Leucine and glycine residues, at the 9th and 10th positions of helical domain of naturally occurring antimicrobial peptide (AMP), Temporin L were substituted with an unnatural amino acid, β-leucine (homovaline) to improve its serum protease stability, haemolytic/cytotoxic properties and reduce the size to some extent. The designed analogue, L9βl-TL showed either equal or improved antimicrobial activity to TL against different microorganisms including the resistant strains. Interestingly, L9βl-TL also exhibited lower haemolytic and cytotoxic activities against human red blood cells and 3T3 cells, respectively. Moreover, L9βl-TL showed antibacterial activity in presence of 25% (v/v) human serum and showed resistance against proteolytic cleavage in presence of it that suggested the serum protease stability of the TL-analogue. L9βl-TL exhibited un-ordered secondary structures in both bacterial and mammalian membrane mimetic lipid vesicles as compared to the helical structures of TL in these environments. However, tryptophan fluorescence studies demonstrated more selective interaction of L9βl-TL with bacterial membrane mimetic lipid vesicles in comparison to non-selective interactions of TL with both kinds of lipid vesicles. Membrane depolarization studies with live MRSA and bacterial membrane-mimetic lipid vesicles suggested a membrane-disrupting mode of action of L9βl-TL. L9βl-TL showed faster bactericidal mechanism compared to TL against MRSA. Interestingly, L9βl-TL was found as more potent than TL either in inhibiting biofilm formation or in eradicating the mature biofilm formed by MRSA. Overall, the present work demonstrates a simple and useful strategy to design of an analogue of TL, with minimal modifications while maintaining its antimicrobial activity with lesser toxicity and higher stability which could be attempted for other AMPs as well.
Keywords: Antimicrobial peptide; MRSA biofilm; Methicillin resistant S. aureus; Temporin L; β-leucine.
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