Potency of all-D amino acid antimicrobial peptides derived from the bovine rumen microbiome on tuberculous and non-tuberculous mycobacteria.

Despite the availability of antibiotics, tuberculosis (TB), caused by , was once again declared the world's leading cause of death from a single infectious agent in 2023. Furthermore, the rising prevalence of drug-resistant strains of , coupled with the limitations of existing therapeutics, underscores the urgent need for new antimicrobial agents that act through different mechanisms, thereby providing novel therapeutic options. From this perspective, antimicrobial peptides (AMPs) derived from the bovine rumen microbiome have shown promise against many resistant pathogens and may therefore offer a promising alternative against TB. Here, we evaluated the efficacy of AMPs from bovine rumen microbiome, namely the & and as well as their amino acid enantiomers, against non-tuberculous ( and and tuberculous ( BCG, ) mycobacteria. In particular, their antimycobacterial activity was assessed against extracellularly and intracellularly replicating H37Rv pathogenic strain. Their innocuity was further studied by determining their respective cytotoxicity against human cell lines and hemolytic activity on human erythrocytes. Finally, their mechanism of action was investigated by a membrane permeabilization assay and a lipid insertion assay surface pressure measurement. Although enantiomers showed increased cytotoxicity to human cell lines, they still offer a good therapeutic window with improved activity compared to their L-form counterparts, especially and which emerged as the best growth inhibitors of all mycobacteria. Remarkably, the enantiomers also demonstrated activity against intramacrophagic replicating H37Rv, with very limited toxicity towards human cells and no hemolytic activity at their respective minimum inhibitory concentration. Membrane permeabilization and monolayer lipid insertion assays suggested that these peptides mostly act by insertion into the mycobacterial membrane resulting in a rapid membranolytic effect. These findings highlight the potential of the enantiomers of Lynronne peptides, as attractive candidates for the development of new anti-TB drugs. Their effective antibacterial properties combined with low toxicity underscore and as building blocks for the development of promising alternatives to conventional antibiotics in the treatment of mycobacterial infections, particularly against .