Antibacterial protection in Marthasterias glacialis eggs: characterization of lysozyme-like activity

Camp. Biochem. Physiol. Vol. 109B. No. 4, pp. 709-713. 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305~0491/94 $7.00 + 0.00

Pergamon 0305-0491(94)00102-2

Antibacterial protection in Marthasterias glacialis eggs: characterization of lysozyme-like activity L. Stabili and P. Pagliara Dipartimento Italy

di Biologia,

Universiti

di Lecce, Via Prov.le

Lecce-Monteroni,

I-73100

Lecce,

Eggs from Marthastherias glacialis exert antibacterial action on marine bacterial strains and show a lysozyme-like activity. This one depends on pH and ionic strength of sample and reacting medium. This hydrolase, purified by gel filtration and ion-exchange chromatography, could be responsible for the bacterial growth inhibitory activity observed. Key words: Lysozyme; Marine bacteria; Eggs; Marthusterias glacialis; Echinoderm; Antibacterial activity; Micrococcus lysodeikticus; Gel filtration. Comp. Biochem. Physiol. 109B, 709-713, 1994,

Introduction The majority of echinoderms are gonochoristic. Asteroids possess multiple gonads with gonoducts leading to interambulacral gonopores between adjacent arms (Brusca and Brusca, 1990). In some of them, spawning and external fertilization involve both eggs and spermatozoa being discharged into the surrounding (aquatic) medium. In seawater, the gametes that are a source of food can be exploited by many organisms including bacteria. Many studies show that among potential reasons for death of marine invertebrate gametes and larvae, predation is considered to be one of the most important ones (Thorson, 1966; Emlet et al., 1987; Young and Chia, 1987). The productivity of the sea is dependent upon the growth of the planktonic community. The qualitative and quantitative changes in this community, therefore, will have far-reaching effects on the total Correspondence to: L. Stabili, Dipartimento

di BioloUniversitA di Lecce, Via Prov.le gia, Lecce-Monteroni, 73100 Lecce, Italy. Tel. 832 320616; Fax 832 351504. Received 14 October 1993; accepted 20 May 1994. 709

economy of the environment. In recent years, marine biologists have suggested that at least some of the observable changes in the planktonic environment may be due to growth-promoting and growth-inhibiting substances secreted, excreted or released by organisms (Nigrelli, 1962). Many studies show the presence of antiviral, antimicrobial, anticoagulant, tumor-inhibitory and neurotropic substances which have been isolated from marine organisms (Nigrelli, 1960; Halstead, 1965; Russel, 1965). One well-known antibacterial substance is lysozyme. The presence of this enzyme is detected by its lytic action on a Gram-positive coccus, Micrococcus lysodeikticus. Lysozyme dissolves certain bacteria by cleaving the polysaccharide component of their cell walls. The function of the cell wall in bacteria is to confer a mechanical support. A bacterial cell devoid of a wall usually bursts because of the high osmotic pressure inside the cell. In echinoderms, a lysozyme-like activity in eggs and larvae from P. lividus was demonstrated by Canicatti (1990). Also in seminal plasma of P. lividus, an antibacterial activity was en-

710

L. Stabili and P. Pagliara

hanced (Stabili and Canicatti, in press). In this paper we report on the occurrence in Marthasterias glacialis eggs of antibacterial compounds and on the partial isolation of a lysozyme-like molecule.

marine strains. Normal growth controls consisted of inoculated Petri dishes incubated with sterile liquid nutrient broth. The percentage of bacteria inhibited by egg homogenate was inferred from the difference between the number of emerging colonies in controls and in tests.

Materials and Methods Animals and collection of eggs Individuals of Marthasterias glacialis (size 20-30 cm) were collected from Santa Caterina (Lecce, Italy) and brought into the laboratory in aerated seawater tanks. Mature eggs were obtained by filtering female gonads in pasteurized seawater (PSW) through four layers of gauze. The eggs were allowed to settle and, after removal of the supernatant, resuspended in PSW to obtain a 50% (v/v) suspension. This suspension was sonicated by a Branson Sonifier and then centrifuged at 12,000 g for 30 min. The resulting supernatant (eggs homogenate) was stored at - 7 0 ° C until use. Bacteria Bacterial strains of Vibrio alginolyticus, Vibrio sp., Vibrio vulnificus, Photobacterium sp., Salmonella sp. and Escherichia coli, isolated from seawater, were furnished by Dr G. Caruso, Istituto Sperimentale Talassografico, CNR-Messina, Italy. Bacteria were always harvested during the exponential growth phase, i.e. after 4 h r culture at 24 or 37°C for the pathogenic strains, under constant rotation at 100 rpm. The medium of culture was represented by sterile nutrient broth (LNB) containing 0.1 g/1 iron phosphate, 1 g/1 yeast extract and 5 g/1 Bacto peptone in PSW, pH 7.5. Antibacterial activity Ten microlitres of bacterial suspension (10 s cells/ml) were incubated with 50 #1 egg homogenate for 30 min at room temperature with gentle stirring at 100 rpm. Serial dilutions of this suspension were plated out in Petri dishes between two layers of nutritive agar (Marine agar 2216E; Difco Laboratories, Detroit, MI). Routinely, 10 -3, 10 -4 and 10 -s dilutions were plated out in triplicate. Emerging colonies were counted after 24 hr incubations at 37°C for the pathogenic strains and at 24°C for

Lysozyme activity To detect lysozyme activity, inoculated Petri dishes were used as standard assay, i.e. 700#1 5mg/ml dried Micrococcus luteus (Sigma, St Louis, MO) was diluted in 7 ml of 0.05 M phosphate buffer (PB)-agarose (1.2%) pH 5.2, then spread on a Petri dish. When the agarose had solidified, 6.3 mm diameter wells were sunk in it and filled with 30 #1 egg homogenate. The diameter of the cleared zone was recorded after overnight incubation at 37°C. Dependence on ionic strength and on pH were investigated. The first parameter was tested in PB, pH 5.2, at I = 0.0175, 0.175 and 1.75. The pH dependence was tested dialysing the sample in PB 0.05M ( I = 0 . 1 7 5 ) at pH values 4.5, 5.2, 6.2, 7.2 and 8.2. Purification of lysozyme About 2ml egg homogenate were applied to a Spherogel-TSK 3000 SW (7.5 x 600 mm) HPLC column (Beckman). Equilibration and elution occurred at 60 ml/hr with PB 0.05 M, pH 5.2. Active fractions, as revealed by standard assay on Petri dishes, were pooled from different runs. Two millilitres of them were loaded on an ion-exchange column (DEAE, Beckman) previously equilibrated with PB 0.05 M, pH 5.2. This column was eluted with a 0 - 0 . 5 M NaC1 gradient in PB 0.05 M, pH 5.2.

Results Antibacterial activity Our data enhance, in vitro, the presence in egg homogenate from Marthasterias glacialis of an antibacterial activity. This activity, as shown in Table 1, was tested on Vibrio alginolyticus, Vibrio vulnificus, Salmonella sp., Escherichia coli, Pseudomonas sp. and Photobacterium sp., isolated from seawater. By way of plate assay, we were able to quantify the degree

Lysozyme-like activity in Marthasterias glacialis eggs

711

Table 1. Result of in vitro experiments showing antibacterial activity of P. lividus egg homogenate Bacterial strain

% G r o w t h inhibition

Vibrio alginolyticus Vibrio vulnifius Photobacterium sp. Pseudomonas sp. Salmonella sp. Eseheriehia eoli

89.6 _ 2.1 78.0 + 5.4 25.6 +_ 5.4 45.0 _+ 11.3 1.2 _+ 1.2 7.2 _ 0.9

A 0.5-

of inhibition produced by egg homogenate on each bacterial strain. Vibrio alginolyticus and Vibrio vulnificus were the most inhibited. Percentages of growth inhibition were, respectively, 89.6 ___2.1% and 78 ___5.4%. Variable percentages of inhibition were instead measured on the other strains. The pathogenic ones, Salmonella sp. and Escherichia coli, were apparently not wholly affected in their growth rate to egg homogenate treatment (1.2 _ 1.2% and 7.2 + 0.9% growth inhibition, respectively). A similar result was obtained with Pseudomonas sp.; Photobacterium sp. was sensitive to egg homogenate, with a percentage of inhibition of 45 ___11.3%.

L ysozyme-like activity Egg homogenate showed also a natural lysozyme-like activity. By the standard assay on Petri dishes we found a diameter of lysis of 9.7 _ 0.4 mm. This activity was influenced by the pH and the ionic strength of the sample and the reacting medium. As shown in Table 2, the largest diameter of lysis was obtained at pH4.2 (13.8_+0.12mm) and when the ionic strength was 0.175 (13.2 + 0.17 mm).

Purification of lysozyme

0

10

20

30

40

50

60

70

Eluate ( m l )

Fig. 1. Gel filtration profile of egg homogenate on Spherogel T S K 3000 SW H P L C column. Sample was 2 m l , equilibration and elution with 0.05 M phosphate buffer, p H 5.2. Protein profile monitored at 280 nm. Peak A = active fractions.

filtration resulted in two peaks of absorbance at 2 8 0 n m (Fig. 1). Lysozyme activity was tested all along the profile and revealed the presence of active fractions related to peak referred to as A. Figure 2 shows the lysis obtained in a Petri dish assay by active fractions eluted. The active fractions from seven runs were pooled, dialysed against water, lyophilized, resuspended in PB 0.05M p H 5 . 2 and loaded into a DEAE-5 PW HPLC column. Elution with a gradient of 0-0.5 M NaC1 pointed out five peaks (Fig. 3). Only the peak referred to as Al was able to lyse Micrococcus cell walls. Figure 4 shows the lysis on a Petri dish of active fractions (3 and 4 corresponding to peak A,).

Submitting 2 ml egg homogenate to gel Table

2. Effect of ionic strength lysozyme-like activity

Ionic strength 0.0175 0.175 1.75

pH 4.2 5.2 6.2 7.2 8.2

and

pH

on

Diameter of lysis (mm) 11.0 ___0.17 13.2 ___0.17 4.0 _ 0 . 1 5 Diameter of lysis (mm) 13.8 12.6 3.8 2.7 3.2

_ 0.12 _+ 0.23 _ 0.22 ___0.0 + 0.0

Fig. 2. Lysozyme activity of active fractions eluted by gel filtration (standard assay on Petri dishes).

712

L. Stabili a n d P. P a g l i a r a

AI -0.5

--' 0.5 .<

i1 /

J_J/ 0

I 15

I 30

J

t 45

Z I

- 0.25

iI

i

i

"r---

60

75

90

105

E l u a t e (ml)

Fig. 3. Ion-exchange chromatography on DEAE 5PW HPLC column. Sample was 250pl of active fractions from gel filtration. Equilibration was 0.05 M phosphate buffer, pH 5.2. Elution with a gradient from 0 to 0.5 M NaC1 (dotted line). Protein profile (continuous line) monitored at 280 nm. Peak AI = active fractions.

Discussion Internal circulating fluids of echinoderms (i.e. coelomic fluid) are generally free from bacteria (Bang and Lemma, 1962; Wardlaw and Unkless, 1978; Kaneshiro and Karp, 1980), and rarely have more than 40 bacteria/ml. Moreover, experimental infection of coelomic fluid of asteroids or echinoids showed that bacterial suspensions are cleared quickly from their body cavity (Bang and Lemma, 1962; Wardlaw and Unkless, 1978; Kaneshiro and Karp, 1980; Yui and Bayne, 1983). This indicates that, in healthy echinoderms, a strong antibacterial activity occurs. As suggested by our

Fig. 4. Lysozyme-likeactivity of active fractions (3 and 4) eluted from ion-exchange chromatography (standard assay on Petri dishes).

results, eggs also possess antibacterial compounds. These are able to inhibit the growth of some marine bacterial strains and to lyse dried Micrococcus cell walls. Thus lysozyme could play an important role in host defence mechanisms. Lysozyme-like enzymes occur in many marine invertebrates (Joll~s et al., 1957). In spite of the large distribution of this enzyme, little is known about its origin and its exact role in defence reaction. Lysozymes of different origins have usually the same bacteriolytic properties as hen egg-white lysozyme: they have a lytic action on Micrococcus lysodeikticus, Bacillus megatherium and Sarcina tiara. However, the substrate for lysozyme is contained not only in Gram-positive bacteria; the lysozyme spectrum of the Gram-negative bacteria has been established by Peterson and Hartsell (1955). Therefore, in our case, it is possible to hypothesize that this hydrolase from egg homogenate could be, at least in part, responsible for the bacterial growth inhibition. However, we cannot exclude that other killing factors are responsible for these events or that both, lysozyme and defence factors, act synergically in antibacterial defence. Our lysozyme had a maximum of activity when the pH of the reaction medium and sample was 4.2 and the ionic strength 0.175, such as some workers showed with other lysozymes (Chang and Carr, 1971). These authors further indicated that, at an ionic strength of 0.2, there was, for hen egg lysozyme, only a peak within a range of acid pH. It's interesting that fertilization envelope extracts from activated eggs of two species of fish, Plecoglossus altivelis and Tribolodon hakonensis, also have the ability to exert antifungal or fungicidal action on the fungus S. parasitica in addition to the bactericidal action (Kudo and Teshima, 1991). Most of the lysozymes have been prepared by classical, rather standardized purification procedures including a prepurification step followed by molecular sieving on Sephadex G-25 or G-75 and ion-exchange chromatography on a cationic resin (CM-cellulose 32), as referred to by Jollrs and Jollrs (1984). A lysozyme was thus isolated from Asterias rubens (Joll+s and Joll+s, 1975). It had a molecular weight

Lysozyme-like activity in Marthasterias glacialis eggs

of 15500 ___1000 and its high sensitivity to ionic strength should particularly be noted. Lysozyme-like molecules have also been isolated in marine bivalves (McHenery et al., 1979); the enzyme of the mussel Mytilus edulis has been purified (Jollrs et al., 1957) and its molecular weight found to be about 18000. We used gel filtration followed by ion-exchange chromatography to isolate the lysozyme-like molecule. In both cases, the biological activity was maintained and appeared in peaks A and Aj, respectively. Therefore, further experiments are necessary to characterize this molecule. Acknowledgements--The authors wish to dedicate this work to the late Professor C. Canicatti (1951-1993), who inspired it. This work was supported by MURST grants.

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