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Modified horseshoe crab peptides target and kill bacteria inside host cells

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Abstract

Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two β-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

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Availability of data and material

Peptide sequences are provided herein. Average data are provided, with processing and curve fits as reported for specific experiments.

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Acknowledgements

This work was supported by funding from the Australian Government scholarships (ASA. Research Training Program Scholarship), the Australian Research Council (Centre of Excellence for Innovations in Peptide and Protein Science CE200100012; DJC. Laureate Fellowship FL150100146; STH. Future Fellowship FT150100398), the National Health and Medical Research Council grants (NL. 1183927; JRW. and BJC. 1098337). NDC is supported as a CZI Imaging Scientist by grant number 2020-225648 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation.

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Authors and Affiliations

  1. Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD, 4072, Australia

    Anna S. Amiss, Peta J. Harvey, David J. Craik, Sónia Troeira Henriques & Nicole Lawrence

  2. Institute for Molecular Bioscience, IMB Centre for Inflammation and Disease Research and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, 4072, Australia

    Jessica B. von Pein, Matthew J. Sweet & Ronan Kapetanovic

  3. Global and Tropical Health Division, Menzies School of Health Research, Darwin, NT, 0811, Australia

    Jessica R. Webb & Bart J. Currie

  4. Australian Cancer Research Foundation/Institute for Molecular Bioscience Cancer Biology Imaging Facility, The University of Queensland, Brisbane, QLD, 4072, Australia

    Nicholas D. Condon

  5. School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Queensland, Australia

    Minh-Duy Phan & Mark A. Schembri

  6. Department of Infectious Diseases and Northern Territory Medical Program, Royal Darwin Hospital, Darwin, NT, 0811, Australia

    Bart J. Currie

  7. Friedrich Miescher Institute for Biomedical Research, 4058, Basel, BS, Switzerland

    Ronan Kapetanovic

  8. Queensland University of Technology, School of Biomedical Sciences, Translational Research Institute, Brisbane, QLD, 4102, Australia

    Sónia Troeira Henriques

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  1. Anna S. Amiss
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  13. Nicole Lawrence
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Contributions

AA, NL, STH, and RK contributed to the study conception and design. Material preparation, data collection and analysis were performed by AA, JP, JW, NL, STH, NC, and PH. Funding and resources were provided by NL, STH, DC, BC, MS, and MS. The manuscript was written by AA, NL, STH, and RK. All authors commented on the previous versions of the manuscript and read and approved the final manuscript. The authors would like to thank Dr Kaustav Gupta at The Institute for Molecular Bioscience, Centre for Inflammation and Disease Research and Australian Infectious Diseases Research Centre, The University of Queensland, for harvesting and culturing bone marrow macrophages.

Corresponding authors

Correspondence to Ronan Kapetanovic, Sónia Troeira Henriques or Nicole Lawrence.

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The authors declare that they do not have any conflicts or competing interests.

Ethical approval

The University of Queensland Institutional animal ethics committee approved the use of primary mouse cells, herein referred to as BMM cells (IMB/123/18). RBCs were collected from healthy adult donors following protocols approved by the Human Research Ethics Committees (University of Queensland approval number 2013000582) Identity of human blood donors was not recorded or reported.

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Amiss, A.S., von Pein, J.B., Webb, J.R. et al. Modified horseshoe crab peptides target and kill bacteria inside host cells. Cell. Mol. Life Sci. 79, 38 (2022). https://doi.org/10.1007/s00018-021-04041-z

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