Rigidification of the <i>Escherichia coli</i> cytoplasm by the human antimicrobial peptide LL-37 revealed by superresolution fluorescence microscopy.
Zhu. Yanyu Y; Mohapatra. Sonisilpa S; Weisshaar. James C JC
Key Findings
- LL‑37 penetrates the bacterial membrane within seconds and reaches a very high intracellular concentration (~90 mM).
- The peptide binds to DNA and ribosomes, creating a dense network of electrostatic links that rigidifies the cytoplasm on a ~30 nm scale.
- Both DNA motion and ribosome diffusion are dramatically reduced, leading to irreversible bacterial growth arrest.
Practical Outcomes
- For those interested in antimicrobial peptide design, the work suggests that effective killing of gram‑negative bacteria requires two features: easy entry through the outer membrane and the ability to lock up the cell’s internal polymers by electrostatic binding. While not a direct protocol for human use, it highlights why high‑dose, positively charged peptides can be powerful antibiotics and why resistance develops slowly.
Summary
The study shows that the human antimicrobial peptide LL‑37 can quickly enter E. coli bacteria, stick to the DNA and ribosomes, and make the cell’s interior stiff and immobile. This stops the bacteria from growing and they never recover, even after washing away excess peptide.
Abstract
Superresolution, single-particle tracking reveals effects of the cationic antimicrobial peptide LL-37 on the <i>Escherichia coli</i> cytoplasm. Seconds after LL-37 penetrates the cytoplasmic membrane, the chromosomal DNA becomes rigidified on a length scale of ∼30 nm, evidenced by the loss of jiggling motion of specific DNA markers. The diffusive motion of a subset of ribosomes is also frozen. The mean diffusion coefficients of the DNA-binding protein HU and the nonendogenous protein Kaede decrease twofold. Roughly 10<sup>8</sup> LL-37 copies flood the cell (mean concentration ∼90 mM). Much of the LL-37 remains bound within the cell after extensive rinsing with fresh growth medium. Growth never recovers. The results suggest that the high concentration of adsorbed polycationic peptides forms a dense network of noncovalent, electrostatic linkages within the chromosomal DNA and among 70S-polysomes. The bacterial cytoplasm comprises a concentrated collection of biopolymers that are predominantly polyanionic (e.g., DNA, ribosomes, RNA, and most globular proteins). In normal cells, this provides a kind of electrostatic lubrication, enabling facile diffusion despite high biopolymer volume fraction. However, this same polyanionic nature renders the cytoplasm susceptible to massive adsorption of polycationic agents once penetration of the membranes occurs. If this phenomenon proves widespread across cationic agents and bacterial species, it will help explain why resistance to antimicrobial peptides develops only slowly. The results suggest two design criteria for polycationic peptides that efficiently kill gram-negative bacteria: facile penetration of the outer membrane and the ability to alter the cytoplasm by electrostatically linking double-stranded DNA and 70S-polysomes.
Study Information
pubmed
2018
2018-12-31T00:00:00.000Z
10.1073/pnas.1814924116
72
75