Abstract Description

Amid the urgent need for new antibiotics, lantibiotics demonstrate promising therapeutic potential. Lantibiotics are peptides that, after ribosomal synthesis, undergo diverse post-translational modifications (PTMs), leading to dehydrated Ser and Thr residues and subsequent lanthionine/methyl-lanthionine formation. Lantibiotics provide an innovative alternative for cell growth inhibition of multidrug-resistant pathogens due to their dual mechanism of action (pore formation and inhibition of cell wall biosynthesis) with low resistance development. Since lantibiotics are mainly produced by Gram-positive organisms and are very effective against closely related Gram-positive bacteria, this study aimed to use genome mining and synthetic biology as a tool to design novel lantibiotics with high activity and specificity towards pathogenic Clostridium and Staphylococcus. Five lantibiotics from the genomes of clostridial species and Staphylococcus capitis (nisinJ) were identified using sequences of known peptides and bioinformatic tools, including NCBI-BLASTp and RiPPMiner. The well-studied lactococcal nisinA lantibiotic biosynthetic pathway was used to heterologously express the identified peptides using Escherichia coli BL21 (DE3). The signal peptide of nisinA was fused to all core peptide sequences to allow modification using the NisC cyclase and NisB dehydratase PTM enzymes, followed by production using an mCherry-fusion expression system. Five expression plasmids were constructed successfully, and transformed into E. coli cells, then tested for protein expression. Following purification using affinity chromatography and cleavage optimization for removal of the mCherry protein and the nisinA leader peptide, the lantibiotics were tested for antimicrobial activity using an agar overlay assay against various bacterial target species. Two out of the five putative clostridial peptides and nisinJ demonstrated antimicrobial activity. This study demonstrated the use of the E. coli mCherry-fusion system and the modification machinery of nisinA to produce novel and functional lantibiotic derivatives. The produced peptides will be used in future studies to analyse the extent of PTM’s and further screening for bioactivity against clinically relevant clostridial and staphylococcus antibiotic resistant species.