Mechanisms of action of escapin, a bactericidal agent in the ink secretion of the sea hare Aplysia californica: rapid and long-lasting DNA condensation and involvement of the OxyR-regulated oxidative stress pathway.
The marine snail Aplysia californica produces escapin, an L-amino acid oxidase, in its defensive ink. Escapin uses L-lysine to produce diverse products called escapin intermediate products of L-lysine (EIP-K), including α-amino-ε-caproic acid, Δ¹-piperidine-2-carboxylic acid, and Δ²-piperidine-2-carboxylic acid. EIP-K and H₂O₂ together, but neither alone, is a powerful bactericide. Here, we report bactericidal mechanisms of escapin products on Escherichia coli. We show that EIP-K and H₂O₂ together cause rapid and long-lasting DNA condensation: 2-min treatment causes significant DNA condensation and killing, and 10-min treatment causes maximal effect, lasting at least 70 h. We isolated two mutants resistant to EIP-K plus H₂O₂, both having a single missense mutation in the oxidation regulatory gene, oxyR. A complementation assay showed that the mutated gene, oxyR(A233V), renders resistance to EIP-K plus H₂O₂, and a gene dosage effect leads to reduction of resistance for strains carrying wild-type oxyR. Temperature stress with EIP-K does not produce the bactericidal effect, suggesting the effect is due to a specific response to oxidative stress. The null mutant for any single DNA-binding protein--Dps, H-NS, Hup, Him, or MukB--was not resistant to EIP-K plus H₂O₂, suggesting that no single DNA-binding protein is necessary to mediate this bactericidal effect, but allowing for the possibility that EIP-K plus H₂O₂ could function through a combination of DNA-binding proteins. The bactericidal effect of EIP-K plus H₂O₂ was eliminated by the ferrous ion chelator 1,10-phenanthroline, and it was reduced by the hydroxyl radical scavenger thiourea, suggesting hydroxyl radicals mediate the effects of EIP-K plus H₂O₂.
Department of Biology and Neuroscience Institute, Georgia State University, Atlanta, Georgia, USA.
Ko-Chun
KC
Ko
Phang C
PC
Tai
Charles D
CD
Derby
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001048:Aplysia / Q000737:chemistry*; D001419:Bacteria / Q000187:drug effects; D004396:Coloring Agents; D004269:DNA, Bacterial / Q000187:drug effects*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000494:pharmacology; D050830:L-Amino Acid Oxidase / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense / Q000235:genetics / Q000502:physiology; D018384:Oxidative Stress / Q000187:drug effects*; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects
D000900:Anti-Bacterial Agents; D004396:Coloring Agents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D016166:Free Radical Scavengers; D007502:Iron Chelating Agents; D012097:Repressor Proteins; C499299:oxyR protein, E coli; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D050830:L-Amino Acid Oxidase; C573011:escapin, Aplysia californica