Optimized LL-37-Derived Peptides Exhibit Antitubercular Activity, Induce Membrane Disruption, and P-Type ATPase Transcriptional Responses in Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major cause of morbidity and mortality worldwide, particularly due to the emergence of drug-resistant strains. Membrane-active antimicrobial peptides (AMPs) represent attractive therapeutic candidates because they target bacterial envelope integrity and disrupt essential cellular processes. We evaluated two rationally designed LL-37-derived peptides: a truncated C-terminally amidated analog (LL37-1) and a modified variant incorporating N-terminal acetylation and a single D-amino acid substitution (D-LL37). Dose–response analysis demonstrated that D-LL37 exhibited greater antimycobacterial potency, with lower inhibitory concentrations of 90% (IC90) and 50% (IC50) values (18.40 ± 0.39 μM and 10.11 ± 0.60 μM, respectively) compared with LL37-1 (25.44 ± 0.36 μM and 15.45 ± 1.40 μM). Fluorescence-based permeability assays revealed partial membrane disruption (36% and 44% at IC90 for LL37-1 and D-LL37, respectively), which was supported by ultrastructural alterations observed by scanning electron microscopy, including bacillary shortening, rough surface formation, cell clusters, and the presence of cellular debris, all of which are consistent with membrane damage. RT-qPCR analysis demonstrated significant upregulation of the P-type ATPase genes ctpF, ctpA, and ctpH following D-LL37 exposure. Collectively, these findings indicate that optimized LL-37-derived peptides exert antitubercular activity associated with envelope perturbation and coordinated activation of ion transport-related stress responses.