The Molecules and Mechanisms Underlying the 2 Antimicrobial Activity of Escapin , an L – Amino Acid 3 Oxidase from the Ink of Sea Hares 4 5

Many marine animals use chemicals to defend themselves and their eggs from predators. 12 Beyond their ecologically relevant functions, these chemicals may also have properties that make 13 them beneficial for humans, including with biomedical and industrial applications. For example, 14 some chemical defenses are also powerful antimicrobial or anti-tumor agents with relevance to 15 human health and disease. One such chemical defense, Escapin, an L–amino acid oxidase in the 16 defensive ink of the sea hare Aplysia californica, and related proteins have been investigated for their 17 biomedical properties. This review details our current understanding of Escapin’s antimicrobial 18 activity, including the array of chemicals generated by Escapin’s oxidation of its major substrates, 19 L–lysine and L–arginine, and mechanisms underlying these molecules’ bactericidal and 20 bacteriostatic effects on planktonic cells and the prevention of formation and removal of bacterial 21 biofilms. Models of Escapin’s effects are presented, and future directions are proposed. 22


Introduction
This review is about Escapin, an L-amino acid oxidase in the ink of a gastropod mollusk, the sea hare Aplysia californica.Escapin and related proteins have been studied in different contexts.First, they have been studied in the context of natural history and chemical ecology, to examine their use as chemical defenses to protect sea hares from predators and their eggs from fouling organisms including bacteria.Second, they have been used in studies of natural products chemistry and drug discovery, in the search for molecules with applications for human health and disease.This review provides a short description of their role ecological roles, then a more extensive treatment of their antimicrobial properties related to human applications.This includes identification of molecules produced by Escapin's oxidation of its substrates L-lysine and L-arginine, and the cellular and molecular mechanisms whereby these molecules act as antimicrobial agents against planktonic and biofilm forms of bacteria.

Escapin in the Context of Chemical Ecology
Gastropod molluscs are renowned for their use of chemical defenses as protection against predators, especially those gastropods with reduced or absent shells such as the Euopisthobranchia and Nudipleura [1].There are many reviews on the subject of chemical ecology and identification of deterrent compounds in these species.Many of these deterrents are diet-derived rather than synthesized de novo, and many are terpenoids [2-7}.Some chemical deterrents are constitutively present, including in the skin and mucus.Other chemical deterrents are released only upon attack by a predator, an example being the ink of sea hares (Figure 1).This ink is actually a mixture of secretions of two glands -the ink gland and the 3 of 14 which contain a quinone cofactor, an example being LodA, an enzyme synthesized by melanogenic marine bacterium Marinomonas mediterranea.A fourth group is the L-aspartate oxidases, which are flavoproteins that use L-aspartate as a substrate.

Escapin's Substrates and Products
Escapin and its homologues prefer as substrates basic amino acids: L-lysine and L-arginine [15,[18][19][20].The principal natural substrate for Escapin in the ink of sea hares is L-lysine, since it is in much higher concentration in ink than is L-arginine [31].Sea hares store Escapin and its substrates in separate reserve pools: Escapin in the ink gland, and L-lysine in the opaline gland [29].Escapin and its substrate are only mixed when the defensive secretion is deployed and released.In fact, Escapin is kept in specific (amber) vesicles in the ink gland, separate from other (purple) vesicles in the ink gland that contain Aplysioviolin, another chemical defense [12,29,32,33].
Studies of the chemistry of Escapin show that Escapin's oxidation of L-lysine produces a mixture of ingredients that changes quickly over time [34], as shown in Figure 4.The first step is Escapin's oxidative deamination of L-lysine (compound 1), which produces an equilibrium mixture of compounds called "escapin intermediate products of lysine" (EIP-K, or just EIP).EIP includes α-keto-

Anti-Predatory Agents.
The efficacy of Escapin's products as a feeding deterrent depends on the identity of the molecules and predators.Hydrogen peroxide is a deterrent against fish and crustaceans [35-37], and EIP is also deterrent against fish [36,37].So far, there is no evidence of synergy between H2O2 and EIP in antipredatory effects.

Antimicrobial Agents.
Escapin's products inhibits the growth of several types of microbes, including Gram-negative and Grampositive bacteria, fungi, yeast, and mold, with minimum inhibitory concentrations of between.25 and 65 µg/ml [18].Homologues of Escapin have been shown to have antitumor properties [5,15,19,38], but Escapin has not been tested for such properties.
Escapin is an effective antimicrobial agent against both planktonic bacteria and biofilms, and, as described in this section, some of its mechanisms of action are known.result.This can be seen in Fig. 5B, in which L-lysine but not L-arginine is an effective substrate in Escapin's bactericidal effects [18].

Another important observation in understanding
Escapin's mechanisms of bactericidal action was a synergistic effect of H2O2 and EIP when L-lysine, but not L-arginine, was used as the substrate [40].Figure 6 shows that a mixture of H2O2 and EIP using L-lysine as the substrate is by far the most effective bactericidal agent of those tested, more than either alone, more than H2O2 and EEP, and more than H2O2 and EIP when Larginine was the substrate [40].The effect can be powerful, reducing the number of cells by more than seven log units in some cases (Fig. 6).This demonstrates that the bactericidal effect is due to molecules other than H2O2 alone.Furthermore, Figure 7  One identified effect of H2O2 and EIP is a rapid and long-lasting DNA condensation [44].A two-min treatment with H2O2 and EIP causes significant DNA condensation(Fig.8) and killing, and 10-min treatment causes a maximal effect that lasts at least 70 h.Consistent with an effect on DNA is that H2O2 and EIP act preferentially on fast-growing cells (i.e. cells in their log-growth phase) vs. cells in their stationary phase [18].Additionally, Escapin's bactericidal effects do not require protein synthesis [18,40].
DNA condensation might occur because of alterations in the oxidation process.Bacteria with a single missense mutation in the oxidation regulatory gene, oxyR, are resistant to EIP and H2O2.This effect is a specific response to oxidative stress (i.e.H2O2), since temperature stress combined with EIP (but not H2O2) does not produce the bactericidal effect [44].Experiments using mutants for several single DNA-binding proteins suggest that EIP and H2O2 function through a combination of DNAbinding proteins [44].Experiments with chelators and scavengers suggest that hydroxyl radicals may mediate these effects [44].
The results to date indicate that the powerful, rapid, and long-lasting bactericidal effect of

Bacterial Biofilms.
In nature, most microbes exist as biofilms rather than as planktonic cells.Biofilms are communities of cells attached to surfaces and encased in a self-produced extracellular matrix called extracellular polymeric substances (EPSs) [45,46].The life cycle of biofilms includes attachment of planktonic cells to a surface, growth of the biofilm, and dispersal (Fig. 9).Biofilms are highly dynamic, with environmental conditions supporting either growth or emigration of bacterial cells from the biofilm.
The structure and composition of EPSs in biofilms is variable but typically includes an abundance of polysaccharides in addition to proteins, nucleic acids, and lipids, held together through physicochemical interactions [47].The EPSs provide biofilms with resistance to environmental perturbations, including to antimicrobials, which makes them a challenge in medical and industrial settings [48][49][50].Consequently, development of antibiofilm strategies is an active field.
Escapin's products have been tested as antimicrobial agents against biofilms of the pathogen Pseudomonas aeruginosa.This included examining inhibition of biofilm formation and disruption of established biofilms, and if EIP and H2O2 acted synergistically [51].In these experiments, chemically synthesized EIP was used as it has similar activities as the products of Escapin's action on L-lysine [40,44].Significant effects were found with very low concentrations, in the micromolar range.For example, in 5 hr assays of inhibition of biofilm formation in microtiter plates, biofilms exposed to 96 µM H2O2 were 30% smaller than controls, biofilms exposed to 3 µM EIP were 25% smaller than controls, and biofilms exposed to the combination of the two (96 µM H2O2 + 3 µM EIP) were 65% smaller than the control.Assays of dispersal of established biofilms by chemical agents involved growing biofilms in flow cells for 20 h, then exposing them to agents for 30 min, and then quantifying biomass using microscopy.These assays show that a combination of EIP at 50 µM + H2O2 at between 0.03 and 3 µM caused 40% clearance of biofilms, while each alone caused very little clearance; thus, over this range of concentrations, EIP + H2O2 had a synergistic effect in disrupting biofilms (Fig. 10).
Together, these results show that micromolar, and in some cases even nanomolar, concentrations of EIP and H2O2 can affect biofilm formation or disruption.These concentrations are significantly lower than those causing bactericidal effects on planktonic bacteria.regulatory elements in P. aeruginosa and suggest that EIP may act by interacting with this network [60].

Conclusions
In response to the global spread of antibiotic resistance, there is a demand for compounds that have the ability to attenuate microbial pathogenicity yet are not biocidal [61].The logic for using compounds with anti-virulence capabilities is to control infections while reducing the strong selection for resistant phenotypes caused by standard antibiotics.This is particularly important for Gramnegative bacteria such as P. aeruginosa for which the presence of outer membranes renders many antibiotics impermeable to the cells.To date, there are a limited number of compounds that have both anti-virulence and anti-biofilm characteristics.A recent review on therapeutics for biofilm eradication argued that a multi-pronged approach is required to effectively manage biofilm infections, due to their complexity at many levels [62].From this standpoint, Escapin has potential as a therapeutic agent and warrants further attention.Progress in evaluating its use as a therapeutic agent requires a greater knowledge of its mechanisms of action.As described in this this review, some of the independent and synergistic actions of EIP and H2O2 are known, but much more detailed information is necessary.A challenge working with EIP is that there is variation in the activity of the mixture, since EIP and H2O2 are reactive and the ratios of the components in the equilibrium mixture may be different with each reaction and over time.To facilitate research into the mechanism of EIP, a consistent mixture of EIP should be available.The development of a synthetic approach to making EIP solved part of this requirement [34].To move beyond the associated uncertainty, a synthetic EIP made from known concentrations of the chemical components needs to be developed.

3 .Figure 1 .
Figure1.An inking sea hare, Aplysia californica.Escapin is found in the ink gland secretion, and its substrate, Llysine, is found in the opaline gland secretion, both of which are mixed and released to form the ink shown emanating from this sea hare.Reproduced with permission Genevieve Anderson.

Figure 3 .
Figure 3.A phylogenetic tree and substrate specificity of L -amino acid oxidases, based on gene sequence, from gastropods, vertebrates, and bacteria.Substrate specificity is indicated by the symbols, where closed circles represent basic amino acids, open circles represent aromatic amino acids, and double circles represent aliphatic amino acids.Size of circles represents relative activity.

Figure 4 .
Figure 4. Summary of the chemistry of the reaction of escapin with L-lysine, including the effects of pH on the relative composition of the molecular species in the equilibrium mixture.This series of chemical reactions produces products that interact with each other to form a diverse group of molecules at low millimolar concentrations within a few seconds of the release of the secretion.Modified from Kamio et al. [34].

4. 2 . 1 .
Planktonic Bacteria.Escapin has both bacteriostatic and bactericidal effects on planktonic cells.The bacteriostatic effect, in which growth is inhibited, is mediated by H2O2 alone, without a contribution from EIP or EEP.This is shown through equal bacteriostatic effects of H2O2 and of Escapin products when Escapin's substrate is either L-lysine or L-arginine (both of which are equally effective substrates in the production of H2O2) (Fig.5A)[18].Homologues of Escapin also appear to be bacteriostatic, at least in large part because of the effects of H2O2[5,15,19,22,39]. Escapin's bactericidal effects, however, are not due only to H2O2, a surprising Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 8 January 2018 doi:10.20944/preprints201801.0047.v1 shows the concentration dependence of this potent synergistic bactericidal effect for E. coli, with a maximum effect of ca. 13 mM EIP-K and 2.5 mM H2O2.Such concentrationresponse relationships where both higher and lower concentrations are less effective than concentration in between is known as the Eagle effect, and it has been reported for a variety of microbes and antimicrobial agents[41][42][43].The synergistic action of co-presented H2O2 and EIP might result from the generation of novel, strongly bactericidal compounds from the chemical reaction between H2O2 and components in EIP.This hypothesis was tested by presenting H2O2 and EIP either simultaneously or sequentially, and then determining if synergy occurred.A short (10 min) cotreatment with H2O2 and EIP was sufficient to generate long-lasting bactericidal effects[40,44], but a 10-min presentation with either H2O2 or EIP only followed by brief rinsing and 10-min treatment of the other showed no synergy[40].This supports the idea that the synergy of H2O2 and EIP is due to novel compounds generated by their chemical interactions, assuming that the effect of either H2O2 alone or EIP alone is long lasting (at least longer than 10 min).

Preprints
(www.preprints.org)| NOT PEER-REVIEWED | Posted: 8 January 2018 doi:10.20944/preprints201801.0047.v1 Escapin's oxidation of L-lysine is that this reaction generates a rich array of highly reactive molecules that affect DNA of fast-growing planktonic bacterial cells.Hydroxyl radicals and possibly other oxidative agents generated by Escapin may interact in a specific way with the oxidation regulatory gene, oxyR.In turn, oxyR interacts with several DNA binding proteins, including Dps and H-NS, causing irreversible DNA condensation and inhibition of DNA unwinding mechanisms, thus arresting the initiation of DNA replication and initiating the degradation of DNA [44].The role of molecules in EIP in this process is less clear.EIP can move across bacterial cell membranes (Mihika T. Kozma, P. C. Tai, and C. D. Derby, unpublished data), so its effects could be effected either by binding to receptor proteins on the bacterial cell membrane or by interacting with intracellular targets.EIP might play a role in stabilizing the oxidative response from H2O2 and thus in inducing the irreversible DNA condensation and degradation.Future studies must investigate not only the independent effects of EIP and H2O2 but also the interactive effects of the combination of the two, because of the synergistic effects of the two components.

Figure 11 .
Figure 11.Effects of EIP + H2O2 on motility of P. aeruginosa after treatment for 2 h at 37°C.Swimming motility was quantified as the area of the motility zone for each treatment.Values are means ± SEM for two replicates for each treatment.Values for treatments were normalized to levels in the untreated controls after the mean of the values for each of the control replicates was determined.Reproduced with permission from Santiago et al. 2016, Antimicrobial Agents and Chemotherapy; published by the American Society for Microbiology.