia
  7. Sunshine Coast Health Institute, Birtinya, QLD, Australia
  8. Centre for Bioinnovation, University of the Sunshine Coast, Sippy Downs, QLD, Australia
  9. University of Melbourne Bio21 Molecular Science and Biotechnology Institute, Bio21 Mass Spectrometry and Proteomics Facility, Parkville, VIC, Australia
  10. Ramaciotti Centre for Cryo-Electron Microscopy, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
  11. HiLIFE Institute of Biotechnology, University of Helsinki, Helsinki, Finland
  12. School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Darlington, NSW, Australia
  13. Centre for Drug Discovery Innovation, The University of Sydney, Darlington, NSW, Australia
  14. Sydney Infectious Diseases Institute, The University of Sydney, Darlington, NSW, Australia
  15. Wellcome Sanger Institute, Hinxton, Cambridgeshire, United Kingdom
  16. Newcastle University Biosciences Institute, Newcastle University, Newcastle, United Kingdom
  17. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia

Gram-negative bacteria resist many approved antibiotics because they have an impermeable outer membrane1. The high-priority pathogen Pseudomonas aeruginosa is infamous for acquiring multidrug resistance and is particularly dangerous for individuals with pre-existing immune deficiencies, such as cystic fibrosis2.
In this study, we show that potent protein antibiotics, called L-type pyocins3, exploit a critical vulnerability in the outer membrane by inhibiting the essential β-barrel assembly machinery (BAM) complex4. We obtained three high-resolution cryo-electron microscopy structures of P. aeruginosa apo-BAM and in two distinct complexes with L-type pyocins, revealing an elaborate multi-step mechanism of action. L-type pyocins first interact with a surface-exposed region of BamA and subsequently deploy a C-terminal peptide that competitively binds β-strand 1, blocking the initiation site of outer-membrane β-barrel protein assembly5. This action occurs at the bacterial cell surface, making L-type pyocins intrinsically insensitive to efflux pumps and periplasmic immunity systems.
To chart the cellular consequences of BAM inhibition, we combined genetic and multi-omics profiling. Within minutes of treatment, P. aeruginosa responds by upregulating microbial competition programs, such as the type VI secretion system, and downregulating translation. Longer term responses include drastic changes in the abundance of outer-membrane proteins, consistent with stress from arrested β-barrel assembly. To investigate how intoxicated cells die, we performed cryo-electron tomography with L-type pyocin-treated P. aeruginosa and observed catastrophic outer membrane disruption, which is lethal for Gram-negative bacteria. Functionally, exposure to L-type pyocins renders P. aeruginosa susceptible to the antibiotic vancomycin, demonstrating immediate potential for combination therapy with approved, but normally ineffective drugs.
In summary, we determined the L-type pyocin cytotoxic mechanism and the cellular consequences of BAM inhibition, from atom to cell4. We validate the BAM complex as a high-value therapeutic target for antibiotics that function without cell entry and define an engineerable system for further development of L-type pyocins as next-generation antibiotics against multidrug-resistant P. aeruginosa.

  1. Pang, Z., Raudonis, R., Glick, B. R., Lin, T.-J. & Cheng, Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnology advances 37, 177-192 (2019). https://doi.org/10.1016/j.biotechadv.2018.11.013
  2. Qin, S. et al. Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal transduction and targeted therapy 7, 199 (2022). https://doi.org/10.1038/s41392-022-01056-1
  3. McCaughey, L. C., Ritchie, N. D., Douce, G. R., Evans, T. J. & Walker, D. Efficacy of species-specific protein antibiotics in a murine model of acute Pseudomonas aeruginosa lung infection. Scientific reports 6, 30201 (2016). https://doi.org/10.1038/srep30201
  4. Munder, F. et al. A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry. bioRxiv, 2025.2009.2018.677229 (2025). https://doi.org:10.1101/2025.09.18.677229
  5. Doyle, M. T. et al. Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding. Cell 185, 1143-1156.e1113 (2022). https://doi.org:https://doi.org/10.1016/j.cell.2022.02.016