Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

Abstract

Plantacyclin B21AG is a circular bacteriocin produced by Lactiplantibacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from a food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 18 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed that Phe8Ala and Trp45Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. The Lys19Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the relative importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.

Functional production of clostridial circularin A in Lactococcus lactis NZ9000 and mutational analysis of its aromatic and cationic residues

“…Moreover, L. lactis , unlike E. faecalis , is a commonly chosen model organism ( Kunji et al, 2003 ) in both laboratory research and industrial applications for various reasons such as easy manipulation and well-known safety. Based on our constructed expression systems, mutagenesis studies using the two-plasmid system (Cir-A + ΔA) proved to be quite efficient and successful, although various challenges have been reported previously at different stages for heterologous production of circular bacteriocins, such as the difficulty of obtaining positive clones ( Kemperman et al, 2003a ; Gor et al, 2020 ) at the molecular cloning level, and the difficulty in achieving antimicrobial production from hosts of different genera at the peptide expression level ( Fernández et al, 2007 ; Kawai et al, 2009 ).…”

“…The cationic and aromatic residues are highly conserved among putative and experimentally confirmed circular bacteriocins ( Vezina et al, 2020 ), and have been demonstrated to play important roles in antimicrobial activity of plantacyclin B21AG ( Gor et al, 2020 ). In terms of the mechanism of the antibacterial activity, it has been reported previously that the aromatic residues (such as Trp) have a specific affinity for the region close to the lipid carbonyl and promote membrane permeation, while the cationic residues (such as Lys) have the affinity to the negative charged lipid phosphate region and are believed to interact with negatively charged cell membrane ( Braun and von Heijne, 1999 ; de Planque et al, 1999 ; Killian and von Heijne, 2000 ; Jiménez et al, 2005 ).…”

“…Among the various RiPP classes [for an overview see Arnison et al (2013) and Montalbán-López et al (2021) ], bacterial head-to-tail cyclized peptides, which are also referred to as circular bacteriocins, form a group characterized by N- to C-terminal ligation of the peptide backbone. Compared with their conventional linear counterparts, circular bacteriocins are highly stable in a broad range of temperatures and pH values, and the lack of free termini in circular bacteriocins decreases their proteolytic degradation by potential exopeptidases ( Borrero et al, 2017 ; Golneshin et al, 2020 ; Gor et al, 2020 ; Vezina et al, 2020 ). In addition, circular bacteriocins generally exhibit a broad antimicrobial spectrum, including activity against food-borne pathogens ( Nakamura et al, 2013 ; Gabrielsen et al, 2014 ; Grande Burgos et al, 2014 ) and clinically relevant multi-drug resistant pathogens ( van Heel et al, 2017 ).…”

“…Mutagenesis studies have been proven efficient in investigating the underlying mechanism. Up to now, extensive mutational analysis has only been performed for enterocin NKR-5-3B ( Perez et al, 2017 ); site-directed mutagenesis has been performed for enterocin AS-48 ( Sánchez-Hidalgo et al, 2008 , 2010 ; Cebrián et al, 2010 ), and for plantacyclin B21AG ( Gor et al, 2020 ). Expanding mutagenesis analyses to other known circular bacteriocins may help significantly in understanding the biosynthesis of circular bacteriocins ( Perez et al, 2018 ).…”

Current Knowledge of the Mode of Action and Immunity Mechanisms of LAB-Bacteriocins

“…N-terminal regions of circular bacteriocins carry several aromatic and/or hydrophobic amino acids suggesting that these residues play a key role in the biosynthesis and probably the antimicrobial activity [96]. Recently, a structural alignment study of different circular bacteriocins revealed the involvement of charged and aromatic conserved amino acids in antimicrobial activity [104]. Indeed, the authors showed that the substitution of phenylalanine and tryptophan residues by alanine by site-directed mutagenesis of the bacteriocin plantacyclin B21AG produced by Lactiplantibacillus plantarum B21 resulted in a strong reduction of the activity compared to the wild type.…”