Title
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
Author
Désiré Madi-Moussa, Barbara Deracinois, Radja Teiar, Yanyan Li, Marius Mihasan, Christophe Flahaut, Sylvie Rebuffat, Françoise Coucheney, and Djamel Drider
Year
2022
Journal
Pharmaceutics 2022, 14, 1921
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.
Full Article
Instrument
JASCO 810 Spectrophotometer
Keywords
Escherichia coli; Antimicrobial activity; Structure-activity relationship; Helical conformation