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

Abstract

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.

Fig 1

DNA condensation in E. coli MC4100. (A) Light micrographs of DNA condensation under different treatments. (a) Control (ddH₂O, used as a solvent in the other treatments); (b) 3 mM H₂O₂; (c) 13.75 mM EIP-K; (d) 13.75 mM EIP-K plus 3 mM H₂O₂. Cells were stained with Hoechst to label DNA, and images were taken by simultaneously presenting UV and transmitted light. Scale bar, 5 μm. (B and C) Quantification of DNA condensation using the length of the major axis of the nucleoid (B) and the form factor, where 1.0 is a perfect circle and 0.0 is a straight line (C). The treatments in panels B and C were the same as in panel A. The values are means and standard errors of the mean (SEM), and the number of cells (n) used for each treatment is shown in the bar. The asterisks indicate that treatments are significantly different from the control. In panel B, there is a significant treatment effect (one-way ANOVA; F_[3,1731] = 389.17; P < 0.001), and post hoc Scheffé tests (α [level of significance] = 0.05) show that (H₂O₂) = (EIP-K) > (control) > (EIP-K plus H₂O₂). In panel C, there is a significant treatment effect (one-way ANOVA; F_[3,1731] = 355.01; P < 0.001), and post hoc Scheffé tests (α = 0.05) show that all treatments differ from each other, except the control and H₂O₂.

Fig 2

Effect of chloramphenicol, an inhibitor of protein synthesis, on DNA condensation in comparison to that of EIP-K plus H₂O₂. The treatments included control (ddH₂O, used as a solvent in the other treatments), 13.75 mM EIP-K plus 3 mM H₂O₂, and 100 μg/ml CAM. The values are means and SEM, and the number of cells (n) for each treatment is shown in the bar. There is a significant treatment effect for major axis length (one-way ANOVA; F_[2,1413] = 526.68; P < 0.001) and form factor (F_[2,1413] = 394.99; P < 0.001). Post hoc Scheffé tests (α = 0.05) show that all treatments differ from each other in major axis length, as well as in form factor. The asterisks indicate that the treatments are significantly different from the control.

Fig 3

Brief treatment with EIP-K plus H₂O₂ causes long-lasting DNA condensation in E. coli MC4100. The treatment was a 10-min incubation in either control (ddH₂O; open bars) or 13.75 mM EIP-K plus 3 mM H₂O₂ (shaded bars), followed by incubation in PBS buffer for either 1.5, 25, or 70 h. The values are means and SEM, and the number of cells (n) for each treatment is shown in the bar. For each treatment time, the control values are significantly greater than the EIP-K plus H₂O₂ values, as indicated by the asterisks (Students t test; P < 0.001).

Fig 4

Effect of time of treatment with 13.75 mM EIP-K plus 3 mM H₂O₂ on bactericidal activity (A) and DNA condensation (B an C) in E. coli MC4100. The values are means and standard errors of the mean for three experiments, each run in duplicate or triplicate. (A) There is a significant effect of treatment time with EIP-K plus H₂O₂ on the number of CFU (one-way ANOVA, F_[7,8] = 83.86; P < 0.0001), and post hoc Scheffé tests (α = 0.05) show that the untreated group (time = 0) is significantly different than all treated groups. (B and C) The negative control, “control 10 min,” is 10-min exposure to ddH₂O, and the number of cells (n) for each treatment is shown in the bars. One-way ANOVA for nucleoid length (B) shows a significant effect of treatment time (F_[5,2433] = 320.35; P < 0.001), and post hoc Scheffé tests (α = 0.05) show that the control group is significantly different than the other groups, as indicated by asterisks. One-way ANOVA for form factor (C) shows a significant effect of treatment time (F_[5,2433] = 394.99; P < 0.001), and post hoc Scheffé tests (α = 0.05) show that the control group has a nucleoid that is significantly rounder than the other groups, as indicated by asterisks.

Fig 5

Bactericidal effect of high concentrations of H₂O₂ with EIP-K on a resistant E. coli strain. White bars, control (untreated; time zero); light-gray bars, treated for 10 min with H₂O₂ at the indicated concentration; dark-gray bars, treated for 10 min with H₂O₂ at the indicated concentration plus 13.75 mM EIP-K. The values are means and standard errors of the mean for three experiments, each run in triplicate. Two-way ANOVA shows a significant treatment effect (F_[2,13] = 96.02; P = 0.0000001), a significant concentration effect (F_[2,13] = 57.15; P = 0.0000001), and a significant treatment-concentration interaction effect (F_[4,13] = 26.87; P = 0.000004). The asterisks indicate that the effect of EIP-K plus H₂O₂ on the resistant strain is significantly affected by the concentration of H₂O₂.

Fig 6

Complementation assay of E. coli MC4100 (A) and E. coli resistant strain 1 with plasmids containing the wild-type or mutant oxyR gene (B), using pET29a as the vector. The conditions were as follows: no vector (None), vector alone, vector plus parental oxyR (oxyR^wt), and vector plus the mutant oxyR gene [oxyR(A233V)]. Each bacterial type was treated with control (ddH₂O), 3 mM H₂O₂, and 13.75 mM EIP-K plus 3 mM H₂O₂. The values are means and SEM for four experiments, each run in duplicate. The asterisks indicate significant differences between cells without any plasmid (None) and any of the vector conditions under EIP-K-plus-H₂O₂ treatment. (A) Two-way ANOVA for the wild-type strains shows that the plasmid type effect is not significant (F_[3,19] = 2.57; P = 0.085), the treatment effect is significant (F_[3,19] = 91.01; P < 0.00000001), and the interaction effect is significant (F_[6,19] = 9.02; P < 0.0001). Given the significant interaction effect, we performed a one-way ANOVA for the EIP-K-plus-H₂O₂ treatment and found a significant effect (F_[3,7] = 46.64; P = 0.00005), and Scheffé post hoc tests show that the bactericidal effect of vector plus oxyR(A233V) is significantly less than that of vector plus wild-type oxyR or vector alone. (B) Two-way ANOVA for resistant E. coli strain RS1 shows a significant plasmid effect (F_[3,21] = 6.33; P = 0.0031), a significant treatment effect (F_[2,21] = 7.38; P = 0.0037), and a significant treatment-plasmid interaction effect (F_[6,21] = 13.85; P = 0.000002). A subsequent one-way ANOVA for EIP-K plus H₂O₂ shows a significant effect (F_[3,9] = 22.11; P = 0.00017), and Scheffé post hoc tests show that the bactericidal effect of vector–wild-type oxyR, but not vector-oxyR(A233V) or vector alone, is greater than that of the control.

Fig 7

Effect of adaptation to H₂O₂ on the bactericidal activity of H₂O₂ plus EIP-K in E. coli MC4100. Bacteria were pretreated with 100 μM H₂O₂ or the same amount of ddH₂O for 1 h, followed by a bactericidal assay using control (ddH₂O), 1 or 3 mM H₂O₂ alone, or 1 or 3 mM H₂O₂ plus 13.75 mM EIP-K. The values are means and SEM for one experiment run in duplicate. Two-way ANOVA shows a significant adaptation effect (F_[1,19] = 16.48; P = 0.003), a significant treatment effect (F_[2,9] = 16.25; P = 0.001), and a significant adaptation-treatment interaction effect (F_[2,9] = 16.21; P = 0.001). The asterisks indicate that H₂O₂ pretreatment reduces the bactericidal effect of H₂O₂ plus EIP-K.

Fig 8

Effects of null mutations of DNA- associated proteins (A) and a Dps null mutant strain (B) on bactericidal activity. (A) Treatment with 13.75 mM EIP-K plus 3 mM H₂O₂ and untreated (number of cells at time zero). The values are means and SEM for two experiments, each run in triplicate. NT3 is the parental strain for the Δhup, ΔhimA, Δhns, and ΔmukB mutants, and ZK126 is the parental strain for the Δdps mutant. Two-way ANOVA shows a significant treatment effect (F_[1,21] = 68.96; P = 0.0000001), a nonsignificant mutant effect (F_[5,21] = 1.27; P = 0.314), and a nonsignificant treatment-mutant interaction effect (F_[6,21] = 1.269; P = 0.313. The lack of asterisks indicates that the mutant strains showed effects similar to those of the wild type under EIP-K-plus-H₂O₂ treatment. (B) Various concentrations of H₂O₂, alone or with 13.75 mM EIP-K, and 13.75 mM EIP-K alone were tested on a Dps null mutant strain and compared with the wild-type (WT) strain or the Dps null mutant. The values are means and SEM for one experiment run in duplicate.

Fig 9

Effect of temperature on the bactericidal effect of EIP-K plus H₂O₂. Bacteria were grown at 37°C to exponential phase and then treated with ddH₂O (control), 13.75 mM EIP-K, 3 mM H₂O₂, or 13.75 mM EIP-K plus 3 mM H₂O₂, each at six temperatures between 0 and 45°C. The values are means and SEM for one experiment run in duplicate. Two-way ANOVA shows a significant treatment effect (F_[3,26] = 253.90; P = 0.0000001), a significant temperature effect (F_[5,26] = 3.25; P = 0.026), and a significant treatment-temperature interaction effect (F_[15,26] = 6.53; P = 0.00002).

Fig 10

Effects of ferrous ion chelators, hydroxyl radical scavengers, or ferrous ions on the bactericidal activity of EIP-K plus H₂O₂. Bacteria were pretreated with a ferrous chelator, 1,10-phenanthroline (100 μM), and hydroxyl radical scavengers, mannitol (100 μM) and thiourea (10 mM), for 30 min, followed by treatment with ddH₂O (control), 13.75 mM EIP-K, 3 mM H₂O₂, or 13.75 mM EIP-K plus 3 mM H₂O₂. EDTA (100 μM) and FeSO₄ (10 μM) were presented at the same time as the treatment with escapin products. The values are means and SEM for two experiments, each run in duplicate. Two-way ANOVA shows a significant treatment effect (F_[3,23] = 145.34; P = 0.0000001), a significant chelator effect (F_[4,23] = 128.50; P = 0.0000001), and a significant treatment-chelator interaction effect (F_[12,23] = 58.57; P = 0.0000001).