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Published on: June 29, 2021
Oxidative stress induced in E. coli by the human antimicrobial peptide LL-37
Heejun Choi1, Zhilin Yang1, James C Weisshaar1,2
1Department of Chemistry, University of Wisconsin-Madison, Madison, WI, United States of America.
Abstract:
Antimicrobial peptides (AMPs) are thought to kill bacterial cells by permeabilizing their membranes. However, some antimicrobial peptides inhibit E. coli growth more efficiently in aerobic than in anaerobic conditions. In the attack of the human cathelicidin LL-37 on E. coli, real-time, single-cell fluorescence imaging reveals the timing of membrane permeabilization and the onset of oxidative stress. For cells growing aerobically, a CellROX Green assay indicates that LL-37 induces rapid formation of oxidative species after entry into the periplasm, but before permeabilization of the cytoplasmic membrane (CM). A cytoplasmic Amplex Red assay signals a subsequent burst of oxidative species, most likely hydrogen peroxide, shortly after permeabilization of the CM. These signals are much stronger in the presence of oxygen, a functional electron transport chain, and a large proton motive force (PMF). They are much weaker in cells growing anaerobically, by either fermentation or anaerobic respiration. In aerobic growth, the oxidative signals are attenuated in a cytochrome oxidase-bd deletion mutant, but not in a -bo3 deletion mutant, suggesting a specific effect of LL-37 on the electron transport chain. The AMPs melittin and LL-37 induce strong oxidative signals and exhibit O2-sensitive MICs, while the AMPs indolicidin and cecropin A do not. These results suggest that AMP activity in different tissues may be tuned according to the local oxygen level. This may be significant for control of opportunistic pathogens while enabling growth of commensal bacteria.
Insights
Antimicrobial peptides like LL-37 induce oxidative stress in E. coli, with activity dependent on oxygen levels. This suggests oxygen-tuned antimicrobial peptide therapies could target pathogens while sparing beneficial bacteria.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Antimicrobial peptides (AMPs) are crucial for innate immunity.
- The mechanism of AMPs, particularly their interaction with bacterial membranes and metabolic state, is not fully understood.
- Some AMPs show differential efficacy under varying oxygen conditions.
Purpose of the Study:
- To investigate the mechanism of action of the antimicrobial peptide LL-37 on E. coli.
- To determine the role of oxygen and cellular respiration in LL-37 activity.
- To elucidate the relationship between oxidative stress, membrane permeabilization, and AMP efficacy.
Main Methods:
- Real-time, single-cell fluorescence imaging.
- CellROX Green and Amplex Red assays to detect oxidative species.
- Growth assays under aerobic and anaerobic conditions.
- Analysis of E. coli mutants with deletions in specific cytochromes.
Main Results:
- LL-37 induces oxidative stress in E. coli periplasm before cytoplasmic membrane permeabilization.
- Oxidative stress signals are significantly enhanced by oxygen, a functional electron transport chain, and proton motive force.
- LL-37's effect on oxidative stress is linked to the cytochrome oxidase-bd complex.
- AMPs LL-37 and melittin exhibit oxygen-sensitive minimum inhibitory concentrations (MICs), unlike indolicidin and cecropin A.
Conclusions:
- LL-37's antimicrobial activity is potentiated by aerobic conditions through induction of oxidative stress.
- The electron transport chain plays a critical role in LL-37-mediated oxidative damage.
- Oxygen levels can modulate AMP efficacy, suggesting potential for targeted therapeutic strategies.
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