Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Abstract

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24–33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance.

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Figure 1: Liposomes composed of cholesterol and sphingomyelin protect monocytes from cholesterol-dependent cytolysins, α-hemolysin (HML) and phospholipase C (PLC).
Figure 2: Cholesterol:sphingomyelin (Ch:Sm) liposomes protect epithelial and endothelial cells from lysis by pneumolysin (PLY) or phospholipase C (PLC).
Figure 3: Liposomes protect monocytes from toxins secreted by S. pyogenes, S. pneumoniae and S. aureus.
Figure 4: A mixture of cholesterol:sphingomyelin (Ch:Sm) + Sm-only liposomes confers survival in mice infected with S. pneumoniae.
Figure 5: A mixture of cholesterol:sphingomyelin (Ch:Sm) + Sm-only liposomes confer survival in mice infected with S. aureus.
Figure 6: A combination of toxin-sequestration with antibiotic treatment to treat fatal S. aureus and S. pneumoniae infections.

References

  1. 1

    Stefani, S. & Goglio, A. Methicillin-resistant Staphylococcus aureus: related infections and antibiotic resistance. Int. J. Infect. Dis. 14. (suppl. 4), S19–S22 (2010).

    Article  Google Scholar 

  2. 2

    Bush, K. et al. Tackling antibiotic resistance. Nat. Rev. Microbiol. 9, 894–896 (2011).

    CAS  Article  Google Scholar 

  3. 3

    Alksne, L.E. & Projan, S.J. Bacterial virulence as a target for antimicrobial chemotherapy. Curr. Opin. Biotechnol. 11, 625–636 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Titball, R.W. Bacterial phospholipases C. Microbiol. Rev. 57, 347–366 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Kadioglu, A., Weiser, J.N., Paton, J.C. & Andrew, P.W. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat. Rev. Microbiol. 6, 288–301 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Bubeck Wardenburg, J., Bae, T., Otto, M., DeLeo, F.R. & Schneewind, O. Poring over pores: α-hemolysin and Panton-Valentine leukocidin in Staphylococcus aureus pneumonia. Nat. Med. 13, 1405–1406 (2007).

    Article  Google Scholar 

  7. 7

    Timmer, A.M. et al. Streptolysin O promotes group A Streptococcus immune evasion by accelerated macrophage apoptosis. J. Biol. Chem. 284, 862–871 (2009).

    CAS  Article  Google Scholar 

  8. 8

    Gonzalez, M.R., Bischofberger, M., Pernot, L., van der Goot, F.G. & Freche, B. Bacterial pore-forming toxins: The (w)hole story? Cell. Mol. Life Sci. 65, 493–507 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Parker, M.W. & Feil, S.C. Pore-forming protein toxins: from structure to function. Prog. Biophys. Mol. Biol. 88, 91–142 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Tweten, R.K. Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins. Infect. Immun. 73, 6199–6209 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Valeva, A. et al. Evidence that clustered phosphocholine head groups serve as sites for binding and assembly of an oligomeric protein pore. J. Biol. cholesterolem. 281, 26014–26021 (2006).

    CAS  Article  Google Scholar 

  12. 12

    Linhartová, I. et al. RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol. Rev. 34, 1076–1112 (2010).

    Article  Google Scholar 

  13. 13

    Olsen, I. & Jantzen, E. Sphingolipids in bacteria and fungi. Anaerobe 7, 103–112 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    CAS  Article  Google Scholar 

  15. 15

    Brown, D.A. & Rose, J.K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533–544 (1992).

    CAS  Article  Google Scholar 

  16. 16

    Harder, T. & Simons, K. Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains. Curr. Opin. Cell Biol. 9, 534–542 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Simons, K. & Gerl, M.J. Revitalizing membrane rafts: new tools and insights. Nat. Rev. Mol. Cell Biol. 11, 688–699 (2010).

    CAS  Article  Google Scholar 

  18. 18

    Klose, C. et al. Yeast lipids can phase-separate into micrometer-scale membrane domains. J. Biol. Chem. 285, 30224–30232 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Huang, J., Buboltz, J.T. & Feigenson, G.W. Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers. Biochim. Biophys. Acta 1417, 89–100 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Dietschy, J.M. & Turley, S.D. Cholesterol metabolism in the central nervous system during early development and in the mature animal. J. Lipid Res. 45, 1375–1397 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Ikonen, E. Cellular cholesterol trafficking and compartmentalization. Nat. Rev. Mol. Cell Biol. 9, 125–138 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Mesmin, B. & Maxfield, F.R. Intracellular sterol dynamics. Biochim. Biophys. Acta 1791, 636–645 (2009).

    CAS  Article  Google Scholar 

  23. 23

    Rittirsch, D., Flierl, M.A. & Ward, P.A. Harmful molecular mechanisms in sepsis. Nat. Rev. Immunol. 8, 776–787 (2008).

    CAS  Article  Google Scholar 

  24. 24

    Stringaris, A.K. et al. Neurotoxicity of pneumolysin, a major pneumococcal virulence factor, involves calcium influx and depends on activation of p38 mitogen-activated protein kinase. Neurobiol. Dis. 11, 355–368 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Spreer, A. et al. Reduced release of pneumolysin by Streptococcus pneumoniae in vitro and in vivo after treatment with nonbacteriolytic antibiotics in comparison to ceftriaxone. Antimicrob. Agents Chemother. 47, 2649–2654 (2003).

    CAS  Article  Google Scholar 

  26. 26

    Wall, E.C. et al. Persistence of pneumolysin in the cerebrospinal fluid of patients with pneumococcal meningitis is associated with mortality. Clin. Infect. Dis. 54, 701–705 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Bakker-Woudenberg, I.A., ten Kate, M.T., Guo, L., Working, P. & Mouton, J.W. Improved efficacy of ciprofloxacin administered in polyethylene glycol-coated liposomes for treatment of Klebsiella pneumoniae pneumonia in rats. Antimicrob. Agents Chemother. 45, 1487–1492 (2001).

    CAS  Article  Google Scholar 

  28. 28

    Brisseau, G.F. et al. Unilamellar liposomes modulate secretion of tumor necrosis factor by lipopolysaccharide-stimulated macrophages. Antimicrob. Agents cholesterolemother. 38, 2671–2675 (1994).

    CAS  Article  Google Scholar 

  29. 29

    Moore, C.L. et al. Prediction of failure in vancomycin-treated methicillin-resistant Staphylococcus aureus bloodstream infection: a clinically useful risk stratification tool. Antimicrob. Agents Chemother. 55, 4581–4588 (2011).

    CAS  Article  Google Scholar 

  30. 30

    Huttunen, R. & Aittoniemi, J. New concepts in the pathogenesis, diagnosis and treatment of bacteremia and sepsis. J. Infect. 63, 407–419 (2011).

    Article  Google Scholar 

  31. 31

    Drulis-Kawa, Z. & Dorotkiewicz-Jach, A. Liposomes as delivery systems for antibiotics. Int. J. Pharm. 387, 187–198 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Allen, R.C., Popat, R., Diggle, S.P. & Brown, S.P. Targeting virulence: can we make evolution-proof drugs? Nat. Rev. Microbiol. 12, 300–308 (2014).

    CAS  Article  Google Scholar 

  33. 33

    McNeela, E.A. et al. Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4. PLoS Pathog. 6, e1001191 (2010).

    Article  Google Scholar 

  34. 34

    Babiychuk, E.B., Monastyrskaya, K., Potez, S. & Draeger, A. Intracellular Ca(2+) operates a switch between repair and lysis of streptolysin O-perforated cells. Cell Death Differ. 16, 1126–1134 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Morton, D.B. Pain and laboratory animals. Nature 317, 106 (1985).

    CAS  Article  Google Scholar 

  36. 36

    Merrill, A.H. Jr., Sullards, M.C., Allegood, J.C., Kelly, S. & Wang, E. Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods 36, 207–224 (2005).

    CAS  Article  Google Scholar 

  37. 37

    Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically – Twenty second information supplement: Approved standard M07–A8. (CLSI, Wayne, PA, USA, 2009).

  38. 38

    Shah, H. How to calculate sample size in animal studies. Natl. J. Physiol. Pharm. Pharmacol. 1, 35–39 (2011).

    Google Scholar 

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Acknowledgements

We gratefully acknowledge the financial support of the University of Bern, Commission for Technology and Innovation (CTI) (16001.1 PFLS-LS to A.D. and E.B.B.), Deutsche Forschungsgemeinschaft (DFG GU 335/16-2 to E.G. and SFB 1112 to B.K.), Federal Ministry of Education and Research (BMBF) (FKZ: 01EO1002 to A.S.) and the Institute of Infection & Global Health, University of Liverpool to A.K.

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B.D.H., D.R.N. and K.A.B. contributed equally to this study. B.D.H., K.A.B., R.Z., M.J.E. and E.G. performed in vivo experiments with S. aureus. D.R.N., S.G. and L.B.-M. performed in vivo experiments with S. pneumoniae. K.M. and J.S. provided bacterial culture supernatants for the in vitro experiments; J.S. performed MBC experiments; B.K. and L.J. performed bio-distribution experiments. M.L., H.W., A.D. and E.B.B. performed in vitro experiments; E.G. and A.K. designed the in vivo experiments with S. aureus and S. pneumoniae, respectively, and provided bacterial culture supernatants for the in vitro experiments. A.K. provided purified pneumolysin for in vitro experiments. E.B.B. designed the in vitro experiments. A.S. provided statistical advice for the paper. A.D. designed the study. E.B.B. designed and coordinated the study and wrote the paper. D.R.N., E.G., A.S., A.K. and A.D. edited and contributed to the writing of the paper. All authors analyzed and discussed the results and commented on the manuscript. E.G., A.K., A.D. and E.B.B. are co-senior authors.

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Correspondence to Eduard B Babiychuk.

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E.B.B. and A.D. are inventors on a patent application pertaining to this work.

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Henry, B., Neill, D., Becker, K. et al. Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice. Nat Biotechnol 33, 81–88 (2015). https://doi.org/10.1038/nbt.3037

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