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Amino Acids

, Volume 50, Issue 5, pp 557–568 | Cite as

Understanding the antimicrobial properties/activity of an 11-residue Lys homopeptide by alanine and proline scan

  • P. Carvajal-Rondanelli
  • M. Aróstica
  • C. A. Álvarez
  • C. Ojeda
  • F. Albericio
  • L. F. Aguilar
  • S. H. Marshall
  • F. Guzmán
Original Article
  • 254 Downloads

Abstract

Previous work demonstrated that lysine homopeptides adopt a polyproline II (PPII) structure. Lysine homopeptides with odd number of residues, especially with 11 residues (K11), were capable of inhibiting the growth of a broader spectrum of bacteria than those with an even number. Confocal studies also determined that K11 was able to localize exclusively in the bacterial membrane, leading to cell death. In this work, the mechanism of action of this peptide was further analyzed focused on examining the structural changes in bacterial membrane induced by K11, and in K11 itself when interacting with bacterial membrane lipids. Moreover, alanine and proline scans were performed for K11 to identify relevant positions in structure conformation and antibacterial activity. To do so, circular dichroism spectroscopy (CD) was conducted in saline phosphate buffer (PBS) and in lipidic vesicles, using large unilamellar vesicles (LUV), composed of 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or bacterial membrane lipid. Antimicrobial activity of K11 and their analogs was evaluated in Gram-positive and Gram-negative bacterial strains. The scanning electron microscopy (SEM) micrographs of Staphylococcus aureus ATCC 25923 exposed to the Lys homopeptide at MIC concentration showed blisters and bubbles formed on the bacterial surface, suggesting that K11 exerts its action by destabilizing the bacterial membrane. CD analysis revealed a remarkably enhanced PPII structure of K11 when replacing some of its central residues by proline in PBS. However, when such peptide analogs were confronted with either DMPG-LUV or membrane lipid extract-LUV, the tendency to form PPII structure was severely weakened. On the contrary, K11 peptide showed a remarkably enhanced PPII structure in the presence of DMPG-LUV. Antibacterial tests revealed that K11 was able to inhibit all tested bacteria with an MIC value of 5 µM, while proline and alanine analogs have a reduced activity on Listeria monocytogenes. Besides, the activity against Vibrio parahaemolyticus was affected in most of the alanine-substituted analogs. However, lysine substitutions by alanine or proline at position 7 did not alter the activity against all tested bacterial strains, suggesting that this position can be screened to find a substitute amino acid yielding a peptide with increased antibacterial activity. These results also indicate that the PPII secondary structure of K11 is stabilized by the interaction of the peptide with negatively charged phospholipids in the bacterial membrane, though not being the sole determinant for its antimicrobial activity.

Keywords

Lysine homopeptide-antimicrobial activity-Ala Pro scanning-membrane rupture 

Notes

Acknowledgements

This work was supported by the Chilean Fondo Nacional de Desarrollo Científico y Tecnológico (Fondecyt), Grant 1140926. Doctoral fellowship Pontificia Universidad Católica de Valparaíso. 

Compliance with ethical standards

Fanny Guzmán, on behalf of all authors, declares that the manuscript has not been submitted to any other journal.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

726_2018_2542_MOESM1_ESM.docx (35 kb)
Supplementary material 1 (DOCX 34 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • P. Carvajal-Rondanelli
    • 1
  • M. Aróstica
    • 2
  • C. A. Álvarez
    • 3
    • 4
  • C. Ojeda
    • 5
  • F. Albericio
    • 6
    • 7
  • L. F. Aguilar
    • 8
  • S. H. Marshall
    • 2
    • 5
  • F. Guzmán
    • 2
  1. 1.Escuela de Alimentos, Facultad de Ciencias Agronómicas y de los AlimentosPontificia Universidad Católica de ValparaísoValparaísoChile
  2. 2.Núcleo de Biotecnología de CuraumaPontificia Universidad Católica de ValparaísoValparaísoChile
  3. 3.Laboratorio de Fisiología y Genética Marina (FIGEMA)Centro de Estudios Avanzados en Zonas Áridas (CEAZA)CoquimboChile
  4. 4.Facultad de Ciencias del MarUniversidad Católica del NorteCoquimboChile
  5. 5.Instituto de BiologíaPontificia Universidad Católica de ValparaísoValparaísoChile
  6. 6.Department of Organic Chemistry and CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and NanomedicineUniversity of BarcelonaBarcelonaSpain
  7. 7.School of ChemistryUniversity of KwaZulu-NatalDurbanSouth Africa
  8. 8.Instituto de QuímicaPontificia Universidad Católica de ValparaísoValparaísoChile

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