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Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms

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Abstract

Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at ‘a’ and/or ‘d’ position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its ‘a’ and ‘d’ positions with d-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its d-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show significant scope for designing antimicrobial agents with selectivity towards microorganisms by substituting leucine residues at ‘a’ and/or ‘d’ positions of a leucine zipper sequence of an antimicrobial peptide with different amino acids.

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Abbreviations

PBS:

Phosphate buffered saline

FITC:

Fluorescin isothiocyanate

Fmoc:

N-(9-fluorenyl) methoxycarbonyl

DIC:

Differential interference contrast

HPLC:

High-performance liquid chromatography

ESI-MS:

Electron spray ionization- mass spectrometry

ANS:

8-Anilinonapthalene-1-sulfonic acid

DiS-C3-5:

3,3′-Dipropylthiadicarbocyanine iodide

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Acknowledgments

CSIR-CDRI communication number of this manuscript is 8728. We are extremely grateful to Dr. S. K. Puri, Parasitology Division, CSIR-CDRI for kindly providing us fresh hRBCs for performing hemolytic activity assays with our peptides. The work was supported by the CSIR network projects NWP 0005 and BioDiscovery (BSC 0120). AA, SA, SS acknowledges the receipt of senior research fellowships from Council of Scientific and Industrial Research (CSIR), India; AK and JKT receipt of senior research fellowships from ICMR and UGC. The authors are extremely thankful to AL Vishwakarma for recoding the flow cytometry profiles and Sanjeev Kanojiya, Sophisticated Analytical Instrumentation Facility (SAIF), CSIR-CDRI for recording the ES-MS mass spectra.

Conflict of interest

The authors declare that they do not have any conflict of interest.

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Authors and Affiliations

  1. Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, 226 031, India

    Aqeel Ahmad, Sarfuddin Azmi, Saurabh Srivastava, Amit Kumar, Jitendra Kumar Tripathi & Jimut Kanti Ghosh

  2. Fermentation Technology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, 226 031, India

    Nripendra N. Mishra & Praveen K. Shukla

Authors
  1. Aqeel Ahmad
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  2. Sarfuddin Azmi
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  3. Saurabh Srivastava
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  4. Amit Kumar
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  5. Jitendra Kumar Tripathi
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  6. Nripendra N. Mishra
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  7. Praveen K. Shukla
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  8. Jimut Kanti Ghosh
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Corresponding author

Correspondence to Jimut Kanti Ghosh.

Additional information

A. Ahmad and S. Azmi contributed equally to the work. S. Srivastava and A. Kumar contributed equally to the work.

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Ahmad, A., Azmi, S., Srivastava, S. et al. Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms. Amino Acids 46, 2531–2543 (2014). https://doi.org/10.1007/s00726-014-1802-3

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  • DOI: https://doi.org/10.1007/s00726-014-1802-3

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