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. 1994 Jul;62(7):2732–2739. doi: 10.1128/iai.62.7.2732-2739.1994

Bactericidal activity of synthetic peptides based on the structure of the 55-kilodalton bactericidal protein from human neutrophils.

B H Gray 1, J R Haseman 1
PMCID: PMC302875  PMID: 8005662

Abstract

Short (10- to 11-mer) hydrophilic peptides based on the structure of the 55-kDa bactericidal protein (BP55, B/PI, and CAP57) from human neutrophil granules were identified from the hydropathy plot of the 456-amino-acid sequence predicted from the nucleotide sequences of cDNA clones for BP55 and B/PI. Peptides corresponding to amino acid residues 90 to 99 (peptide #90-99), 86 to 99, or 90 to 102 of BP55 were bactericidal toward 5 x 10(6) Pseudomonas aeruginosa cells at 0.6 x 10(-5) to 1.5 x 10(-5) M and killed an Escherichia coli rough strain at 3 x 10(-5) M. The #90-99 peptide with a cysteine added at the amino terminus (C#90-99) was approximately 10 times more active than #90-99, killing P. aeruginosa at 1.5 x 10(-6) M. Peptides representing amino acid residues 27 to 37, 118 to 127, and 160 to 170 and the first 10 amino acids of the signal sequence for BP55 were not bactericidal. When coupled to either keyhole limpet hemocyanin or ovalbumin protein carriers through the thiol group, the C#90-99 peptide was not diminished on a molar basis in its capacity for killing of P. aeruginosa. Two other relatively hydrophilic peptides with an added amino-terminal cysteine, peptides C#227-236 and C#418-427, were not bactericidal at 1.2 x 10(-4) M or at 100 times the effective bactericidal concentration of C#90-99. The C#90-99 peptide killed E. coli at 1.5 x 10(-5) M, or at 10 times the concentration required to kill an equal number of P. aeruginosa cells. Although Pseudomonas cepacia and Staphylococcus aureus were resistent to killing by the parent BP55 molecule, they were susceptible to the C#90-99 and #90-99 peptides in the same concentration range as was E. coli. When all peptides were compared for the ability to neutralize E. coli O55:B5 endotoxin in a Limulus amoebocyte lysate assay, the C#227-236, C#418-427, and #160-170 peptides completely inhibited gelation at a 10(-4) M concentration. All other synthetic peptides, including bactericidal peptide #90-99 and its congeners, lacked endotoxin-neutralizing activity at the highest concentration tested (4.5 x 10(-4) M). A hybrid of the C#227-236 and #90-99 peptides (CHybrid) was identical to the C#227-236 peptide component in effectiveness for carrying out endotoxin neutralization and was fivefold better than the #90-99 peptide in its capacity for killing P. aeruginosa.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chen B., Polunovsky V., White J., Blazar B., Nakhleh R., Jessurun J., Peterson M., Bitterman P. Mesenchymal cells isolated after acute lung injury manifest an enhanced proliferative phenotype. J Clin Invest. 1992 Nov;90(5):1778–1785. doi: 10.1172/JCI116052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Elsbach P., Weiss J. Bactericidal/permeability increasing protein and host defense against gram-negative bacteria and endotoxin. Curr Opin Immunol. 1993 Feb;5(1):103–107. doi: 10.1016/0952-7915(93)90088-a. [DOI] [PubMed] [Google Scholar]
  3. Farley M. M., Shafer W. M., Spitznagel J. K. Lipopolysaccharide structure determines ionic and hydrophobic binding of a cationic antimicrobial neutrophil granule protein. Infect Immun. 1988 Jun;56(6):1589–1592. doi: 10.1128/iai.56.6.1589-1592.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ganz T., Selsted M. E., Lehrer R. I. Defensins. Eur J Haematol. 1990 Jan;44(1):1–8. doi: 10.1111/j.1600-0609.1990.tb00339.x. [DOI] [PubMed] [Google Scholar]
  5. Gazzano-Santoro H., Parent J. B., Grinna L., Horwitz A., Parsons T., Theofan G., Elsbach P., Weiss J., Conlon P. J. High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-terminal fragment to the lipid A region of lipopolysaccharide. Infect Immun. 1992 Nov;60(11):4754–4761. doi: 10.1128/iai.60.11.4754-4761.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gray P. W., Flaggs G., Leong S. R., Gumina R. J., Weiss J., Ooi C. E., Elsbach P. Cloning of the cDNA of a human neutrophil bactericidal protein. Structural and functional correlations. J Biol Chem. 1989 Jun 5;264(16):9505–9509. [PubMed] [Google Scholar]
  7. Hartree E. F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972 Aug;48(2):422–427. doi: 10.1016/0003-2697(72)90094-2. [DOI] [PubMed] [Google Scholar]
  8. Heumann D., Gallay P., Betz-Corradin S., Barras C., Baumgartner J. D., Glauser M. P. Competition between bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein for lipopolysaccharide binding to monocytes. J Infect Dis. 1993 Jun;167(6):1351–1357. doi: 10.1093/infdis/167.6.1351. [DOI] [PubMed] [Google Scholar]
  9. Hoess A., Watson S., Siber G. R., Liddington R. Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A resolution. EMBO J. 1993 Sep;12(9):3351–3356. doi: 10.1002/j.1460-2075.1993.tb06008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hovde C. J., Gray B. H. Characterization of a protein from normal human polymorphonuclear leukocytes with bactericidal activity against Pseudomonas aeruginosa. Infect Immun. 1986 Oct;54(1):142–148. doi: 10.1128/iai.54.1.142-148.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hovde C. J., Gray B. H. Physiological effects of a bactericidal protein from human polymorphonuclear leukocytes on Pseudomonas aeruginosa. Infect Immun. 1986 Apr;52(1):90–95. doi: 10.1128/iai.52.1.90-95.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kelly C. J., Cech A. C., Argenteanu M., Gallagher H., Shou J., Minnard E., Daly J. M. Role of bactericidal permeability-increasing protein in the treatment of gram-negative pneumonia. Surgery. 1993 Aug;114(2):140–146. [PubMed] [Google Scholar]
  13. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lehrer R. I., Lichtenstein A. K., Ganz T. Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol. 1993;11:105–128. doi: 10.1146/annurev.iy.11.040193.000541. [DOI] [PubMed] [Google Scholar]
  16. Little R. G., Kelner D. N., Lim E., Burke D. J., Conlon P. J. Functional domains of recombinant bactericidal/permeability increasing protein (rBPI23). J Biol Chem. 1994 Jan 21;269(3):1865–1872. [PubMed] [Google Scholar]
  17. Mannion B. A., Weiss J., Elsbach P. Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli. J Clin Invest. 1990 Mar;85(3):853–860. doi: 10.1172/JCI114512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marra M. N., Wilde C. G., Collins M. S., Snable J. L., Thornton M. B., Scott R. W. The role of bactericidal/permeability-increasing protein as a natural inhibitor of bacterial endotoxin. J Immunol. 1992 Jan 15;148(2):532–537. [PubMed] [Google Scholar]
  19. Marra M. N., Wilde C. G., Griffith J. E., Snable J. L., Scott R. W. Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity. J Immunol. 1990 Jan 15;144(2):662–666. [PubMed] [Google Scholar]
  20. Morita T., Ohtsubo S., Nakamura T., Tanaka S., Iwanaga S., Ohashi K., Niwa M. Isolation and biological activities of limulus anticoagulant (anti-LPS factor) which interacts with lipopolysaccharide (LPS). J Biochem. 1985 Jun;97(6):1611–1620. doi: 10.1093/oxfordjournals.jbchem.a135218. [DOI] [PubMed] [Google Scholar]
  21. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  22. Ooi C. E., Weiss J., Doerfler M. E., Elsbach P. Endotoxin-neutralizing properties of the 25 kD N-terminal fragment and a newly isolated 30 kD C-terminal fragment of the 55-60 kD bactericidal/permeability-increasing protein of human neutrophils. J Exp Med. 1991 Sep 1;174(3):649–655. doi: 10.1084/jem.174.3.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ooi C. E., Weiss J., Elsbach P., Frangione B., Mannion B. A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein. J Biol Chem. 1987 Nov 5;262(31):14891–14894. [PubMed] [Google Scholar]
  24. Pereira H. A., Erdem I., Pohl J., Spitznagel J. K. Synthetic bactericidal peptide based on CAP37: a 37-kDa human neutrophil granule-associated cationic antimicrobial protein chemotactic for monocytes. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4733–4737. doi: 10.1073/pnas.90.10.4733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rustici A., Velucchi M., Faggioni R., Sironi M., Ghezzi P., Quataert S., Green B., Porro M. Molecular mapping and detoxification of the lipid A binding site by synthetic peptides. Science. 1993 Jan 15;259(5093):361–365. doi: 10.1126/science.8420003. [DOI] [PubMed] [Google Scholar]
  26. SHINEFIELD H. R., RIBBLE J. C., BORIS M., EICHENWALD H. F. Bacterial interference: its effect on nursery-acquired infection with Staphylococcus aureus. I. Preliminary observations on artificial colonzation of newborns. Am J Dis Child. 1963 Jun;105:646–654. [PubMed] [Google Scholar]
  27. Schumann R. R., Leong S. R., Flaggs G. W., Gray P. W., Wright S. D., Mathison J. C., Tobias P. S., Ulevitch R. J. Structure and function of lipopolysaccharide binding protein. Science. 1990 Sep 21;249(4975):1429–1431. doi: 10.1126/science.2402637. [DOI] [PubMed] [Google Scholar]
  28. Shafer W. M., Martin L. E., Spitznagel J. K. Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate. Infect Immun. 1984 Jul;45(1):29–35. doi: 10.1128/iai.45.1.29-35.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shafer W. M., Shepherd M. E., Boltin B., Wells L., Pohl J. Synthetic peptides of human lysosomal cathepsin G with potent antipseudomonal activity. Infect Immun. 1993 May;61(5):1900–1908. doi: 10.1128/iai.61.5.1900-1908.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Siefferman C. M., Regelmann W. E., Gray B. H. Pseudomonas aeruginosa variants isolated from patients with cystic fibrosis are killed by a bactericidal protein from human polymorphonuclear leukocytes. Infect Immun. 1991 Jun;59(6):2152–2157. doi: 10.1128/iai.59.6.2152-2157.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Spitznagel J. K. Antibiotic proteins of human neutrophils. J Clin Invest. 1990 Nov;86(5):1381–1386. doi: 10.1172/JCI114851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tobias P. S., Mathison J. C., Ulevitch R. J. A family of lipopolysaccharide binding proteins involved in responses to gram-negative sepsis. J Biol Chem. 1988 Sep 25;263(27):13479–13481. [PubMed] [Google Scholar]
  33. Ulevitch R. J. Recognition of bacterial endotoxins by receptor-dependent mechanisms. Adv Immunol. 1993;53:267–289. doi: 10.1016/s0065-2776(08)60502-7. [DOI] [PubMed] [Google Scholar]
  34. Wasiluk K. R., Skubitz K. M., Gray B. H. Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa. Infect Immun. 1991 Nov;59(11):4193–4200. doi: 10.1128/iai.59.11.4193-4200.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Weiss J., Elsbach P., Olsson I., Odeberg H. Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes. J Biol Chem. 1978 Apr 25;253(8):2664–2672. [PubMed] [Google Scholar]
  36. Weiss J., Elsbach P., Shu C., Castillo J., Grinna L., Horwitz A., Theofan G. Human bactericidal/permeability-increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria. J Clin Invest. 1992 Sep;90(3):1122–1130. doi: 10.1172/JCI115930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weiss J., Hutzler M., Kao L. Environmental modulation of lipopolysaccharide chain length alters the sensitivity of Escherichia coli to the neutrophil bactericidal/permeability-increasing protein. Infect Immun. 1986 Feb;51(2):594–599. doi: 10.1128/iai.51.2.594-599.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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