Deepening the study of the two-peptide lantibiotic lichenicidin: bioengineering, toxicity and mode of action

Many novel peptides have recently been discovered and characterized that have been shown to be effective against clinically relevant strains, including multi-drug resistant bacteria. Lantibiotics are presented as an alternative to the more traditional antibiotics. The present study aimed to clarify aspects of the biosynthesis, structure, mode of action and toxicity of lichenicidin. Lichenicidin is a two peptide (Bliα and Bliβ) lantibiotic produced by B. licheniformis with bioactivity against Staphylococcus aureus and Listeria monocytogenes. It was the first lantibiotic to be produced totally in vivo in Escherichia coli. This system was used to produce variants of Bliα and Bliβ.The production levels, bioactivity and structure of such variants was compared with that of the native peptides. Variants with slightly improved bioactivity were identified and should be further characterized. Bliα Glu26 residue was found to be important for bioactivity and could not be replaced by other residues, even if negatively charged. In general, Ser and Thr can be replaced with each other, even in the positions involved in ring formation. Regarding Bliβ biosynthesis, the hexapeptide sequence is essential to the proteolysis step and to ensure the specificity of the modifying enzyme. In addition Bliβ biosynthetic enzymes were used to produce and secrete other peptides (including non-lantibiotics) in E. coli, opening new perspectives for the biotechnological application of these enzymes. Lichenicidin production in E. coli was improved by replacing the original genetic determinants and regulatory regions of Bacillus with those from E. coli and by producing Bliα and Bliβ separately. Lichenicidin purification method was also optimized to ensure high yields of pure peptides. In this process, it was found that Bliβ production is limited by the proteolytic step that occurs in the extracellular environment. Regarding antimicrobial activity, lichenicidin minimal inhibitory concentration (MIC) was determined against methicillin-sensitive and methicillin-resistant S. aureus strains (MSSA and MRSA), being higher for the latter. Separately, Bliα and Bliβ have bioactivity, although Bliβ activity is higher than Bliα, both are inferior to their synergistic activity. Time-kill assays showed that, at the MIC, lichenicidin inhibits MSSA in less than 3 h. For higher concentrations, rapid killing occurs, suggesting a mode of action consistent with a pore formation mechanism of action. In lipid membrane models (LUV), Bliβ is able to induce leakage. Bliα has low affinity for S. aureus membranes, but it strongly binds to lipid II-containing LUV, contributing to accelerate cell lysis. In addition, Bliα and Bliβ stabilize each other when binding to S. aureus cells. Furthermore, lichenicidin is not toxic against human erythrocytes and fibroblasts. This work gathers new and relevant information on lichenicidin, thus contributing to a better assessment of its possible biotechnological and therapeutic applications.