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 Mycobacterium tuberculosis, 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 M. tuberculosis, 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 Lynronne 1, 2 & 3 and P15s as well as their all-D amino acid enantiomers, against non-tuberculous (M. abscessus, M. marinum and M. smegmatis) and tuberculous (M. bovis BCG, M. tuberculosis) mycobacteria. In particular, their antimycobacterial activity was assessed against extracellularly and intracellularly replicating M. tuberculosis 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 via surface pressure measurement. Although all-D 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 Lynronne 2D_all and P15sD_all which emerged as the best growth inhibitors of all mycobacteria. Remarkably, the all-D enantiomers also demonstrated activity against intramacrophagic replicating M. tuberculosis 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 all-D enantiomers of Lynronne peptides, as attractive candidates for the development of new anti-TB drugs. Their effective antibacterial properties combined with low toxicity underscore Lynronne 2D_all and P15sD_all as building blocks for the development of promising alternatives to conventional antibiotics in the treatment of mycobacterial infections, particularly against M. tuberculosis.