Elucidation of the Antimycobacterial Activity of D-Form Human Lactoferricin 1–11 (D-Form hLF 1–11) Against Mycobacterium smegmatis Through Proteomics and Imaging Analysis

Background/Objectives: Tuberculosis (TB), caused by Mycobacterium tuberculosis complex, remains a major global health challenge. Recently, D-enantiomer of human lactoferricin 1–11 (D-form hLF 1–11), a short peptide derived from the N-terminal region of lactoferrin, has demonstrated potent antimycobacterial activity. However, its direct mechanism of action has not yet been elucidated. Methods & Results: In the present study, M. smegmatis was employed as a model organism to investigate the mechanism underlying D-form hLF 1–11 activity. Initially, the minimum inhibitory concentration (MIC) was determined and the results revealed growth inhibition at 400 µg/mL. Live/dead fluorescence staining demonstrated mycobactericidal activity, as indicated by increased propidium iodide (PI) uptake relative to the untreated control. Scanning electron microscopy and high-resolution fluorescence microscopy revealed membrane disruption and substantial morphological deformation, along with a time-dependent accumulation of the peptide at the membrane and inside the cells. Furthermore, label-free quantitative proteomic analysis of peptide-treated cells revealed extensive metabolic alterations in carbon metabolism, acetyl-CoA-dependent lipid biosynthesis, oxidative stress defense, translational machinery, and energy production systems. Conclusions: Collectively, these findings provide mechanistic insights into the antimycobacterial activity of D-form hLF 1–11 against M. smegmatis.