Lichenicidin

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that arise as an alternative to the traditional antibiotics. Lichenicidin is active against clinically relevant bacteria and it was the first lantibiotic to be fully produced in vivo in the Gram-negative host Escherichia coli. Here, we present the results of a library of lichenicidin mutants, in which the mutations were generated based on the extensive bibliographical search available for other lantibiotics. The antibacterial activity of two-peptide lantibiotics, as is lichenicidin, requires the synergistic activity of two peptides. We established a method that allows screening for bioactivity which does not require the purification of the complementary peptide. It is an inexpensive, fast and user-friendly method that can be scaled up to screen large libraries of bioengineered two-peptide lantibiotics. The applied system is reliable and robust because, in general, the results obtained corroborate structure-activity relationship studies carried out for other lantibiotics.

Lantibiotics are promising candidates to address the worldwide problem of antibiotic resistance. They belong to a class of natural compounds exhibiting strong activity against clinically relevant Gram-positive bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Lichenicidin is a class II two-peptide lantibiotic. The presence of the two mature peptides, Bliα and Bliβ, is necessary for full activity against target bacteria. This work aims at clarifying the synergistic activity of both peptides in their interaction with the target membranes. The effect of lichenicidin was tested against S. aureus cells and large unilamellar vesicles. Lichenicidin increases the net surface charge of S. aureus, as shown by zeta-potential measurements, without reaching electroneutralization. In addition, lichenicidin causes cell surface perturbations that culminate in the leakage of its internal contents, as observed by atomic force microscopy. Bliα seems to have low affinity for S. aureus, however, it contributes to increase the affinity of Bliβ, because together they present higher affinity than separately. In contrast, Bliα seems to provide an anchoring site for lichenicidin in lipid II-containing membranes. Interestingly, Bliβ alone can induce high levels of membrane leakage, but this effect appears to be faster in the presence of Bliα. Based on this information, we propose a mechanism of action of lichenicidin.

Lantibiotics are a promising class of natural antimicrobial peptides. Lichenicidin is a two-peptide lantibiotic in which two mature peptides act synergistically to exhibit full bioactivity. Considering the two-peptide lantibiotics described so far, only cytolysin has been deeply characterized in terms of toxicity towards eukaryotic cells and it was found to be hemolytic and cytotoxic. This work aimed to improve the production of lichenicidin in vivo and characterize its antibacterial activity and toxicity against human cells. Peptides were purified and minimal inhibitory concentration (MIC) was determined against several strains; a time-kill assay was performed with Staphylococcus aureus. The hemolytic effect of lichenicidin was evaluated on blood samples from healthy donors and its toxicity towards human fibroblasts. The quantity of purified peptides was 1 mg/l Bliα and 0.4 mg/l Bliβ. MIC for methicillin-sensitive and resistant S. aureus (MSSA and MRSA) strains were 16-32 µg/ml and 64-128 µg/ml, respectively. At the MIC, lichenicidin took less than 3 h to eliminate MSSA, indicating a strong bactericidal effect. It induces cell lysis at the highest concentration, an effect that might be potentiated by Bliβ. Lichenicidin was not cytotoxic to human erythrocytes and fibroblasts. In this work, we evaluated the therapeutic potential of lichenicidin as a possible antimicrobial alternative.