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Modification of chicken avian β-defensin-8 at positively selected amino acid sites enhances specific antimicrobial activity

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Abstract

Antimicrobial peptides (AMPs), essential components of innate immunity, are found in a range of phylogenetically diverse species and are thought to act by disrupting the membrane integrity of microbes. In this paper, we used evolutionary signatures to identify sites that are most relevant during the functional evolution of these molecules and introduced amino acid substitutions to improve activity. We first demonstrate that the anti-microbial activity of chicken avian β-defensin-8, previously known as gallinacin-12, can be significantly increased against Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Salmonella typhimurium phoP− mutant and Streptococcus pyogenes through targeted amino acid substitutions, which confer increased peptide charge. However, by increasing the AMP charge through amino acid substitutions at sites predicted to be subject to positive selection, antimicrobial activity against Escherichia coli was further increased. In contrast, no further increase in activity was observed against the remaining pathogens. This result suggests that charge-increasing modifications confer increased broad-spectrum activity to an AMP, whilst positive selection at particular sites is involved in directing the antimicrobial response against specific pathogens. Thus, there is potential for the rational design of novel therapeutics based on specifically targeted and modified AMPs.

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Acknowledgment

We acknowledge Dr. Kieran Meade and Paul Cormican for helpful comments. This research was supported by the Food Institutional Research Measure (FIRM) Grant 01/R&D/D/135 from the Irish Department of Agriculture, Food and Rural Development. The authors declare no conflicting interests.

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Authors and Affiliations

  1. Education and Research Centre, St. Vincent’s University Hospital, Dublin 4, Ireland

    Rowan Higgs, Sarah Cahalane, Andrew T. Lloyd & Cliona O’Farrelly

  2. Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland

    David J. Lynn

  3. School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland

    Iñigo Alaña & Chandralal M. Hewage

  4. Conway Institute, University College Dublin, Dublin 4, Ireland

    Iñigo Alaña, Chandralal M. Hewage & Cliona O’Farrelly

  5. Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland

    Tharappel James

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  1. Rowan Higgs
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  2. David J. Lynn
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  3. Sarah Cahalane
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  4. Iñigo Alaña
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  5. Chandralal M. Hewage
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  6. Tharappel James
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  7. Andrew T. Lloyd
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  8. Cliona O’Farrelly
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Corresponding author

Correspondence to Cliona O’Farrelly.

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Supplementary Fig. 1(A)

RP-HPLC chromatogram of A) AvBD8. The deprotected synthetic linear peptides were loaded onto a C18 RP-HPLC column in 0.1% trifluoroacetic acid and a gradient elution was performed using 0–100% 0.1% TFA (solution A) and acetonitrile (solution B) at ml/min flow rate. The flow through was monitored at 220 nm absorbency for the peptides (JPEG 49 kb)

High resolution image file (TIF 489 kb)

Supplementary Fig. 1(B)

RP-HPLC chromatogram of B) AvBD8ps+ (JPEG 50 kb)

High resolution image file (TIF 491 kb)

Supplementary Fig. 1(C)

RP-HPLC chromatogram of C) AvBD8ns+ (JPEG 83 kb)

High resolution image file (TIF 1437 kb)

Supplementary Fig. 1(D)

RP-HPLC chromatogram of D) AvBD8ps− (JPEG 49 kb)

High resolution image file (TIF 477 kb)

Supplementary Fig. 1(E)

RP-HPLC chromatogram of E) AvBD8ns− (JPEG 83 kb)

High resolution image file (TIF 1437 kb)

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Higgs, R., Lynn, D.J., Cahalane, S. et al. Modification of chicken avian β-defensin-8 at positively selected amino acid sites enhances specific antimicrobial activity. Immunogenetics 59, 573–580 (2007). https://doi.org/10.1007/s00251-007-0219-5

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  • DOI: https://doi.org/10.1007/s00251-007-0219-5

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