Bacteriostatic and cytolytic activity of purple fluid from the sea hare

Yamazaki, Masatoshi; Ohye, Hirokazu; Kisugi, Jun; Kamiya, Hisao

doi:10.1016/0145-305x(90)90030-i

Cited by 24 publications

(14 citation statements)

References 18 publications

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“…Unlike previous reports of similar proteins isolated from sea hares (Kisugi et al, 1989;Yamazaki et al, 1990;Melo et al, 1998Melo et al, , 2000, we demonstrate that substrates determined the bactericidal or bacteriostatic effects of escapin. Bacteria cultured in M9 and glucose medium without high levels (50·mmol·l -1 ) of selected amino acids or without Tryptone Peptone supplements were not killed (Figs·7, 8).…”

Section: Discussioncontrasting

confidence: 50%

“…The existence of persister cells in our cultures of E. coli may explain why several sea hare homologues of escapin, including aplysianin-E (Kisugi et al, 1989), aplysianin-P (Yamazaki et al, 1990) and dactylomelin-P isolated from purple fluid of Aplysia dactylomela (Melo et al, 2000), have been characterized as bacteriostatic and not bactericidal, whereas we found escapin to be bactericidal. To test this idea, we extracted and purified dactylomelin-P from Aplysia dactylomela and found that it was also bactericidal under our assay conditions; K.-C. Ko and H. Yong, unpublished data).…”

Section: Discussioncontrasting

confidence: 48%

“…Several of these proteins, including three from sea hares, are known to be L-amino acid oxidases (Iijima et al, 2003;Jimbo et al, 2003;Butzke et al, 2004), but the cofactors responsible for oxidase activity have not been experimentally demonstrated. These studies differ in their findings as to whether the proteins are bacteriostatic or bactericidal (Yamazaki et al, 1990;Otsuka-Fuchino et al, 1992;Melo et al, 1998;. In addition to the interesting biological roles these proteins may play for their host organisms, some may also have practical industrial or medical uses, yet none have been successfully expressed in high output systems such as bacteria (Obara et al, 1999;Torii et al, 2000;Petzelt et al, 2002;Butzke et al, 2004;Cummins et al, 2004).…”

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confidence: 99%

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Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing l-amino acid oxidase from ink of the sea hare Aplysia californica

Yang

Johnson

et al. 2005

Journal of Experimental Biology

108

122

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SUMMARY A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica was cloned and sequenced, and is the first sea hare antimicrobial protein to be functionally expressed in E. coli. Sequence analysis suggested that this protein is a flavin-containing l-amino acid oxidase (LAAO), with one predicted potential glycosylation site, although the glycosylation could not be experimentally confirmed. This protein, which we call `escapin', has high sequence similarity to several other gastropod proteins. Escapin was verified by NMR, mass spectroscopy and HPLC to have FAD as its flavin cofactor. Escapin's antimicrobial effects, bacteriostasis and bactericidal, were determined using a combination of two assays: (1) incubation of bacteria on solid media followed by assessment of inhibition by direct observation of zones of inhibition or by turbidity measurements; and (2) incubation of bacteria in liquid media followed by counting viable colonies after growing on agar plates. Native escapin inhibited the growth of Gram-positive and Gram-negative bacteria, including marine bacteria (Vibrio harveyiiand Staphylococcus aureus) and pathogenic bacteria(Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa). Escapin also inhibited the growth of yeast and fungi, with different efficacies. Escapin's antimicrobial activity was concentration dependent and did not decrease when stored for more than 5 months at room temperature. Escapin was bacteriostatic and not bactericidal in minimal media (e.g. salt media) with glucose, yeast extract, and a mixture of 20 amino acids each at 50 μmol l-1, but was bactericidal in media enriched with Tryptone Peptone. Escapin was also strongly bactericidal in media with l-lysine at concentrations as low as 3 mmol l-1 and slightly bactericidal in 50 mmol l-1l-arginine, but not in most other amino acids even at 50 mmol l-1. Escapin had high oxidase activity (producing hydrogen peroxide) with either l-arginine or l-lysine as a substrate and little to no oxidase activity with other l-amino acids. Hydrogen peroxide alone (without escapin or amino acids) was strongly bacteriostatic but poorly bactericidal, similar in this respect to l-arginine but different from l-lysine in the presence of escapin. Together these results suggest that there are multiple mechanisms to escapin's antimicrobial effects, with bacteriostasis resulting largely or entirely from the effects of hydrogen peroxide produced by escapin's LAAO activity, but bactericidal effects resulting from lysine-dependent mechanisms not directly involving hydrogen peroxide. Recombinant escapin expressed in bacteria was also active against Gram-positive and Gram-negative bacteria,suggesting that glycosylation is not essential for antimicrobial activity.

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Section: Discussioncontrasting

confidence: 50%

Section: Discussioncontrasting

confidence: 48%

mentioning

confidence: 99%

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Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing l-amino acid oxidase from ink of the sea hare Aplysia californica

Yang

Johnson

et al. 2005

Journal of Experimental Biology

108

122

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“…Mollusks rely predominantly on cellular defense reactions in which invading microorganisms are encapsulated by blood cells or phagocytosed (14). The presence of antimicrobial activity in Mollusca has been reported from the mucus of the giant snail Achatina fulica (31,40), from the egg mass and purple fluid of the sea hare Aplysia kurodai (35,93), and from the body wall of the sea hare Dolabella auricularia (32). Work on marine mollusks has focused on the mussels Mytilus edulis and Mytilus galloprovincialis.…”

Section: Molluscamentioning

confidence: 99%

Antimicrobial Peptides from Marine Invertebrates

Tincu

Taylor

2004

Antimicrob Agents Chemother

299

135

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“…Beckwith et al (8) reported that the small peptides, dolastatins, isolated from Dollabella auricularia, demonstrate an antineoplastic effect and induce apoptosis in some human lymphoma cell lines. Several researchers (9)(10)(11)(12)(13)(14) have isolated aplysianin A, E and P from the albumen gland, the egg and the purple fluid, respectively, of Aplysia kurodai. Aplysianins are high molecular weight glycoproteins which have antibacterial and antineoplastic effects the same as dolastatins and inhibit the synthesis of deoxyribonucleic acid and ribonucleic acid, just as an effect of a chemotherapeutic drug.…”

Section: Discussionmentioning

confidence: 99%

Acute Liver Damage with Characteristic Apoptotic Hepatocytes by Ingestion of Aplysia kurodai, a Sea Hare.

et al. 1998

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A case of acute liver damage by ingestion ofAplysia kurodai, a sea hare is reported. A 40-yearold man, complaining of vomiting and pyrexia after eating a sea hare, was admitted. Laboratory data showed mild liver damage with sustained elevations of aminotransferases. Microscopic findings in the liver biopsy specimen revealed characteristic apoptotic hepatocytes accompanied by mitotic hepatocytes. It is suggested that bioactive substances in the sea hare might induce such apoptosis of hepatocytes in the liver. (Internal Medicine 37: 927-929, 1998)

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Bacteriostatic and cytolytic activity of purple fluid from the sea hare

Cited by 24 publications

References 18 publications

Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing l-amino acid oxidase from ink of the sea hare Aplysia californica

Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing l-amino acid oxidase from ink of the sea hare Aplysia californica

Antimicrobial Peptides from Marine Invertebrates

Acute Liver Damage with Characteristic Apoptotic Hepatocytes by Ingestion of Aplysia kurodai, a Sea Hare.

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