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

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.

In vitro Susceptibility and Evaluation of Techniques for Understanding the Mode of Action of a Promising Non-antibiotic Citrus Fruit Extract Against Several Pathogens

“…The morphological similarity between these two structures suggests the possibility that blebs were the source of the microvesicles detected as free forms. Blebs seem to be a common bacteria response to antimicrobials, as previously described for S. enterica (Kumaresan et al, 2015), S. aureus (Carvajal-Rondanelli et al, 2018) and other Gram-negative and Gram-positive bacteria (Takenouchi and Nishino, 1991; Tagai et al, 2011; Ryu et al, 2014; Prudêncio et al, 2015; Zhang et al, 2016). On the other hand, in this study only the Gram-negative species collapsed when exposed to the product, suggesting a different response of the two kinds of bacterial envelopes to BIOCITRO ® .…”

Dissection of the Structural Features of a Fungicidal Antibody-Derived Peptide

“…T11F is a peptide that exhibits antimicrobial activity and that, in solution, exists in a dimeric form stabilised by the presence of a Cys residue at the N-terminus and by the regular alternation of charged and uncharged residues [6]. Alanine scanning is a standard and systematic approach to identify the contribution of amino acid residues to the peptide’s function and conformation [10,11]. Previous studies showed that alanine substitution of the positively charged residues caused a decrease in candidacidal activity, while a more dramatic reduction in activity resulted from cysteine replacement [6].…”

Identification of Antimicrobial Peptides from the Microalgae Tetraselmis suecica (Kylin) Butcher and Bactericidal Activity Improvement

“…Alanine substitution has been commonly used to probe the contribution of each residue in the structure and antimicrobial activity of peptides because the substitution of a side chain residue by methyl groups provides an effective strategy for detecting the side chains responsible for secondary structure and activity [21,43]. In this study, AQ-1766 and its alanine analogs showed similar secondary structures, with a slight tendency to form helical structures, but with distortions in the CD spectra probably due to the presence of the aromatic residues, in particular tryptophan, as previously described for other peptides [34,44].…”

“…Firstly, their antibacterial activity was higher than the one of the reference peptide (AQ-1766), particularly against Gram+ bacteria. This effect can be related to the increase of the peptide’s positive net charge, associated with the lysine side chain, which is able to interact with the negatively charged bacterial membranes [43,47]. Secondly, a notorious change in the CD spectra of the lysine analogs was observed with respect to the AQ-1766 peptide and their alanine counterparts, which also suggests the lysine influence in the secondary structure.…”