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Molecules and Mechanisms Underlying the Antimicrobial Activity of Escapin, an L-Amino Acid Oxidase from the Ink of Sea Hares.

Introduction

This review is about escapin, an L-amino acid oxidase in the ink of a gastropod mollusc, 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 evolutionary origins and diversity and their ecological roles, followed by 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 bacteria and biofilms of bacteria.

Chemical Defenses in Gastropods

Escapin is one of many chemical defenses in gastropod molluscs, which are renowned for their use of chemicals as protection against predators. This is especially true for those gastropods with reduced or absent shells, such as the Euopisthobranchia and Nudipleura (Jorger et al., 2010). Much is known about the identity and chemical ecology of deterrent compounds in these species. Many of these deterrents are diet-derived rather than synthesized de novo, and many are terpenoids (Avila, 1995; Cimino et al., 1999; Kamiya et al., 2006; Paul et al., 2007; Benkendorff, 2010; Bornancin et al., 2017). 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 (Fig. 1). This ink is actually a mixture of secretions of two glands, the ink gland and the opaline gland, which are simultaneously released into the mantle cavity, mixed there, and then expelled through a siphon toward the site of predatory attack (Walters and Erickson, 1986). The ink gland secretes a deep purple ink, and the opaline gland releases a whitish opaline that polymerizes on contact with water to become highly viscous. The feeding-deterrent properties of sea hare ink have been reviewed elsewhere (Carefoot, 1987; Johnson and Willows, 1999; Derby, 2007; Derby and Aggio, 2011).

Escapin and Its Homologs

L-amino acid oxidases such as escapin are one type of amino acid oxidase. Amino acid oxidases are enzymes that oxidize amino acids and in the process produce hydrogen peroxide ([H.sub.2][O.sub.2]) and ammonium. Amino acid oxidases are found broadly in animals and microbes and differ in substrate specificity and function. Phylogenetic analysis of amino acid oxidases based on gene sequences identified several clusters or types (Campillo-Brocal et al., 2015b). One group is the L-amino acid oxidases (LAAOs), which are flavin-dependent enzymes that oxidize L-amino acids. A second group is the D-amino acid oxidases, which also use flavins as cofactors but use D-amino acids as substrates. A third group is the L-aspartate oxidases, which are flavoproteins using L-aspartate as a substrate. A fourth group is the L-lysine [epsilon]-oxidases and related proteins, which contain a quinone cofactor; an example of this oxidase is LodA, which is synthesized by the melanogenic marine bacterium Marinomonas mediterranea (Campillo-Brocal et al., 2015a, b).

The LAAOs are found in diverse phylogenetic groups besides the gastropods. For example, in the vertebrates, venomous snakes use LAAOs as active ingredients in their venom (Ponnudurai etal., 1994), and some fish use LAAOs as toxins and antimicrobials in their mucus (Jung et al., 2000; Kitani et al., 2008, 2015). Bacteria also use LAAOs as antimicrobials (Kamiya et al., 2006; Campillo-Brocal et al., 2015b). The LAAOs of vertebrates, gastropods, and bacteria appear to have evolved separately (Hughes, 2010; Campillo-Brocal et al., 2015b; Kasai et al., 2015).

Regarding the LAAOs in gastropods, each sea hare species expresses several LAAOs with an organ-specific expression pattern, and an organ can express more than one type of LAAO (Petzelt et al., 2002; Iijima et al., 2003a, b; Butzke et al., 2004, 2005; Johnson et al., 2006). For example, orthologs of escapin found in the ink gland of other sea hares include dactylomelin-P in Aplysia dactylomela (Melo et al., 2000) and APIT (Aplysia punctata ink toxin), cyplasin-L, and cyplasin-S in A. punctata (Petzelt et al., 2002; Butzke et al., 2004, 2005). Aplysianin-A is a paralog of escapin, found in the albumen gland and egg masses of Aplysia californica (Cummins et al., 2004); and orthologs of aplysianin-A are present in Aplysia kurodai (Jimbo et al., 2003). Other homologs are found in gastropods in addition to sea hares (Obara et al., 1992; Ehara et al., 2002; Hathaway et al., 2010).

The LAAOs in the albumen gland and egg mass of sea hares have antimicrobial activity (Kamiyaetal, 1986, 2006; Yamazaki et al., 1989a, b, c; Iijima et al., 2003b; Jimbo et al., 2003; Cummins et al., 2004). Because sea hares lay eggs in strings on the substrate, using antimicrobials to prevent fouling is an obvious advantage and one that is accomplished by many invertebrates through diverse molecules (Barbieri et al., 1997; Benkendorff et al., 2001; Benkendorff, 2010). Because LAAOs can also be aversive to omnivores (Aggio and Derby. 2008; Nusnbaum and Derby, 2010a, b), their presence in egg masses could have the additional function of deterring some animals from ingesting the eggs.

It has been speculated that escapin and its orthologs in the ink gland of sea hares evolved from antimicrobial paralogs in other tissues (Derby. 2007). Such an evolutionary scenario might explain why LAAOs in ink glands have an antimicrobial function, in addition to their more derived antipredatory function. Another possibility is that LAAOs act as a salve by coating the sea hare's skin during an attack, subsequently preventing infections to any injured skin. Placing antimicrobial molecules in the skin itself would be a more direct way for preventing such infections, but to date, no LAAOs have been reported in the skin or body wall of Aplysia spp., although antimicrobial peptides have been reported in the skin and body wall of the sea hare Dolabella auricularia (Iijima et al., 2003a, b).

The Chemistry of Escapin

Escapin and its homologs prefer as substrates the basic amino acids L-lysine and L-arginine (Jimbo et al., 2003; Butzke et al., 2004, 2005; Yang et al., 2005). The principal natural substrate for escapin in the ink of sea hares is L-lysine, because it is in much higher concentration in ink than is L-arginine (Kicklighter et al., 2005). Sea hares store escapin and its substrates in separate reserve pools, with escapin in the ink gland and l-lysine in the opaline gland (Johnson et al., 2006). Escapin and its substrates are only mixed when the defensive secretion is deployed and released. In fact, in the ink gland, escapin is kept in amber vesicles, separate from the purple vesicles that contain aplysioviolin. another chemical defense (Johnson et al., 2006; Kamio et al., 2010a. b: Derby and Aggio. 2011).

Studies of the chemistry of escapin show that its oxidation of l-lysine produces a mixture of molecules (shown in Fig. 2) whose composition changes quickly over time (Kamio et al., 2009). The first step is escapin's oxidative deamination of l-lysine, which produces an equilibrium mixture of compounds called "escapin intermediate products of l-lysine" (EIP-K, or just EIP). Some of the compounds in EIP react non-enzymatically with [H.sub.2][O.sub.2] to yield a mixture of compounds called "escapin end products of l-lysine" (EEP-K, or EEP). The pH of Aplysia californica ink is ca. 5.0 at full strength, in contrast to a pH of ca. 8.0 for seawater (Shabani et al., 2007). This is significant because pH affects the equilibrium among escapin's reaction products. A kinetic analysis of escapin showed that incubation of escapin and l-lysine at natural concentrations produces millimolar concentrations of [H.sub.2][O.sub.2], ammonia, and other reaction products within seconds (Kamio etal., 2009). The chemistry of escapin's oxidation of l-arginine has not been studied in as much detail as that of l-lysine.

An important technical development in the study of the effects of escapin's product is the ability to synthesize [[DELTA].sup.1]-piperidine-2-carboxylic acid (compound 3 in Fig. 2) (Kamio et al., 2009). This has allowed us to compare results using EIP generated from escapin's hydrolysis of l-lysine with results based on "synthetic EIP" (i.e., [[DELTA].sup.1]-piperidine-2-carboxylic acid). In the case of synthetic EIP, one can at least begin experiments with a known concentration of compound, even though for either escapin-generated EIP or synthetic EIP, an equilibrium mixture will develop in aqueous solution, as described above.

Antipredatory Effects of Escapin

Escapin can act as a feeding deterrent against potential predators. 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 (Aggio and Derby, 2008; Nusnbaum and Derby, 2010a, b), and EIP is a deterrent against fish (Nusnbaum and Derby, 2010a, b). So far, there is no evidence of synergy between [H.sub.2][O.sub.2] and EIP in its antipredatory effects.

Antimicrobial Effects of Escapin

Bacteria have a life cycle that allows them to exist in both planktonic and biofilm phases. As shown in Figure 3, mature biofilms release planktonic bacteria under certain conditions, and these planktonic bacteria swim and eventually settle on new surfaces. There, they irreversibly attach and form micro-colonies that can eventually differentiate into mature biofilms. So, understanding the effects of antimicrobials on bacteria requires examination of these life phases.

Effects of escapin on planktonic bacteria

Escapin's products inhibit the growth of gram-negative and gram-positive bacteria, fungi, yeast, and mold (Yang et al., 2005). The products of homologs of escapin have been shown to have antitumor properties (Yamazaki, 1993; Jimbo et al., 2003; Butzke et al., 2004; Kamiya et al., 2006), but escapin has not been tested for such properties. The mixture of EIP and [H.sub.2][O.sub.2] is an effective antimicrobial agent against both planktonic bacteria and biofilms, and, as described in this subsection, some of its mechanisms of action are known.

The products of escapin have both bacteriostatic and bactericidal effects on planktonic cells. The bacteriostatic effect, in which growth is inhibited, is mediated by [H.sub.2][O.sub.2] alone, without a contribution from EIP or EEP. This is shown through equal bacteriostatic effects of [H.sub.2][O.sub.2] and of escapin products when escapin's substrate is either l-lysine or l-arginine, each an equally effective substrate in the production of [H.sub.2][O.sub.2] (Fig. 4A) (Yang et al., 2005). Products of homologs of escapin also appear to be bacteriostatic, at least in large part due to the effects of [H.sub.2][O.sub.2] (Ehara et al., 2002; Jimbo et al., 2003; Butzke et al., 2004; Kanzawa et al., 2004; Kamiya et al., 2006). Escapin's bactericidal effects, however, are not due only to [H.sub.2][O.sub.2]. This is shown in Figure 4B, in which l-lysine, but not l-arginine, is an effective substrate in escapin's bactericidal effects (Yang et al., 2005).

Another important observation in understanding escapin's mechanisms of action against planktonic bacteria is a synergistic effect of [H.sub.2][O.sub.2] and EIP when l-lysine, but not l-arginine, is used as the substrate (Ko et al., 2008). Figure 5 shows that a mixture of [H.sub.2][O.sub.2] and EIP, when using l-lysine as the substrate, is by far the most effective bactericidal agent of those tested: more than either [H.sub.2][O.sub.2] or EIP alone, more than [H.sub.2][O.sub.2] and EEP alone or together, and more than [H.sub.2][O.sub.2] and EIP when l-arginine was the substrate (Ko et al., 2008). The effect can be powerful, reducing the number of cells by more than seven log units in some cases (Fig. 5). This demonstrates that the bactericidal effect against planktonic bacteria is due to molecules other than [H.sub.2][O.sub.2] alone. Furthermore, there is a concentration dependence to this potent synergistic bactericidal effect for Escherichia coli, with a maximum effect of ca. 13 mmol [L.sup.-1] EIP and 2.5 mmol [L.sup.-1] [H.sub.2][O.sub.2]. These are relatively high concentrations of EIP and [H.sub.2][O.sub.2], but they are reducing the number of colonies by many log-fold. Such a concentration-response relationship, where both higher and lower concentrations are less effective than ones in between, is known as the Eagle effect, and it has been reported for a variety of other antimicrobial agents and microbes (Stevens et al., 1988; Fleischhacker et al., 2008).

The synergistic action of co-presented [H.sub.2][O.sub.2] and EIP might result from the generation of novel strongly bactericidal compounds from the chemical reaction between [H.sub.2][O.sub.2] and components in EIP. This hypothesis was tested by presenting [H.sub.2][O.sub.2] and EIP either simultaneously or sequentially and then determining whether synergy occurred. A short (10-min) co-treatment with [H.sub.2][O.sub.2] and EIP was sufficient to generate long-lasting bactericidal effects (Ko et al., 2008, 2012), but a 10-min presentation with either [H.sub.2][O.sub.2] or EIP only, followed by brief rinsing and 10-min treatment of the other substance, showed no synergy (Ko et al., 2008). This supports the idea that the synergy of [H.sub.2][O.sub.2] and EIP is due to novel compounds generated by their chemical interactions, assuming that the effect of either [H.sub.2][O.sub.2] alone or ED? alone is long-lasting (at least longer than 10 min).

One identified effect of [H.sub.2][O.sub.2] and EIP on planktonic bacteria is a rapid and long-lasting DNA condensation (Fig. 6, top row) (Ko et al., 2012). A 2-min treatment with [H.sub.2][O.sub.2] and EIP causes significant DNA condensation, and a 10-min treatment causes a maximal effect that lasts at least 70 h. Consistent with an effect on DNA is that [H.sub.2][O.sub.2] and EIP act preferentially on fast-growing cells (i.e., cells in their log-growth phase) compared to cells in their stationary phase (Yang et al., 2005). Additionally, the bactericidal effects of escapin's products do not require protein synthesis (Yang et al., 2005; Ko et al., 2008).

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 [H.sub.2][O.sub.2]. This effect is a specific response to oxidative stress (i.e., [H.sub.2][O.sub.2], because temperature stress combined with EIP (but not [H.sub.2][O.sub.2]) does not produce the bactericidal effect (Ko et al., 2012). Experiments using mutants for several single DNA-binding proteins suggest that EIP and [H.sub.2][O.sub.2] may function through a combination of DNA-binding proteins (Ko et al., 2012). Experiments with chelators and scavengers suggest that hydroxy 1 radicals may mediate these effects (Ko et al., 2012).

The results to date indicate that the powerful, rapid, and long-lasting bactericidal effect of escapin's oxidation of l-lysine is due to the generation of a mixture of highly reactive molecules that affect the 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 may interact with several DNA-binding proteins, including Dps (DNA-binding protein from starved cells) and H-NS (histone-like nucleoid structuring protein), causing irreversible DNA condensation and inhibition of DNA-unwinding mechanisms, thus arresting the initiation of DNA replication and initiating the degradation of DNA (Ko et al., 2012). The role of molecules in EIP in this process is less clear. EIP can move across bacterial cell membranes (M. T. Kozma [Georgia State University], PCT, and CDD, unpubl. data), so EIP's effects could result either because of its 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 [H.sub.2][O.sub.2] and thus in inducing the irreversible DNA condensation and degradation. Future studies must investigate not only the independent effects of EIP and [H.sub.2][O.sub.2] but also the interactive effects of the combination of the two, due to the synergistic effects of the two components.

Effects of escapin on 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 (Costerton et al., 1999; Houry et al., 2012). The life cycle of biofilms includes attachment of planktonic cells to a surface, growth of the biofilm, and dispersal, as shown in Figure 3. Biofilms are highly dynamic, with environmental conditions supporting either growth or emigration of bacterial cells from the biofilm. The structure and composition of extracellular polymeric substances in biofilms are variable but typically include an abundance of polysaccharides in addition to proteins, nucleic acids, and lipids, held together through physicochemical interactions (Flemming and Wingender, 2010). The extracellular polymeric substances provide biofilms with resistance to environmental perturbations, including to antimicrobials, which makes them a challenge in medical and industrial settings (Donlan, 2008; Hart and Rather, 2008; Simoes et al., 2010). 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. These studies include examination of the effects of EIP and [H.sub.2][O.sub.2], alone and in combination, on the inhibition of biofilm formation and disruption of established biofilms (Santiago et al., 2016). Significant effects were found with very low (micromolar) concentrations of EIP and [H.sub.2][O.sub.2]. For example, in 5-h assays of inhibition of biofilm formation in microtiter plates, biofilms exposed to 96 [micro]mol [L.sup.-1] [H.sub.2][O.sub.2] were 30% smaller than controls, biofilms exposed to 3 [micro]mol [L.sup.-1] EIP were 25% smaller than controls, and biofilms exposed to the combination of the two (96 [micro]mol [L.sup.-1] [H.sub.2][O.sub.2] + 3 [micro]mol [L.sup.-1] EIP) were 65% smaller than the control (Santiago et al., 2016). 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 confocal microscopy. These assays show that a combination of EIP at 50 [micro]mol [L.sup.-1] + [H.sub.2][O.sub.2] at between 0.03 and 3 [micro]mol [L.sup.-1] caused 40% clearance of biofilms, while each alone caused very little clearance (Santiago et al., 2016). Thus, over this range of concentrations. EIP + [H.sub.2][O.sub.2] had a synergistic effect in disrupting biofilms (Fig. 6, bottom row). Together, these results show that micromolar and, in some cases, even nanomolar concentrations of EIP and [H.sub.2][O.sub.2] can inhibit formation of biofilms and can cause disruption of existing biofilm. These concentrations are significantly lower than those causing bactericidal effects on planktonic bacteria. This concentration difference might be because the assays for planktonic bacteria require effects of much greater magnitude (i.e., at least several log-unit reductions in number of colonies) than the assays for biofilm (i.e., percent reduction in biomass). More studies are needed to explore this issue.

Model of escapin's effects on bacteria

A model of the effects and underlying mechanisms of escapin's products on the life stages of bacteria is presented in Figure 3. The combination of EIP + [H.sub.2][O.sub.2] has two effects on planktonic bacteria. First, it causes DNA condensation and subsequent death of planktonic bacteria at higher concentrations. Second, it causes an increase in swimming motility, though not swarming or other types of motility (Santiago et al., 2016). These effects could lead to a reduction in the colonization of surfaces by planktonic bacteria and subsequent reduction in the formation of biofilms. Impaired adhesion of bacteria by EIP + [H.sub.2][O.sub.2] could also contribute to its negative effect on biofilm formation. Once bacteria are established as mature biofilms, their biomass can be reduced by EIP + [H.sub.2][O.sub.2]. Several mechanisms are possible at this stage, involving either intracellular or extracellular factors. An essential component of a microbial biofilm is the extracellular matrix, which physically anchors the community to the substratum. The matrix is composed of diverse biopolymers and ions, which contribute to its structural integrity. A matrix building block used by diverse biofilm-forming microorganisms is extracellular DNA (Ibanez de Aldecoa et al., 2017). The mixture of EIP + [H.sub.2][O.sub.2] promotes DNA condensation of planktonic bacteria (Ko et al., 2012), raising the possibility that it could influence extracellular DNA concentrations and three-dimensional structure in the biofilm matrix, resulting in biofilm disruption. Alternatively, EIP, with its mixture of cationic and amphipathic structures, could act as a surfactant, and surfactants are known to be able to contribute to biofilm detachment (Diaz De Rienzo et al., 2016; Silva et al., 2017). Additionally, the negatively charged functional groups of several EIP components (see Fig. 2) could work as chelators, interacting with calcium ions in the biofilm matrix and reducing its stability (van der Waal and van der Sluis, 2012; Flemming, 2016). Another potential mode of action of EIP + [H.sub.2][O.sub.2] on biofilms is by affecting cellular signaling networks that influence biofilm detachment. One of the surprising findings of our work was the low concentrations of [H.sub.2][O.sub.2] that affect biofilm disruption (Santiago et al., 2016). While no work to date has focused on sub-micromolar concentrations of [H.sub.2][O.sub.2]. nitric oxide concentrations in the same range can promote biofilm dispersal (Barraud et al., 2015). Nitric oxide targets cyclic di-GMP signaling in Pseudomonas aeruginosa (Barraud et al., 2009; Kim and Lee, 2016), and [H.sub.2][O.sub.2] may work similarly and be potentiated by EIP. EIP and [H.sub.2][O.sub.2] can enhance swimming motility while decreasing biofilm formation. These traits have been inversely linked via cyclic di-GMP signaling pathways and related regulatory elements in P. aeruginosa, and this suggests that EIP may act by interacting with this network (Li et al., 2017). The model presented in Figure 3 incorporates these ideas, and although it is based on limited data, it provides a conceptual framework from which future experiments can be launched.

Conclusions

In response to the global spread of antibiotic resistance, there is a demand for compounds that have the ability to attenuate microbial pathogenicity and yet are not biocidal (Vale et al., 2016). The logic for using compounds with antivirulence capabilities is to control infections while reducing the strong selection for resistant phenotypes caused by standard antibiotics. This is particularly important for gram-negative bacteria such as Pseudomonas 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 antivirulence and antibiofilm characteristics. A recent review on therapeutics for biofilm eradication argued that a multipronged approach is required to effectively manage biofilm infections, because of their complexity at many levels (Koo et al., 2017). 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 review, some of the independent and synergistic actions of EIP and [H.sub.2][O.sub.2] 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, because EIP and [H.sub.2][O.sub.2] are reactive and because 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 (Kamio et al., 2009). To move beyond the associated uncertainty, a synthetic EIP made from known concentrations of the chemical components needs to be developed.

Acknowledgments

The experimental work on the antimicrobial aspects of escapin was supported by the Georgia Research Alliance and Georgia State University, and work on chemical ecology and antipredatory effects of escapin was supported by grants from the National Science Foundation. We thank our students and collaborators for their substantial contributions to the experimental work and ideas presented in this review.

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CHARLES D. DERBY (1,2,*), ERIC S. GILBERT (2), AND PHANG C. TAI (2)

(1) Neuroscience Institute, Georgia State University, Atlanta, Georgia 30303; and (2) Department of Biology, Georgia State University, Atlanta, Georgia 30303

(*) To whom correspondence should be addressed. E-mail: cderby@gsu.edu.

Abbreviations: EEP. escapin end products of L-lysine; EIP. escapin intermediate products of L-lysine; LAAO. L-amino acid oxidase.

Received 21 February 2018: Accepted 7 June 2018; Published online 30 July 2018.
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Author:Derby, Charles D.; Gilbert, Eric S.; Tai, Phang C.
Publication:The Biological Bulletin
Date:Aug 1, 2018
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