Bacteria´s inner and outer membrane permeabilization by de novo antimicrobial peptide P1 through spectroscopic methods

ESPECHE, JUAN CARLOS; MATURANA, PATRICIA; MARTINEZ MELINA; MAFFIA PAULO; HOLLMANN AXEL

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofisica; 2019

Sociedad Argentina de Biofisica

n the present work, we evaluate the interaction of the antimicrobial peptide 1 (P1) with bacterial membranes as well as its ability to permeabilize the inner and outer membrane of Gram positive and Gram negative bacteria. P1 is a cationic peptide with 21 amino acids (WPKWWKWKRRWGRKKAKKRRG), designed identifying short putative active regions from AMP databases. Its antimicrobial activity was previously tested in Staphylococcus aureus and Escherichia coli strains having gotten a MIC value of 32 and 128 respectively.Inicially, we evaluated the interaction of P1 with bacterial envelope by using Zeta Potential in suspensions containing the mentioned strains. In both cases, zeta potential became less negative after peptide incubation confirming the ability of the peptides to bound into the cell envelope. However, the effect becomes more noticeable in S. aureus. in a good agridemnt with MIC dataNext, in order to determine the ability of the peptide to permeabilize the outer membrane of these bacteria, fluorescence methods were used. Results have shown that P1 is able to disrupt both strains? outer membranes reaching higher values and faster kinetics in the case of E. coli. In the case of S. aureus kinetics are slightly slower.Inner membrane permeabilization was assessed only for E. coli using absorbance. It was found that the kinetics for the disruption of this membrane is reached successfully by the peptide. In this case, it required longer time lapses for the permeabilization to happen.From the results we got, we can conclude that the peptide is able to permeabilize both outer and inner membrane of E. coli, reaching the first effect in much shorter times (from seconds to a pair of minutes) than the second (1 hour approximately). This difference could be due to the fact that the peptide needs to permeabilize the outer membrane first in order to reach the inner. Differences observed in disruption speed for the membrane in S. aureus (Gram positive bacteria) could be explained since this bacteria have a thicker cell wall, that might hinder the reach of the peptide to the membrane. Furthermore, a relation between concentration of peptide and membrane damage was stablished for both membranes.