Structure of Lacticaseicin 30 and Its Engineered Variants Revealed an Interplay between the N-Terminal and C-Terminal Regions in the Activity against Gram-Negative Bacteria

Abstract

Lacticaseicin 30 is one of the five bacteriocins produced by the Gram-positive Lacticaseibacillus paracasei CNCM I-5369. This 111 amino acid bacteriocin is noteworthy for being active against Gram-negative bacilli including Escherichia coli strains resistant to colistin. Prediction of the lacticaseicin 30 structure using the Alphafold2 pipeline revealed a largely helical structure including five helix segments, which was confirmed by circular dichroism. To identify the structural requirements of the lacticaseicin 30 activity directed against Gram-negative bacilli, a series of variants, either shortened or containing point mutations, was heterologously produced in Escherichia coli and assayed for their antibacterial activity against a panel of target strains including Gram-negative bacteria and the Gram-positive Listeria innocua. Lacticaseicin 30 variants comprising either the N-terminal region (amino acids 1 to 39) or the central and C-terminal regions (amino acids 40 to 111) were prepared. Furthermore, mutations were introduced by site-directed mutagenesis to obtain ten bacteriocin variants E6G, T7P, E32G, T33P, T52P, D57G, A74P, Y78S, Y93S and A97P. Compared to lacticaseicin 30, the anti-Gram-negative activity of the N-terminal peptide and variants E32G, T33P and D57G remained almost unchanged, while that of the C-terminal peptide and variants E6G, T7P, T52P, A74P, Y78S, Y93S and A97P was significantly altered. Finally, the N-terminal region was further shortened to keep only the first 20 amino acid part that was predicted to include the first helix. The anti-Gram-negative activity of this truncated peptide was completely abolished. Overall, this study shows that activity of lacticaseicin 30, one of the rare Gram-positive bacteriocins inhibiting Gram-negative bacteria, requires at least two helices in the N-terminal region and that the C-terminal region carries amino acids playing a role in modulation of the activity. Taken together, these data will help to design forthcoming variants of lacticaseicin 30 as promising therapeutic agents to treat infections caused by Gram-negative bacilli.

The Editorial Position on ‘Recent Advances in Multifunctional Antimicrobial Peptides as Preclinical Therapeutic Studies and Clinical Future Applications’

“…In addition, ten mutations were introduced to obtain several engineered variants. In comparison to the original AMP, they observed no differences for the N-terminal peptide and variants E32G, T33P, and D57G preserving the antibacterial effects, but demonstrated a reduced antimicrobial activity against Gram-negative bacteria for the C-terminal peptide and the E6G, T7P, T52P, A74P, Y78S, Y93S, and A97P variants [20]. Finally, the authors synthesized an N terminal domain without the first 20 amino acids in the first helix, showing that this truncated peptide did not have antimicrobial activity [20].…”

“…In comparison to the original AMP, they observed no differences for the N-terminal peptide and variants E32G, T33P, and D57G preserving the antibacterial effects, but demonstrated a reduced antimicrobial activity against Gram-negative bacteria for the C-terminal peptide and the E6G, T7P, T52P, A74P, Y78S, Y93S, and A97P variants [20]. Finally, the authors synthesized an N terminal domain without the first 20 amino acids in the first helix, showing that this truncated peptide did not have antimicrobial activity [20]. Another approach used by Etayash et al involved chemically modifying AMP IDR1018 by covalently linking it with a short-chain PEG (PEG6) and a glucose moiety (N-acetyl glucosamine: GlcNAc) and assessing the impact of this chemical modification on the peptide's antimicrobial and biological properties.…”

“…Also, different studies are presented that dealt with improving antimicrobial activities and reducing their adverse effects using structure-function relationship studies, engineered variations, chemical modifications, and nanoparticle incorporation [20][21][22]. To identify the structural requirements of AMP activity directed against Gram-negative bacteria, Madi-Moussa and collaborators heterologously produced a series of variants of Lacticaseicin 30 including the N-terminal (amino acids 1 to 39) and central and C-terminal (amino acids 40 to 111) domains in E. coli and assayed their antibacterial activities.…”

Bacteriocins and Bacteriophages as Dual Biological Players for Food Safety Applications

“…In recent years, LAB-bacteriocins have become of increased interest because of their GRAS status [17] and because of their use as biopreservative agents of choice in the food industry to replace chemical agents [18]. Except in some cases of Gram-negative bacteria inhibition [19][20][21][22][23], LAB-bacteriocins are most often active against Gram-positive target strains, which are phylogenetically related to the producing strains [8]. In fact, the absence of activity across Gram-negative bacteria could be explained by the outer membrane, which impedes their access to the inner membrane, where they exert their activity [24].…”