INFECTION AND IMMUNITY, JUlY 1987, p. 1594-15990019-9567/87/071594-06$02.00/0Copyright © 1987, American Society for Microbiology

Activities of Aeromonas hydrophila Hemolysins and TheirInteraction with Erythrocyte Membranes

SHUNJI KOZAKI,l* KEIKO KATO,' TSUTOMU ASAO,2 YOICHI KAMATA,' AND GENJI SAKAGUCHI'Department of Veterinary Science, College ofAgriculture, University of Osaka Prefecture, Sakai-shi, Osaka 591,1 and

Osaka Prefectural Institute of Public Health, Higashinari-ku, Osaka 537,2 Japan

Received 22 December 1986/Accepted 30 March 1987

The activity of hemolysin produced by Aeromonas hydrophila CA-li, isolated from an environmental source,was more sensitive to temperature than that of hemolysin produced by strain AH-1, isolated from a diarrhealcase. CA-il hemolysin failed to elicit hemolysis below 10°C. Immunoblotting analyses showed that bothhemolysins formed into oligomers in rabbit erythrocyte membrane even when no hemolysis occurred,suggesting that the binding and the subsequent oligomerization are temperature independent. Sodiumsalicylate inhibited lysis of rabbit erythrocytes by both hemolysins, but selected monosaccharides andoligosaccharides did not. Thin-layer immunostaining indicated that both hemolysins bound to phosphoglycer-ides with net negative charge but weakly to the ones with no net negative charge. Neither sphingomyelin norlysophosphoglyceride reacted with the hemolysins, whereas the hemolysins bound to free fatty acids. Theseresults suggest that the binding of either hemolysin to the membrane component, probably phospholipid,requires both negative charge of the polar head group and suitable hydrophobicity of the nonpolar tails.

Aeromonas hydrophila is widely distributed in the aquaticenvironment and has been implicated in human acute diar-rhea (1, 9, 11, 12, 16, 18, 24, 26). Some workers suggested aclose relation between hemolysin production and entero-toxigenicity of isolates from clinical and environmentalsources (9, 11, 16, 26). In previous studies (2, 3), we purifiedand characterized two hemolysins which are biologicallysimilar but immunologically distinct. Since both hemolysinscause fluid accumulation in infant mouse intestines andrabbit ileal loops and elicit cytotoxic effects on Vero cells,they are considered as a cytotoxic enterotoxin (2, 3). Theenteropathogenic action of the hemolysins seems to becorrelated with lysis of erythrocytes, but its mode of actionis not fully understood. Howard and Buckley (17) reportedthat hemolysin (aerolysin) bound to glycoprotein in erythro-cyte membrane and formed functional holes, resulting indisruption of erythrocytes. However, this observation waslimited only to rat erythrocytes possessing a relatively highsensitivity to aerolysin.

This study was undertaken to reexamine the sensitivitiesof erythrocytes from different species of animals to the twohemolysins. Some cytolytic toxins produced by gram-positive bacteria seem to cause disruption of erythrocytes bygeneration of functional holes, resulting from oligomeriza-tion of toxin (7). We demonstrated that A. hydrophilahemolysin forms into oligomers on erythrocyte membranes.This report also describes the interaction of hemolysin withthe erythrocyte membrane and its components, especiallyphospholipids.

MATERIALS AND METHODS

Preparation of hemolysins and antisera. The hemolysins ofA. hydrophila strains AH-1 and CA-11 were purified byindividual methods as described earlier (2, 3). Antiseraagainst the purified hemolysins were each obtained fromrabbits immunized by a previously described method (3).

* Corresponding author.

Determination of hemolytic activity. Hemolytic activitywas determined by procedures described earlier (3). In brief,a sample (1 ml) of a hemolysin solution, diluted in 0.01 MTris hydrochloride buffer (pH 7.2) containing 0.9% NaCl(TBS), was mixed with an equal volume of a 1% (vol/vol)erythrocyte suspension. After incubation for 90 min, unlessotherwise stated, the mixture was centrifuged, and theabsorbance of the supernatant was read at 540 nm. Thehemolysis caused with 0.05% saponin was regarded as 100%.To examine the effect of enzymatic digestion of erythrocyteson hemolytic activity, a 20% suspension of rabbit erythro-cytes was treated with trypsin or chymotrypsin (SigmaChemical Co., St. Louis, Mo.) at 1 mg/ml at 37°C for 6 h.After incubation, the erythrocytes were washed twice withTBS. To determine the inhibitory effect of sodium salicylateon hemolytic activity, a mixture of hemolysin (5 to 20 ng/ml)and a 1% rabbit erythrocyte suspension was incubated at25°C in the presence of sodium salicylate at different con-centrations. The data were expressed as the mean of dupli-cate or triplicate experiments.

Preparation of 86Rb-labeled rabbit erythrocytes. Washederythrocytes (about 5 x 109/ml) were incubated with 86RbCl(50 IxCi/ml; Amersham Laboratories, Buckinghamshire, Eng-land) at 37°C for 3 h and then at 4°C for 20 h (15). The labelederythrocytes were washed four times in TBS and resus-pended to 1% in TBS. To an 86Rb-labeled erythrocytesuspension (0.5 ml) was added an equal volume of ahemolysin solution. The mixtures were incubated undervarious conditions and centrifuged. The supernatant (0.6 ml)was transferred to a polystyrene tube, and its radioactivitywas determined with an Nal-well-type scintillation counter.The specific radioactivity released was calculated by thefollowing equation: % Maximal release = [(hemolysin-induced release - spontaneous release)/(maximal release -

spontaneous release)] x 100.The 86Rb release caused with 0.05% saponin was regarded

as 100%. Spontaneous release, determined by counting theradioactivity released from the erythrocytes in buffer with-out hemolysin, was less than 5% of the maximum. The datawere expressed as the mean of duplicate experiments.

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Preparation of hemolysin-treated erythrocyte membranes.A hemolysin solution at 40 ng/ml (20 ml) was added to anequal volume of a 1% rabbit erythrocyte suspension, and themixtures were incubated for 30 min at 4 and 37°C. The lysederythrocytes incubated at 37°C were collected by centrifu-gation at 16,000 x g and 4°C for 30 min. The unlysederythrocytes, treated at 4°C, were disrupted in 0.008 Msodium barbital hydrochloride buffer (pH 7.4), containing 0.2mM CaCl2 (barbital buffer), and centrifuged. The pellet waswashed three times and suspended in 0.2 ml of barbitalbuffer. The suspension was dissolved in an equal volume of0.01 M Tris hydrochloride buffer (pH 6.8) containing 1%sodium dodecyl sulfate (SDS), 20% glycerol, and 50 mMdithiothreitol by heating for about 5 min at 100°C, and a 20-pdportion was subjected to SDS-polyacrylamide gel electro-phoresis (PAGE). To exclude the possibility of an artifactresulting from binding of the toxin after cell lysis, weprepared toxin-treated erythrocytes under osmotic protec-tion. When the hemolysin was incubated with a rabbiterythrocyte suspension in TBS containing 30 mM Dextran 4(Mr, ca. 4,000; Serva Feinbiochemica, Heidelberg, FederalRepublic of Germany) at 37°C for 30 min, no hemolysisoccurred. The mixture was washed twice with TBS-Dextran4 and then suspended in barbital buffer. The erythrocytemembrane, collected by centrifugation and solubilized, wasalso subjected to SDS-PAGE.SDS-PAGE and immunoblotting. SDS-PAGE was carried

out in 5 to 10% linear gradient gels (19). After electrophore-sis, proteins were transferred from the gel to nitrocellulosepaper (TM-4; Toyo Roshi Co., Ltd., Tokyo, Japan) by theblotting method of Bumette (10). The nitrocellulose paperwas treated with 3% bovine serum albumin (Boehringer,Mannheim, Federal Republic of Germany) in 0.075 M phos-phate buffer (pH 7.3) containing 0.075 M NaCl (phosphate-buffered saline), to block unoccupied sites. The paper wasincubated with rabbit antihemolysin (1:200 dilution) in phos-phate-buffered saline containing 3% bovine serum albuminfor 30 min. After treating with sheep anti-rabbit immuno-globulin G conjugated with peroxidase (1:1,000 dilution;Cooper Biochemical Inc., West Chester, Pa.), the nitrocel-lulose paper was exposed to the substrate solution (0.05%3,3'-diaminobenzidine and 0.003% H202 in phosphate-buffered saline). The reaction was stopped by a rinse withdistilled water. Molecular weight was estimated with ahigh-molecular-weight calibration kit (Pharmacia Fine Chemi-cals, Uppsala, Sweden) including thyroglobulin (Mr,330,000), ferritin (half unit; Mr, 220,000), bovine serumalbumin (Mr, 67,000), catalase (Mr, 60,000), lactate dehydro-genase (Mr, 36,000), and ferritin (Mr, 18,500). These molec-ular standards, transferred to a piece of paper, were coloredby staining with 0.1% amido black lOB.TLC immunostaining. Thin-layer chromatography (TLC)

immunostaining was performed by the method of Higashi etal. (14) with a slight modification. Phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphati-dylinositol, cardiolipin, sphingomyelin, and phosphatidicacid were each developed on a silica gel-coated plastic plate(Polygram, Sil G; Marcherey-Nagel, Doren, Federal Repub-lic of Germany) with methylacetate-1-propanol-chloro-form-methanol-0.25% KCI (25:25:25:10:9 by vol); lysophos-phatidylcholine and lysophosphatidylethanolamine weresimilarly developed with chloroform-methanol-water(65:35:4); cholesterol, cholesterol esters, free fatty acids,and triacylglycerols were developed with n-hexane-diethylether-acetic acid (80:30: 1); and cerebrosides and gangliosideGM, were developed with chloroform-methanol-0.2%

TABLE 1. Sensitivities of erythrocytes from various animalspecies to A. hydrophila hemolysinsHemolysin (ng/mI) required for 50%o hemolysis at temp:

Species AH-1 CA-11l0°C 25°C 37°C lO1C 25°C 37°C

Rat 7.1 2.1 1.1 610 3.9 1.9Guinea pig 10.5 3.0 1.7 390 4.5 2.0Rabbit 7.3 4.6 7.8 420 3.8 3.8Sheep 24.0 33.0 62.0 >1,000 31.0 37.5Cow 83.0 31.5 59.0 >1,000 27.5 27.0Horse 67.0 20.0 48.0 >1,000 5.5 5.2

CaC12 2H20 (55:45:10). The plates were treated with 1%egg albumen (Difco Laboratories, Detroit, Mich.) to mini-mize nonspecific reactions, and then the hemolysin (1pug/ml), in 0.01 M phosphate buffer (pH 7.2) containing 0.15M NaCl and 3% polyvinylpyrrolidone, was allowed to reactfor 2 h at 37°C. After washing, binding to lipids was detectedwith rabbit antihemolysin and anti-rabbit immunoglobulin Gconjugated with peroxidase. The substrate solution con-tained 0.05% 4-chloro-1-naphthol and 0.01% H202 in 0.05 MTris hydrochloride buffer (pH 7.4) containing 0.2 M NaCl.Amino acid analyses. Amino acid analyses were performed

by the method of Moore et al. (22). AH-1 and CA-11hemolysins were each dialyzed against distilled water andlyophilized. The lyophilized materials were hydrolyzed for24, 48, and 72 h at 110°C with 6 N HCl (the low-boiling-pointHCl for amino acid analysis; Wako Pure Chemical Indus-tries, Osaka, Japan) in sealed glass tubes after evaporationand degassing. The hydrolysates were dried with an evapo-rator and then dissolved in 0.02 N HCl. Amino acid analyseswere performed with a Hitachi 835 amino acid analyzer.Half-cystine was determined as cysteic acid by the per-formic-acid oxidation method of Moore (21). The tryptophancontent was determined after alkaline hydrolysis followed byspectrophotometric determination (13). The amounts ofserine and threonine were estimated by extrapolation to zerohydrolysis time, and the amounts of all other amino acidswere calculated from the average values of all the samples.The experiments were repeated twice. The numbers ofresidues per molecule of two hemolysins were each esti-mated based on Mr 50,000.

Chemicals. Oligosaccharides (mannan and a-methyl man-noside) were purchased from Sigma; monosaccharides (D-mannose, glucose, galactose, and a-L-fucose) and lipidswere from Wako Pure Chemical Industries.

RESULTS

Effects of temperature on hemolytic activities on variouserythrocytes. The dose responses of AH-1 and CA-11hemolysins were measured at 37°C with rabbit erythrocytesas target cells. The concentrations of AH-1 and CA-11hemolysins causing 50% hemolysis were 7.8 and 3.8 ng/ml,respectively (Table 1). The rates of lysis of rabbit erythro-cyte with both hemolysins were temperature dependentalthough the manner in which the lysis occurred was dif-ferent. At 25°C, both hemolysins lysed rabbit erythrocytes tothe same extent. The activity of CA-11 hemolysin wasimpaired at lower temperatures, but that of AH-1 hemolysinwas not; CA-11 hemolysin lysed few rabbit erythrocytes attemperatures below 10°C, whereas AH-1 hemolysin lysederythrocytes so even at 4°C (Fig. 1). The two hemolysinslysed erythrocytes from five animal species to different

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0Ea)I

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20C 37C

50-15C 25C

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60 120Incubation Time (min)

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4C60 120

FIG. 1. Lysis of rabbit erythrocytes with hemolysins at differenttemperatures. A hemolysin solution (20 ng/ml) was mixed with anequal volume of a 1% erythrocyte suspension, and the mixtureswere incubated for 5 to 120 min. A, AH-1 hemolysin; 13, CA-11hemolysin.

extents. Rat, guinea pig, and rabbit erythrocytes were sen-sitive to both hemolysins at 25 and 37'C. The sensitivities ofhorse erythrocytes to both hemolysins at 25 and 37'C werequite different; the erythrocytes were sensitive to CA-11hemolysin but resistant to AH-1 hemolysin (Table 1). Al-though we examined an excess amount (1 ,ug/ml), we couldnot determine the doses of CA-11 hemolysin required for50% hemolysis of sheep, cow, or horse erythrocytes at 100C(Table 1). In general, the erythrocytes of different animalspecies appeared to be divided by hemolysin sensitivity intotwo groups, sensitive and resistant. The hemolysin-induced86Rb release from rabbit erythrocytes was also dependentupon temperature. At the optimal temperature (25'C forAH-1 and 37'C for CA-11 hemolysins), 86Rb release oc-curred more rapidly than hemoglobin release, reaching pla-teau in 30 min. Even at 100C, at which temperature CA-11hemolysin failed to cause hemolysis, 86Rb release was ob-served, indicating that CA-11 did some damage to rabbiterythrocyte membranes (Fig. 2). The presence of 30 mMDextran 4 in the incubation buffer had no influence onhemolysin-induced 86Rb release from rabbit erythrocytes at25°C.

Properties of rabbit erythrocyte membrane-associated he-molysins. In SDS-PAGE, AH-1 hemolysin migrated as asingle band with Mr 50,000, as did CA-11 hemolysin. The

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Incubation Time (min)FIG. 2. 86Rb release from rabbit erythrocytes with hemolysins at

different temperatures. The mixtures of hemolysin and erythrocyte(as described in the legend of Fig. 1) were incubated for 1 to 120 min.A, AH-1 hemolysin; B, CA-11 hemolysin. Radioactivity of maximalrelease was 2.8 x 104 cpm.

FIG. 3. Detection of rabbit erythrocyte membrane-associatedhemolysin by immunoblotting. Hemolysins (lane 1, AH-1; lane 5,CA-11) and erythrocyte membrane treated with hemolysin (lanes 2,3, 4, 6, 7, and 8) were subjected to SDS-PAGE. Lanes: 2 and 6,treated at 37'C; 3 and 7, treated at 4'C; 4 and 8, treated at 37'C in thepresence of Dextran 4; 9, erythrocytes without treatment withhemolysin. After blotting, strips of paper were incubated with rabbitantibody to hemolysin AH-1 (lanes 1 to 4) or CA-11 (lane 5 to 9).

native hemolysins were also confirmed by immunoblotting tomigrate to the same positions. When the erythrocyte mem-brane lysed with AH-1 hemolysin was solubilized and thensubjected to SDS-PAGE and immunoblotting, a major bandand a minor one (Mrs approximately 365,000 and 355,000,respectively) were seen. The same results were obtainedwith erythrocyte membranes treated with AH-1 hemolysin at4°C or in the presence of Dextran 4. With CA-11 hemolysinattached to erythrocytes, the two bands were also detectedby immunoblotting. No band was detected in erythrocyteswithout treatment with hemolysin (Fig. 3). Furthermore,when erythrocytes of the other animal species listed in Table1 were treated with AH-1 or CA-11 hemolysin, immunoblotssimilar to those shown in Fig. 3 were obtained (data notshown).

Effects of various substances on hemolytic activity. Sodiumsalicylate inhibited not only hemolysis (Fig. 4) but also the86Rb release (data not shown) of both hemolysins. In addi-tion, treatment of rabbit erythrocytes with trypsin orchymotrypsin had no effect on lysis with either hemolysin.Monosaccharides (final concentration; 60 mM) and oligosac-charides (125 p,gIml) failed to affect the hemolytic activity ofeither hemolysin.

Demonstration of binding of hemolysins to lipids by TLCimmunostaining. Direct binding of AH-1 and CA-11 hemo-

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12.5 25.0 50.0 12.5 25.0 50.0Sodium Salicylate (mM)

FIG. 4. Inhibitory effects of sodium salicylate on the hemolyticactivities of hemolysins. A, AH-1 hemolysin; B, CA-11 hemolysin.A mixture of a hemolysin solution and an erythrocyte suspensionwas incubated at 25'C in the presence of sodium salicylate. Thehemolysin solutions tested were at 5 (O), 10 (A), and 20 (0) ng/ml.

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1 2 3 4 5 6FIG. 5. Binding of hemolysins to phosphoglycerides, detected

by TLC immunostaining. A, AH-1 hemolysin; B, CA-11 hemolysin.Lipids in amounts of 10 jig were developed on a TLC plate. Lanes:1, phosphatidylcholine; 2, phosphatidylethanolamine; 3, phosphati-dylserine; 4, phosphatidylinositol; 5, cardiolipin; 6, phosphatidicacid.

lysins to various lipids (each at 10 ,ug) was examined. AH-1hemolysin bound firmly to phosphatidylserine, phosphati-dylinositol, cardiolipin, and phosphatidic acid, whereas itbound only weakly to phosphatidylcholine and phosphatid-ylethanolamine (Fig. 5A). No binding of AH-1 hemolysin tolysophosphatidylcholine, lysophosphatidylethanolamine,sphingomyelin, cerebrosides, or ganglioside GM1 was ob-served. Among neutral lipids, free fatty acids and cholesterolshowed binding with AH-1 hemolysin; the binding of thehemolysin to the former was stronger than that to the latter.AH-1 hemolysin did not bind to cholesterol esters ortriacylglycerols (Fig. 6). CA-11 hemolysin was also found tobind to phospholipids and neutral lipids in the same patternsas those of AH-1 hemolysin (Fig. 5B and 6).Amino acid analyses. The amino acid compositions of

AH-1 and CA-11 hemolysins are shown in Table 2. AH-1hemolysin contained more aspartic acid and histidine resi-dues than did CA-11 hemolysin, whereas the latter containedmore methionine residues. The absence of cysteic acidsuggests that neither hemolysin possesses any disulfide bondin its molecule.

smallest amount of AH-1 hemolysin for lysis. The activity ofthis hemolysin on rat erythrocytes at 37°C was found to beabout 7 and 60 times as high as those on rabbit and oninsensitive cow and sheep erythrocytes, respectively. Wealso found that rat, guinea pig, and rabbit erythrocytespossessed similar sensitivities to CA-11 hemolysin at tem-peratures between 10 and 37°C. Aerolysin seems to resembleAH-1 hemolysin more closely than CA-11 hemolysin; theisoelectric points of aerolysin (5.39 and 5.46) reported byBuckley et al. (8) were nearly the same as those of AH-1hemolysin (5.43 to 5.58) (2, 3). AH-1 and CA-11 hemolysinswere compared at different temperatures; CA-11 hemolysinwas more markedly influenced, and its activity disappearedbelow 10°C.The 86Rb release from rabbit erythrocytes with AH-1 and

CA-11 hemolysins occurred within a few minutes, precedinghemoglobin release. Even when no hemolysis occurred inthe presence of Dextran 4 or at a relatively low temperature,the 86Rb release was observed. These findings may suggestthat both hemolysins generate transmembrane pores whichpermit rapid passage of cations. Since lysis of rabbit eryth-rocytes with each hemolysin was inhibited by Dextran 4 asan osmotic protectant, the pores generated on erythrocytemembrane may be of about 3-nm diameter (6). This is inaccordance with previous observations (17).Such pore-forming toxins as staphylococcal ot-toxin and

streptolysin 0 have been reported to aggregate on themembrane (7). We therefore examined the shape ofhemolysin bound to erythrocytes. Immunoblotting analysisshowed that hemolysins associated with lysed rabbit eryth-rocyte membrane formed oligomers with Mrs of 355,000 and365,000, being likely to be heptamers or octamers. Wecannot explain at present how the hemolysins changed totwo different forms. It may be considered that such oligo-mers resulted solely from aggregation of hemolysin but notfrom association of hemolysin with some erythrocyte-membrane component, since prolonged aging or concentra-tion by ultrafiltration of the hemolysins induced the sameoligomers migrating to the same positions in SDS-PAGE andimmunoblotting (unpublished data). The oligomerizationtook place also on erythrocytes treated with either

DISCUSSION

Some workers reported that erythrocytes from variousmammals differ greatly in sensitivity to A. hydrophilahemolysin (aerolysin) and that rat and mouse erythrocytesare most sensitive to it (5, 17). In the present investigation,of all erythrocyte types tested, rat erythrocytes required the

_-CtoLesteroL esters-TriaCylglycerOLS

-Free fatty acids

Cholesterol

1 2FIG. 6. Binding of hemolysins to neutral lipids, detected by TLC

immunostaining. Lanes: 1, AH-1 hemolysin; 2, CA-11 hemolysin.Lipids (10 ,ug each) were developed.

TABLE 2. Amino acid composition of A. hydrophila hemolysinsResidues/molecule of hemolysin'

Amino acidAH-1 CA-11

Lysine 25 25Histidine 9 6Arginine 20 18Tryptophan 14 17Cysteic acid 0 0Aspartic acid 61 42Threonine 31 38Serine 37 44Glutamic acid 41 49Proline 26 28Glycine 45 47Alanine 34 29Valine 35 31Methionine 3 8Isoleucine 25 22Leucine 28 23Tyrosine 20 26Phenylalanine 14 17

a Based on the molecular weight of 50,000.

A

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hemolysin at 4°C, indicating that the binding and the subse-quent oligomerization are temperature independent.The activities of AH-1 and CA-11 hemolysins on rabbit

erythrocytes were not affected by any selected monosac-charide or oligosaccharide. Judging from this, a carbohy-drate might play little role in hemolysis. Amino acid analysesshowed that neither hemolysin contained any cysteic acid.These results support the observation by Bernheimer et al.(5) that the activity of aerolysin was not affected by sulfhy-dryl inhibitors. No significant differences between thehemolysins were found from the amino acid compositiondata, however. On the basis of these results, the differencesin hemolytic activity along with a certain immunologicalcross-reactivity (3) between the two hemolysins may be dueto diversity in the amino acid sequence. Howard andBuckley (17) suggested that a glycoprotein liberated from raterythrocytes by trypsin treatment may be the receptor ofaerolysin. They stated also that rabbit erythrocytes containlittle or no such glycoprotein and that hemolysis of rabbiterythrocytes with aerolysin may be due to its direct interac-tion with the lipid bilayers (17). Their statements are con-sistent with our present results in that rabbit erythrocytestreated and untreated with a protease were lysed with eitherhemolysin to the same extent, which suggests that thehemolysins, like staphylococcal a-toxin (7), may interactwith the lipid bilayers of the erythrocyte membrane.Sodium salicylate effectively inhibited both hemolysins.

Since sodium salicylate is considered to interact with thephospholipid bilayer and to alter the membrane potential(20), the present results may indicate that the hemolysinbinds to phospholipids in the erythrocyte membrane. Aprevious report (4) showed that aerolysin was not inacti-vated with any lipids tested in aqueous solution. Testing alipid in an aqueous solution for interaction, however, mightbring forth an erroneous result due mainly to some uniquephysicochemical property of the lipid such as micellar for-mation. Recently, the TLC immunostaining technique hasbeen developed to examine a test material for direct bindingto lipid components (14, 25). We employed this technique todemonstrate the binding of hemolysins to lipids. On the TLCplate, hemolysins bound firmly to phosphoglycerides withnet negative charge but weakly to the ones with no netelectric charge at neutrality. Neither sphingomyelin norlysophosphoglyceride, which removes one fatty acid mole-cule from a phosphoglyceride, reacted with the hemolysin,whereas free fatty acids bound to the hemolysins. Cere-brosides or ganglioside GM, did not react to the hemolysins.These observations suggest that the binding to lipid requiresboth a negative charge on the polar head group and suitablehydrophobicity of the nonpolar tails. We speculate that A.hydrophila hemolysins bind to membrane components(probably phospholipid) with net negative charge, enter themembrane, and interact with the nonpolar tails of phospho-lipids by hydrophobic-hydrophobic interaction, eventuallyforming functional channels and disrupting membrane per-meability. No satisfactory explanation has been given, how-ever, to such irreconcilable findings as the fact that the twohemolysins, binding to the same phospholipids, exhibitedhemolytic activities different from each other on erythro-cytes of different animal species and at different tempera-tures. Though a previous report (23) showed that there aremarked variations in the phospholipid contents in erythro-cytes from various mammalian species, such differencesseem to be unrelated to sensitivity to hemolysin. Differencesin the integrity of the target lipid bilayers may affect sensi-tivity to hemolysin. Further investigation with artificial

membrane consisting of phospholipids is required to clarifythe differences in the mode of action of A. hydrophilahemolysins.

ACKNOWLEDGMENTS

We thank Kohei Oda and Yoshihisa Nakano, Department ofAgricultural Chemistry in our University, for their helpful advices.

This investigation was supported by a grant-in-aid for cooperativeresearch (no. 61304030) from the Ministry of Education, Science,and Culture of Japan.

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Aeromonas hydrophila. J. Med. Microbiol. 10:317-323.2. Asao, T., Y. Kinoshita, S. Kozaki, T. Uemura, and G. Sakaguchi.

1984. Purification and some properties of Aeromonas hydro-phila hemolysin. Infect. Immun. 46:122-127.

3. Asao, T., S. Kozaki, K. Kato, Y. Kinoshita, K. Otsu, T. Uemura,and G. Sakaguchi. 1986. Purification and characterization of anAeromonas hydrophila hemolysin. J. Clin. Microbiol. 24:228-232.

4. Bernheimer, A. W., and L. S. Avigad. 1974. Partial character-ization of aerolysin, a lytic exotoxin from Aeromonas hydro-phila. Infect. Immun. 9:1016-1021.

5. Bernheimer, A. W., L. S. Avigad, and G. Avigad. 1975. Inter-actions between aerolysin, erythrocytes, and erythrocyte mem-branes. Infect. Immun. 11:1312-1319.

6. Bhakdi, S., N. Mackman, J. Nicaud, and I. B. Holland. 1986.Escherichia coli hemolysin may damage target cell membranesby generating transmembrane pores. Infect. Immun. 52:63-69.

7. Bhakdi, S., and J. Tranum-Jensen. 1985. Membrane damage bychannel-forming proteins: staphylococcal a-toxin, streptolysin-0 and the C5b-9 complement complex. Biochem. Soc. Symp.50:221-233.

8. Buckley, J. T., L. N. Halasa, K. D. Lund, and S. Maclntyre.1981. Purification and some properties of the hemolytic toxinaerolysin. Can. J. Biochem. 59:430-435.

9. Burke, V., M. Gracey, J. Robinson, D. Peck, J. Beaman, and C.Bundell. 1983. The microbiology of childhood gastroenteritis:Aeromonas species and other infective agents. J. Infect. Dis.148:68-74.

10. Burnette, W. N. 1981. "Western blotting": electrophoretictransfer of proteins from sodium dodecyl sulfate-polyacry-lamide gels to unmodified nitrocellulose and radiographic detec-tion with antibody and radioiodinated protein A. Anal. Bio-chem. 112:195-203.

11. Cumberbatch, N., M. J. Gurwith, C. Langston, R. B. Sack, andJ. L. Brunton. 1979. Cytotoxic enterotoxin produced by Aero-monas hydrophila: relationship of toxigenic isolates to diarrhealdisease. Infect. Immun. 23:829-837.

12. George, W. L., M. M. Nakata, J. Thompson, and M. L. White.1985. Aeromonas-related diarrhea in adults. Arch. Intern. Med.145:2207-2211.

13. Goodwin, T. W., and R. A. Morton. 1946. The spectrophoto-metric determination of tyrosine and tryptophan in proteins.Biochem. J. 40:628-632.

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