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Sci Rep
2014 Mar 12;4:4293. doi: 10.1038/srep04293.
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Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells.
Avitabile C
,
D'Andrea LD
,
Romanelli A
.
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Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.
Figure 1. Superimposition of CD spectra recorded at a fixed incubation time of the peptides (5 μM) with different amounts of E.coli cells in phosphate buffer (blue: 84 μL, green 112 μL, red: 140 μL; purple 168 μL; grey 196 μL).(a) magainin 2, 2 h incubation; (b) cecropin A, 2 h incubation; (c) magainin 2, 4 hours incubation, (d) cecropin A, 4 h incubation. Aliquotes of cells were withdrawn from suspension of cells at 2 OD. These spectra are representative of one set of experiments and are obtained after subtraction of the CD contribute of E.coli cells incubated for the same time as peptides in buffer.
Figure 2. Superimposition of the CD spectra obtained incubating E.coli cells at 0.1 OD with 5 μM peptides at different times.Panels a and b report spectra respectively for magainin 2 and cecropin A in a 2 hours time period; panels c and d show spectra of the peptides (magainin 2 panel c and cecropin A panel d) after incubation times of 2, 4 and 6 hours. These spectra are representative of one set of experiments and are obtained after subtraction of the CD contribute of E.coli cells incubated for the same time as peptides in buffer. The spectra of the peptides in buffer (without cells) are reported in panels c and d.
Figure 3. Variation of the CD signal at 222 nm vs time obtained subtracting the cell signal to the cells + peptides mixtures signal.E.coli cells at 0.1 OD were incubated with the peptides cecropin A (squares) and magainin 2 (circles) for 6 hours. Points are obtained as the mean (±SEM) of three experiments.
Figure 4. Superimposition of CD spectra recorded for magainin 2 (5 μM) (a) and cecropin A (5 μM) (b) at increasing E.coli LPS concentration, as indicated by the arrow.These spectra are representative of one set of experiments. Variation of the CD signal at 222 nm recorded within 6 hours for peptides (5 μM)/LPS mixtures at 1:1 molar ratio for magainin 2 (c) and cecropin A (d). The CD spectra of LPS were subtracted.
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