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.

Keywords: Escherichia coli; antimicrobial activity; helical conformation; structure-activity relationship.

Figure 1

Mass spectrometry analyses of 6his-tag-depleted Ser-lacticaseicin 30 (average molecular mass 12,339.2 Da). (A) Linear positive MALDI-TOF mass spectrum illustrating the [M + H]⁺ and [M + 2H]²⁺ mass signals obtained. The measured MALDI-TOF-MS molecular mass was evaluated as 12,349.2 Da. (B) ESI mass spectrum and deconvoluted molecular mass (UniDec DScore: 54.16) revealing a molecular mass of 12,340 Da. (C) Peptide mapping of the 6his-tag-depleted Ser-lacticaseicin 30 after tryptic hydrolysis. Blue lines correspond to identified peptides along the lacticaseicin 30 sequence following the bioinformatic retreatment of RP-HPLC-MS/MS data using PEAKS Studio Xpro and the UniProtKB/Swiss-Prot-TrEMBL protein databases restricted to Lactobacillus (https://www.uniprot.org/, accessed on April 2022).

Figure 2

AlphaFold2 predicted structure of lacticaseicin 30 showing five helices (various shades of red) connected by coil regions (green). Five predicted H-bonds (blue) stabilize the position of the three helices.

Figure 3

(A) Circular dichroism spectra of lacticaseicin 30 at pH 7 (blue) and pH 5 (red) recorded in the absence or presence of 10 mM SDS micelles. (B) The secondary structure content (%) of lacticaseicin 30 at pH 7 and 5 was predicted using the BestSel Software.

Figure 4

Amino acid sequence and predicted three-dimensional structure of lacticaseicin 30. The predicted helical regions (helix 1 to helix 5) are underlined. Truncated forms of lacticaseicin 30 are shown by colored dotted lines (red: N-ter-Lacticaseicin 30, orange: N-ter-H1-Lacticaseicin 30 and blue: C-ter-Lacticaseicin 30). Mutated amino acids of variants are in red. Lacticaseicin 30 variants generated by directed mutagenesis are designated by a number between 1 and 10 (1: E6G, 2: T7P, 3: E32G, 4: T33P, 5: T52P, 6: D57G, 7: A74P, 8: Y78S, 9: Y93S and 10: A97P). Activities in the same range as native lacticaseicin 30 and total absence of activity are shown by + and −, respectively; * and “:” indicate successively a gradual decrease of activity.