Hemolytic mechanism of cytolysin produced from V. vulnificus

Life Sciences, Vol. 53, pp. 571-577 Printed in the USA

Pergamon Press

HEMOLYTIC MECHANISM OF CYTOLYSIN PRODUCED FROM V. VULNIFICUS

Hyung-Rho Kim~), Hye-Won Rho, Mi-Hee Jeong, Jin-Woo Park, Jong-Suk Kim, Byung-I-lyun Park, Uh-Hyun Kim and Seok-Don Park* Department of Biochemistry, Chonbuk National University Medical School, Chonju 560-182, Republic of Korea * Department of Dermatology, Wonkwang University School of Medicine, Iri 570-749, Republic of Korea (Received in final form June 1, 1993) Summary The characteristics of hemolytic action of cytolysin produced from v. were investigated in mouse erythrocytes. The cytolysin bound erythrocyte membranes in temperature-independent manner and then lysed cells temperature-dependently. Hemoglobin release by the cytolysin was completely inhibited by the presence of raffinose or melezitose, but K÷ release was not affected. The cytolysin-induced hemolysis was always accompanied with the conversion of membrane-bound cytolysin into an oligomer of 210 kDa, corresponding to a tetramer of native cytolysins. Nonesterified cholesterol inactivated the cytolysin by converting active monomeric cytolysin into inactive oligomer. The results suggest that the cytolysin lyses erythrocytes due to the formation of small pores on erythrocyte membrane by cholesterol-mediated oligomerization of the cytolysin. vulnificus

t ' i b r i o v u l n i f i c u s is an estuarine bacterium that has been associated with septicemia and serious wound infection in persons who are immunocompromised or who have underlying diseases such as cirrhosis or hemochromatosis (I). A variety of factors, including an extracellular cytolysin (2), an elastolytic protease (3), the presence of a polysaccharide capsule (4), resistance to the bactericidal effects of sera (5), resistance to phagocytosis (6) and the ability to acquire iron from transferrin (7) have been implicated as possible virulence determinants for 7. v u l n i f i c u s in animal models. Kreger and Lockwood (8) demonstrated the existence of cytolysin in culture medium of 7. v u l n i f i c u s possessing cytotoxicity for cultured mammalian cells, vascular permeability factor and lethal activity.

Hemolysis caused by bacterial hemolysins may be of two types; colloid-osmotic hemolysis by staphylococcal a-toxin (9) or E. c o l i a-hemolysin (10), and noncolloid-osmotic hemolysis by thiol-activated hemolysins such as streptolysin O (11). A two-step process has been suggested for hemolysis by v. r u l n i f i c u s cytolysin; a temperature-independent membrane binding step and a temperature-dependent cell disruption step (12, 13). The mechanism through which 7. r u l n i f i c u s 1) To whom correspondence should be addressed. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.

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cytolysin damages erythrocyte membranes is, however, still unclear. We have therefore sought to isolate the cytolysin from erythrocyte membranes and to characterize the membrane-bound cytolysin with respect to its biochemical properties and to its capacity to generate functional membrane damages. In this paper, we present data indicating that the cytolysins are oligomerized at the membrane surface to form small pores which lead to cell lysis.

Materials and Methods Bacterial strain and culture: A virulent strain of vibrio v u l n i f i c u s E4125 was kindly supplied by Dr. M.H. Kothary (Department of Microbiology, Virulence Assessment Branch, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington D.C.). The strain was cultivated in heart infusion diffusate broth (Difco) at 37°C for 4 h as described by Kreger et al. (14). Purification of cytolysin: The cytolysin was homogeneously purified from the culture supernatant by ammonium sulfate fractionation, calcium-phosphate gel adsorption, quaternary methylamine anion exchange chromatography and octyl-Sepharose CL-4B chromatography as described by Kim et al. (15). Assay of hemolytic activity: The hemolytic activity against mouse erythrocytes was determined by the method of Bernheimer and Schwartz (16). The cytolysin was diluted with phosphate-buffered saline (67 mM NazHPO4, 77 mM NaCI, pH 7.4) containing 1 mg/ml of bovine serum albumin (PBS-BSA). One ml of the cytolysin was mixed with 1 ml of 0.7% mouse erythrocyte suspensions in PBS-BSA. The mixture was incubated at 37°C for 30 min and centrifuged. The absorbance of hemoglobin in the supernatant was measured at 545 nm. One hemolytic unit (HU) is defined as that amount which liberates half of the hemoglobin in the erythrocyte suspensions. Isolation of membrane-bound cytolysin: One ml of 2% mouse erythrocyte suspensions in PBS-BSA was incubated with 60 or 600 HU of the cytolysin at 0°C or 37°C for 30 min. After complete lysis of erythrocytes by the addition of 3 ml of distilled water, erythrocyte membranes were pelleted at 15,000xg for 10 min and washed three times with 5 mM phosphate buffer (pH 8.0). Washed membrane fractions (5 mg of protein per ml) were electrophoresed. Preparation of antiascitic fluid: Antiascitic fluid was raised in mice by the injection of the cytolysin according to the immunization procedure of Tung (17). The mice (6-8 weeks old) were injected intraperitoneally with 0.2 ml of purified cytolysin (2 a g ) emulsified with 9 volumes of complete adjuvant on days of 0, 14, 21 and 28. SDS-PAGE and immunoblotting: Samples were denatured with the same volume of 0.5 M Tris buffer (pH 6.8) containing 4% SDS, 20% glycerol and 0.05% bromophenol blue at the room temperature or by boiling for 5 min. SDS-PAGE was performed in 7.5% slab gel according to Laemmli (18). Proteins were stained with Coomassie blue or transferred to nitrocellulose paper. Blots were incubated with ascitic antibodies (dilution 1:200) followed by alkaline phosphatase-conjugated goat anti-mouse IgG (dilution 1:1000) (19). Determination of K~ concentration: K÷ was measured with a potassium electrode (Orion Research, Cambridge, MA, U.S.A.) according to manufacturer's instructions. Determination of protein:

Protein was determined by the method of Bradford (20) using bovine

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serum albumin as standard.

Results Fig. 1 shows the effect of the incubation temperature on hemolytic and binding activities of the cytolysin . erythrocyte membranes. The hemolysis by the cytolysin was temperaturedependent and marked after 2 rain lag period at 37°C, whereas no hemolysis was observed at 0°C. The result that the cytolysin did not lyse erythrocytes at 0°C prompted us to determine whether the cytolysin binds to erythrocytes at 0°C. After incubation of the cytolysin (60 I-IU) with 2% erythrocyte suspensions in the presence of 50 mM raffinose to prevent hemolysis, the hemolytic activity remaining unbound in the supernatant was determined by incubating with fresh erythrocytes at 37°C. The binding of the cytolysin to erythrocyte membranes occurred as rapidly at 0°C as at 37"C, indicating that binding process is temperature-independent. However, hemolysis occurred even at 0°C when erythrocytes were incubated with the cytolysin at high concentration (Fig. 2). The amount of the cytolysin required for hemolysis at 0°C was 300 times greater than that at 37°C. FIG. 1

1(~) 8o

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80 I

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.~ 40

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Effect of incubation temperature on hemolytic

d and binding activities of the cytolysin to

d

2o

i

0 0

2 4 IncubationTime (min)

~

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6

mouse erythrocytes. A 2% suspension of mouse erythrocytes in PBS-BSA was incubated with the cytolysin (60 HU/ml) at 37°C (©, R) or 0°C ( 0 , II) for the indicated time. For binding assay, the reaction mixture was supplemented with 50 mM raffinose to prevent hemolysis. Hemolytic activity is expressed as % of released hemoglobin during incubation and binding activity as % of hemolytic activity remaining unbound in the supernatant.

1()()

FIG. 2

80"

Hemolysis of mouse erythrocytes as a function of the cytolysin concentration. A 2% suspension of erythrocytes in PBS-BSA was incubated with cytolysin at 37°C (O) or 0°C (O) for 30 min in a total volume of 1 ml. Hemolytic activity is expressed as % release of hemoglobin.

60" o

E

#

40"

20"

f~ .1

1

10

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CytolysinConcentration(HU/ml) If hemolysis by the cytolysin is the result of a colloid-osmotic process, the addition of

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large, nonpenetrating molecules to the extracellular medium should retard hemoglobin release from the damaged cells without inhibiting pore formation. A study of the comparative release of K÷ and hemoglobin was undertaken to characterize the nature of the membrane lesion by incubating erythrocytes with the cytolysin in the presence of various sized carbohydrates as osmotic protectants at 50 mM concentration for balancing the osmotic pressure generated by hemoglobin (21). Although osmotic protectants present in the extracellular medium did not inhibit K÷ release, the effect of carbohydrates on hemoglobin release was varied with molecular size from no inhibition of hemolysis with glucose to complete inhibition with raffinose and melezitose. Sucrose partially inhibited hemoglobin release (Fig. 3). The results indicate that the cytolysin produces small pores on erythrocyte membranes, which are freely permeable to molecules smaller than sucrose.

FIG. 3

Glucose

Sucrose

I

Raffinose

Melezitose i

l)

20

40

60

80

Effect of various sized carbohydrates on the release of K÷ (D) and hemoglobin ( 1 ) from mouse erythrocytes by the cytolysin. The cytolysin (10 HU) was added to 0.35% suspension of erythrocytes in PBS-BSA containing 50 mM carbohydrates as indicated in a total volume of 10 ml. Incubation was carried out at 37°C for 30 min. Each value denotes the mean ± S.E.M.

100

Release (%) In order to characterize the binding nature of the cytolysin to erythrocyte membranes, erythrocytes were incubated with the cytolysin at 0°C or 37*<2 for 30 min and their membrane fraction was solubilized and subjected to SDS-PAGE immunoblotting. When erythrocytes were incubated with the cytolysin at low concentration, the cytolysin bound to membrane at 0°C was exclusively a monomeric form of 51 kDa, whereas membrane-bound cytolysin at 37°C was oligomeric form of 210 kDa corresponding to a tetramer of native cytolysin (Fig. 4, lanes 1, 2). When erythrocytes were incubated with the cytolysin at high concentration at 0°C, under which condition complete hemolysis occurred, oligomeric form was also found in addition to monomeric form (Fig. 4, lane 3). The result suggests that oligomerization is an essential step for hemolysis, whether it is temperature-dependent or concentration-dependent.

1

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51 k D a - - - ~

2

3

4

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Fig. 4 SDS-PAGE immunoblotting of membranebound cytolysin. Membranes from erythrocyte suspension treated with the cytolysin at low concentration of 60 HU/ml at 0°C (lane 1) or 37°C (lane 2), and with the cytolysin at high concentration of 600 HU/ml at 0"C (lane 3) or 37*(2 (lane 4) were subjected to SDS-PAGE immunoblotting without boiling denaturation. Lane 5 denotes the native cytolysin.

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Many bacterial hemolysins are known to be inhibited by the addition of lipids such as phospholipids (22), gangliosides (23) and cholesterol (12, 24) which constitute the binding sites in erythrocyte membranes. The V. v u l n i f i c u s cytolysin was also rapidly inactivated by cholesterol at 37"C, but the inactivation was markedly retarded at 0"C (Fig. 5). The inactivation by cholesterol appears due to the conversion of active monomeric cytolysin into inactive oligomer of 210 kDa (Fig. 6, lane 2). Most of the newly formed oligomers were reconverted to monomeric form by boiling for 5 min under the non-reducing condition, indicating that no disulfide bond was formed during oligomerization (Fig. 6, lane 4). 1001

FIG. 5

80 -

Effect of cholesterol on hemolytic activity of the cytolysin. The cytolysin (200 HU) was incubated with cholesterol (4 xzg) at 37°C (O) or 0°C ( 0 ) for the indicated time in a total volume of 20 xtl and its residual hemolytic activity was assayed.

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20-

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IncubationTime (rain)

1

2

3

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210 kDa

FIG. 6 SDS-PAGE of cholesterol treated-cytolysin. The cytolysin (200 HU) was incubated for 60 min at 37°C without (lane 1, 3) or with (lane 2, 4) cholesterol (4 ttg). Samples were denatured at room temperature for lanes 1 and 2, or by boiling for lanes 3 and 4.

51 k D a - - - - ~

Discussion The binding of It. v u l n i f i c u s cytolysin to erythrocytes showed similar patterns at 0"C and 37°C; the binding process is thus temperature-independent. But the cell disruption is temperature-dependent process and invariably accompanied by oligomerization of cytolysin. When mouse erythrocytes were treated with the cytolysin at 37°C, erythrocytes were lysed and membrane-bound cytolysin was assembled into oligomer of 210 kDa, corresponding to a tetramer of native cytolysin. Extracellular osmotic protectants could prevent the hemolysis but not the leakage of K÷, indicating that cell disruption is colloid-osmotic process. The intracellular K* could not pass through the normal erythrocyte membrane, but was able to permeate after the formation of pores by cytolysin. Whereas, hemolysis and oligomerization were not observed at 0"C; the cytolysin bound to erythrocytes at 0°C remained exclusively as a monomer of 51 kDa

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and there was no leak of K~ (data not shown). These results suggest that 7. v u l n i f i c u s cytolysin binds in monomeric form to membranes and then oligomerization occurs upon their subsequent collision during lateral diffusion in the lipid bilayer, in a manner analogous to that established for staphylococcal a-toxin (9). It is understandable that lateral movement in the membrane plane will be retarded at low temperature. However at high concentration of the cytolysin, hemolysis as well as oligomerization occurred even at 0°C. One possible explanation for concentration-dependent hemolysis at low temperature is that bound cytolysin molecules are close enough to overcome the hindrance of oligomerization. The data suggest that the formation of cytolysin oligomer on erythrocyte membranes is an essential step for hemolysis. In colloid-osmotic hemolysis, the initial pore size of the erythrocyte membranes formed by hemolysins such as staphylococcal a-toxin or E. c o l i a-hemolysin is small. The effective diameter of the pore formed by E. c o l i a-hemolysin is about 2-3 nm (I0) and that formed by staphylococcal a-toxin is 2.5 nm (9). When small pores are formed by hemolysins, small molecules such as K÷ and Na ÷ can penetrate the membrane but hemoglobin with an effective diameter of 4.8 nm cannot pass through the pores and its release can only occur by osmotic cell lysis. Inhibition of hemolysis by sucrose, raffinose, and melezitose suggests that hemolysis by the cytolysin is produced in a colloid-osmotic manner due to the formation of smaller pore than that of E. c o l i a - h e m o l y s i n . The effect of osmotic protectants on hemoglobin release by the cytolysin varies with their molecular sizes from no inhibition by glucose to complete inhibition by raffinose and melezitose. The diameter of monosaccharides, disaccharides, and trisaccharides are 0.72, 0.92, and 1.14 nm respectively (25). It was therefore assumed that the effective diameter of the membrane pore formed by the cytolysin is about 0.92 nm, which allows the penetration of small molecules such as K÷ and glucose but not that of large molecules such as raffinose, melezitose and hemoglobin. One of the common properties of thiol-activated toxins such as streptolysin O, tetanolysin or pneumolysin is inactivation by cholesterol and certain related sterols (24, 26). g. v u l n i f i c u s cytolysin is also inhibited by cholesterol, but it is different from other thiol-activated cytolysins by being stable to oxygen and sulfhydryl blocking agents (12). The unique observation in this study is that cholesterol alone can induce the oligomerization of V. v u l n i f i c u s cytolysin. The cytolysin oligomer formed by cholesterol had molecular weight of 210 kDa, which was identical to tetramer formed on erythrocytes. When our samples were boiled before electrophoresis, most of the oligomers were reconverted to monomeric form of 51 kDa. The oligomer formed by pore-forming toxins are so stable that they are not dissociated even in SDS unless heated to temperatures close to 100*C (9, 27). It is known that purified V. v u l n i f i c u s cytolysin boiled with SDS exhibits minor band (MW > 200,000) in addition to major band (2). Our preparation of cytolysin showed same pattern, but this minor band was a discrete band with much higher molecular weight from our oligomer formed by erythrocyte membranes or cholesterol. The inactivation of the cytolysin with concomitant formation of oligomer by cholesterol indicates that the cholesterol-induced inactivation is due to the consumption of active monomers by oligomerization occurred prior to membrane binding. On the other hand, esterified cholesterols such as cholesteryl laurate and cholesteryl myristate did not inactivate the cytolysin, showing the importance of 3'-OH group of cholesterol for the inactivation (15). The result suggests that non-esterified cholesterol is the possible membrane constituent responsible for the cytolysin-membrane interaction which leads to the disruption of cell through the formation of transmembrane pores. Acknowledgement This work was supported by a grant from Korea Science and Engineering Foundation, Republic of Korea.

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