Enhanced Silkworm Cecropin B Antimicrobial Activity against Pseudomonas aeruginosa from Single Amino Acid Variation

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen causing severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis. Several clinically isolated P. aeruginosa strains were found to be resistant to three or more antimicrobial classes indicating the importance of identifying new antimicrobials active against this pathogen. Here, we characterized the antimicrobial activity and the action mechanisms against P. aeruginosa of two natural isoforms of the antimicrobial peptide cecropin B, both isolated from the silkworm Bombyx mori. These cecropin B isoforms differ in a single amino acid substitution within the active portion of the peptide, so that the glutamic acid of the E53 CecB variant is replaced by a glutamine in the Q53 CecB isoform. Both peptides showed a high antimicrobial and membranolytic activity against P. aeruginosa, with Q53 CecB displaying greater activity compared with the E53 CecB isoform. Biophysical analyses, live-cell NMR, and molecular-dynamic-simulation studies indicated that both peptides might act as membrane-interacting elements, which can disrupt outer-membrane organization, facilitating their translocation toward the inner membrane of the bacterial cell. Our data also suggest that the amino acid variation of the Q53 CecB isoform represents a critical factor in stabilizing the hydrophobic segment that interacts with the bacterial membrane, determining the highest antimicrobial activity of the whole peptide. Its high stability to pH and temperature variations, tolerance to high salt concentrations, and low toxicity against human cells make Q53 CecB a promising candidate in the development of CecB-derived compounds against P. aeruginosa.

Insect Cecropins, Antimicrobial Peptides with Potential Therapeutic Applications

“…It has been demonstrated that the antimicrobial activity of Cec AMPs relies on the structure they assume in the presence of bacterial cells. Circular dichroism analyses showed that in aqueous solution, Cecs have a random coiled structure but adopt α-helical conformations upon interaction with microbial membranes, where they exert a lytic effect [53,58,84,86]. Although some aspects remain unclear, it is currently accepted that Cec peptides do not interact with specific receptors but initially associate with the bacterial membrane along the axes of the α-helical domains parallel to the lipid bilayer surface.…”

“…Finally, in a study evaluating the interaction between different B. mori natural Cec B variants and live Gram-negative Pseudomonas aeruginosa , it was suggested that Cecs might first affect the outer bacterial membrane, enabling the translocation of the peptide to the inner membrane, resulting in the disorganization of both lipid bilayers [53].…”

“…Several natural Cecs and synthetic derivatives have shown a high stability to heat treatments and/or pH variations (e.g., [53,98,107]) . In addition, they usually maintained their antimicrobial activity in complex biological fluids, mimicked in vitro by using high concentrations of serum, as well as in the presence of elevated levels of divalent cations such as Ca 2+ and Mg 2+ , which show 1–2 and 0.5 mM concentrations in human saliva, respectively, and might reduce or inhibit AMP effectiveness (e.g., [37,53,107,128]).…”

“…Several natural Cecs and synthetic derivatives have shown a high stability to heat treatments and/or pH variations (e.g., [53,98,107]) . In addition, they usually maintained their antimicrobial activity in complex biological fluids, mimicked in vitro by using high concentrations of serum, as well as in the presence of elevated levels of divalent cations such as Ca 2+ and Mg 2+ , which show 1–2 and 0.5 mM concentrations in human saliva, respectively, and might reduce or inhibit AMP effectiveness (e.g., [37,53,107,128]). Similarly, natural Cecs and Cec-analogs were also active when analyzed in the presence of high concentrations of Na + , typical of airway surface fluids from patients affected by cystic fibrosis, who often suffer lung infections from bacteria such as P. aeruginosa , A. baumanni , or S. aureus (e.g., [49,53,98,102,129]).…”

Emerging therapies against infections with Pseudomonas aeruginosa

“…Murepavadin is a specific weapon against P. aeruginosa , which sets it apart from the large pipeline of natural and synthetic antimicrobial peptides that act against multiple taxa, including P. aeruginosa . Several novel peptides with broad antimicrobial activity—for example, DGL13K 49 , Mel4 50 , melimine 50 , cecropin B 51 , LBP-2 52, 53 , Pse-T2 54 , 6K-F17 55 , MDP1 56 , and MDP2 56 —have recently been described.…”