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.

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.

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

Bacteriocins produced by lactic acid bacteria (LAB-bacteriocins) may serve as alternatives for aging antibiotics. LAB-bacteriocins can be used alone, or in some cases as potentiating agents to treat bacterial infections. This approach could meet the different calls and politics, which aim to reduce the use of traditional antibiotics and develop novel therapeutic options. Considering the clinical applications of LAB-bacteriocins as a reasonable and desirable therapeutic approach, it is therefore important to assess the advances achieved in understanding their modes of action, and the resistance mechanisms developed by the producing bacteria to their own bacteriocins. Most LAB-bacteriocins act by disturbing the cytoplasmic membrane through forming pores, or by cell wall degradation. Nevertheless, some of these peptides still have unknown modes of action, especially those that are active against Gram-negative bacteria. Regarding immunity, most bacteriocin-producing strains have an immunity mechanism involving an immunity protein and a dedicated ABC transporter system. However, these immunity mechanisms vary from one bacteriocin to another.

Expression of five class II bacteriocins with activity against Escherichia coli in Lacticaseibacillus paracasei CNCM I-5369, and in a heterologous host.

Five open reading frames viz orf010, orf12, orf023, orf030 and orf038 coding class II bacteriocins in Lacticaseibacillus paracasei CNCM I-5369 strain previously isolated from an Algerian dairy product, were found to be expressed after 24 h of growth. The strain has also shown anti-E. coli activity in a narrow pH range between 4.5 and 5. Then, expression and purification of these bacteriocins was conducted in the heterologous host E. coli. This strategy enabled us to purify the peptide encoded by orf030 in large quantities, in contrast to other peptides that were produced but required to be released from the insoluble fraction following 4 M urea and desalting treatments. All peptides heterologously produced were characterized by MALDI TOF Mass spectrometry and successfully tested for their anti-E. coli activity. Furthermore, in silico transcriptional analysis was determined by Findterm tool and with Bagel4 software permitted to locate potential promoters and co-transcription events.

Lacticaseicin 30 and Colistin as a Promising Antibiotic Formulation against Gram-Negative ß-Lactamase-Producing Strains and Colistin-Resistant Strains.

Antimicrobial resistance is a global health concern across the world and it is foreseen to swell if no actions are taken now. To help curbing this well announced crisis different strategies are announced, and these include the use of antimicrobial peptides (AMP), which are remarkable molecules known for their killing activities towards pathogenic bacteria. Bacteriocins are ribosomally synthesized AMP produced by almost all prokaryotic lineages. Bacteriocins, unlike antibiotics, offer a set of advantages in terms of cytotoxicity towards eukaryotic cells, their mode of action, cross-resistance and impact of microbiota content. Most known bacteriocins are produced by Gram-positive bacteria, and specifically by lactic acid bacteria (LAB). LAB-bacteriocins were steadily reported and characterized for their activity against genetically related Gram-positive bacteria, and seldom against Gram-negative bacteria. The aim of this study is to show that lacticaseicin 30, which is one of the bacteriocins produced by Lacticaseibacillus paracasei CNCM I-5369, is active against Gram-negative clinical strains (Salmonella enterica Enteritidis H10, S. enterica Typhimurium H97, Enterobacter cloacae H51, Escherichia coli H45, E. coli H51, E. coli H66, Klebsiella oxytoca H40, K. pneumoniae H71, K. variicola H77, K. pneumoniae H79, K. pneumoniae H79), whereas antibiotics failed. In addition, lacticaseicin 30 and colistin enabled synergistic interactions towards the aforementioned target Gram-negative clinical strains. Further, the combinations of lacticaseicin 30 and colistin prompted a drastic downregulation of mcr-1 and mcr-9 genes, which are associated with the colistin resistance phenotypes of these clinical strains. This report shows that lacticaseicin 30 is active against Gram-negative clinical strains carrying a rainbow of mcr genes, and the combination of these antimicrobials constitutes a promising therapeutic option that needs to be further exploited.