Structural and functional characterization of mycobactericidal ubiquitin-derived peptides in model and bacterial...

Area of Science:

• Biochemistry
• Microbiology
• Cell Biology

Background:

• Macrophages utilize bactericidal ubiquitin-derived peptides (Ub-peptides) to eliminate mycobacteria within lysosomes.
• Understanding the structural and functional interactions of Ub-peptides with mycobacteria is crucial for elucidating host defense mechanisms.

Purpose of the Study:

• To characterize the structure and function of the bactericidal Ub-peptide Ub2 in interaction with mycobacteria.
• To investigate the mechanism by which Ub-peptides exert their mycobactericidal effects on bacterial membranes.

Main Methods:

• Utilized sodium dodecyl sulfate micelles and phospholipid vesicles to study Ub2 conformation and stability.
• Employed point mutations to assess the impact of peptide charge on bactericidal activity.
• Applied differential scanning calorimetry to analyze Ub2 interaction with model membranes.
• Investigated Ub2 effects on intact mycobacteria, including membrane integrity, transmembrane potential, and localization.

Main Results:

• Ub2 adopts a β-sheet conformation dependent on its primary sequence when interacting with lipid environments.
• Reduced net charge in Ub2 mutants significantly diminished its bactericidal activity.
• Ub2 inserts into and perturbs phospholipid vesicles, disrupts the mycobacterial membrane integrity, and equilibrates transmembrane potential.
• Ub2 was found within both the membrane and cytoplasm of treated mycobacteria.

Conclusions:

• The study elucidates the structural basis for Ub-peptide bactericidal activity against mycobacteria.
• Ub-peptides function by directly disrupting the mycobacterial membrane, highlighting their role in innate immunity.
• Identified additional bactericidal Ub-peptides, expanding the understanding of this class of antimicrobial agents.