Vol. 25, No. 3

INFECTION AND IMMUNITY, Sept. 1979, p. 939-942 0019-9567/79/09-0939/04$02.00/0

Production of Indirect Hemolysin by Yersinia enterocolitica and Its Properties MISAO TSUBOKURA,* KOICHI OTSUKI, ITSUO SHIMOHIRA, AND HIROSHI YAMAMOTO Department of Veterinary Microbiology, Faculty ofAgriculture, Tottori University, Tottori, 680, Japan

Received for publication 29 May 1979

Hemolysis was demonstrated on blood agar by adding lecithin from strains of biovar 1 Yersinia enterocolitica. Hemolytic activity was also observed in culture filtrates containing lecithin. No hemolytic activity was detected from any blood agar or culture filtrate without lecithin or after incubation at 37°C. Crude indirect hemolysin was prepared by ammonium sulfate fractionation from culture filtrates, and two enzymes, phospholipase A and lipase, were fractionated by gel filtration. Hemolytic substances were recognized as lysolecithin and fatty acid when lecithin was decomposed by phospholipase A and lipase, respectively. In a chance observation we noted that strains of biovar 1 (typed by Wauters) Yersinia enterocolitica showed hemolytic activity when cultured on a blood agar plate containing lecithin. In this paper, a description is given of the hemolytic activity of hemolysin produced by Y. enterocolitica and its properties. MATERIALS AND METHODS Strains used. A total of 202 strains of Y. enterocolitica were used in the investigation. Of them, a lecithinase-positive Vache strain (05A) and a lecithinasenegative MYO(X) strain (03), which had been supplied by S. Winblad of Lund University, Malmo, Sweden, were predominantly used. Media and tests of biological activity. Hemolytic activity was assessed utilizing rabbit blood agar containing 0.1% egg yolk lecithin (Wako Pure Chemicals, Osaka, Japan). Organisms were streaked on the medium and incubated at 250C for 48 h. Hemolysis was determined by the existence of a clear zone around the streak culture. Nutrient agar containing 1% egg yolk lecithin was used for the detection of lecithinase. Lecithinase-positive strains were identified by production of a sheen zone surrounding the streak culture. Lard medium (8) and Tween 80 medium (1) were used for the detection of lipase. The former medium was prepared from a nutrient agar containing 0.2% lard (Wako Pure Chemicals) melted by boiling water and 0.005% neutral red (Wako Pure Chemicals). Lipasepositive strains were identified by a red color around the streak culture. The latter medium was prepared from heart infusion agar (Nissui Seiyaku, Tokyo, Japan) containing 0.2% Tween 80 (Wako Pure Chemicals). A positive reaction was identified by the appearance of an opaque zone around the streak culture. Titration of hemolytic activity. Hemolytic activity (indirect hemolysin) was titrated essentially by the methods of Yanagase et al. (19) and Wakui and Kawachi (18). Serial twofold dilutions of the test materials were made in saline, and a 0.5-mi sample, 0.2 ml

of 0.01% lecithin, and 0.3 ml of a 1% rabbit erythrocyte suspension were mixed. The mixtures were shaken and allowed to stand for 2 h at 370C. After incubation overnight at 50C, hemolytic titers were calculated from the highest dilution (complete hemolysis). Titration of the hemolytic substance (direct hemolysin) was made by serial twofold dilution of the sample in saline, and each 0.5 ml was mixed with an equal amount of 1% erythrocyte suspension; then the mixtures were treated the same way as mentioned above. Assay ofcrude indirect hemolysin (CIH). Vache strain, producing a clear hemolytic zone on lecithin blood agar, was subcultured into tryptosoya broth (Nissui Seiyaku) or brain heart infusion broth (Nissui Seiyaku) and incubated at 25°C for 4 days. A cell-free culture filtrate was prepared by centrifuging the broth culture at 7,000 x g for 40 min at 5°C. Ammonium sulfate was added to the filtrate to produce 60% saturation, and it was allowed to stand overnight at 5°C. The resulting precipitate was collected and dissolved in a small amount of 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2) solution. This solution was dialyzed (cellophane tube) against 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2) solution and Iyophilized. The Lyophilized material was designated CIH. The CIH was dissolved in distilled water to 100 or 200 hemolytic units. Physical and chemical properties of CIH. (i) Heat stability. The CIH was heated at 60, 100, and 121°C for 15 or 30 min. (ii) Sensitivity to trypsin. A 0.1-mil portion of 2% trypsin (1:250; Difco Laboratories, Detroit, Mich.) was added to 0.9 ml of CIH solution and incubated for 1 h at 37°C, and 1 ml of 0.1% trypsin inhibitor (Miles Laboratories Ltd., Stoke Poges, U. K.) solution was added. Samples of phospholipase A (from bee venom; Sigma Chemical Co., St. Louis, Mo.) and lipase (from Candida cyclindracea; Sigma Chemical Co.) were used as controls. Gel filtration. The CIH was passed through a Sephadex G-100 column (2.5 by 30 cm) and eluted

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with 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2) at a flow rate of 25 ml/h. Volumes of 5 ml were collected by a fraction collector. As a control, phospholipase A and lipase were also filtered by the same procedure. Thin-layer chromatography (TLC). Ten milliliters of CIH and 1 ml of 1% lecithin were mixed, incubated at 37"C for 2 h, and lyophilized. Lipids were extracted from the lyophilized material by the method of Bligh and Dyer (2). The chloroform layer was dried in vacuo and dissolved in a small amount of chloroform. This material was applied to thin-layer plates and developed with chloroform-methanol-water (65: 25:4, vol/vol/vol) as solvent. Lecithin and lysolecithin were developed on the same plate as controls. The plate was dried and sprayed with 0.0012% Rhodamine 6GO (Chroma, Stuttgart, Germany) and was observed immediately under ultraviolet irradiation. A part of the accord with the spot was scratched and extracted with chloroform. The chloroform-soluble part was dried in vacuo, dissolved or suspended in saline, and titrated for hemolytic activity. Gas chromatography. A Shimazu FID-type column (4 mm by 2 m) was used. Conditions of analysis were as follows: temperature, 60 to 2300C; packing, silicone OV-17; support, celite 545,80 mesh; and carrier gas, N2. Lipids isolated by TLC were dried over anhydrous Na2SO4, and 2 ml of methanolic solution containing 1% p-toluenesulfonic acid was added; then the mixture was refluxed for 1 h to yield the fatty acid methyl esters. Their composition was analyzed by gas chromatography. Cell toxicity of the hemolytic substance. The hemolytic substance extracted from the thin-layer plate was dissolved or suspended in Eagle minimum essential medium (Nissui Seiyaku) and tested for toxicity to cultured cells. A 0.5-ml amount of hemolytic substance (32 hemolytic units) was poured into tubes containing cover slips (5 by 32 mm) loaded with monolayers of HeLa cells. After 2 h of incubation at 370C, the cover slips were washed with Eagle minimum essential medium. They were observed for cytopathogenic effects with or without Giemsa staining.

INFECT. IMMUN.

medium was measured for hemolytic activity. The results obtained are shown in Fig. 1. Hemolytic activity was detected in the culture filtrate from strain Vache in tryptosoya and brain heart infusion broths after 24 h of culture and reached a maximum titer after 3 or 4 days. After that, activity declined slowly. High hemolytic activity of medium no. 1 from Vibrio parahaemolyticus, as previously determined by Yanagase et al. (19), and peptone water were scarcely detected. Shaking the culture produced insignificant amounts of hemolysin. Despite much effort, no hemolytic activity was observed in the culture filtrates after cultivation at 370C. Culture filtrates from strain MYO(X) did not contain hemolysin. Physical and chemical properties of CIM. Hemolytic activity was retained at 600C for 30 min, but was inactivated at 1000C for 15 min TABLE 1. Relationship between hemolytic activity and production of lecithinase and lipase Enzyme

Enzyme production

Lecithinase

+ -

Lipase Tween 80

+ -

Lard

+ -

Hemolytic activity +_

85a 0

0 117

74 11 55 30

2 115 2 115

a Number of strains. 32

16

az: RESULTS w Hemolytic activity of Y. enterocoliticam Accidentally, it was noted that some strains of Y. enterocolitica showed hemolytic activity when cultured on blood agar containing lecithin. Ii 8 Then experiments were performed on the rela- w 4 tionship between hemolytic activity and production of lecithinase and lipase by 202 strains of Y. 2 enterocolitica. Eighty-five strains produced lec0 ithinase and hemolysin when lecithin was added to blood agar (Table 1). Lipase production dif7 1 3 0 4 9 2 8 6 5 fered with the type of medium and did not always correlate with hemolytic activity. HeDAYS AFTER CULTIVATION molytic activity was demonstrated at an incuFIG. 1. Hemolytic activity of culture filtrates from bation temperature of 250C, but not at 370C. Vache strain. Symbols: 0, Tryptosoya broth; A, brain Hemolytic activity ofculture filtrate. Two heart infusion broth; (3, shaking culture (tryptosoya strains, Vache and MYO(X), were cultured in broth); O. medium 1; x, peptone water; *, 37°C (trypliquid medium containing 3% lecithin, and the tosoya broth). -

HEMOLYSIN OF Y. ENTEROCOLITICA

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(Fig. 2A). The activity of CIH was lost by trypsin treatment (Fig. 2B).

Gel filtration. Hemolytic activity was divided into two fractions, Fr I and Fr II, by cz: Sephadex G-100. These fractions eluted like li- Ipase and phospholipase A, respectively (Fig. 3). TLC of hemolytic substances. Reacting CIH with lecithin yielded two spots (A, Rf 0.9; and B, Rf 0.15) as detected by TLC. The same two spots were observed after treatment of lecithin with phospholipase A. Lipase produced only spot A. Both spots A and B had hemolytic activity (Fig. 4). 5 15 20 Analysis by gas chromatography of spot A FRACTION NUMBER isolated after the CIH treatment detected three FIG. 3. Gel filtration of CIH by Sephadex G-100. fatty acids, myristic, palmitic, and stearic acids. Cell toxicity of the hemolytic substance. Symbols: 0, CIH; 0, phospholipase A; (3, lipase. Cell toxicity of lysolecithin (spot B on TLC) and fatty acids (spot A on TLC) were examined using Rf cultured HeLa cells. An effect of lysolecithin was 1.0 observed by detachment from glass on the nons32 32 02 A tained cell sheet. Shrinking of nuclei, poor staining of cytoplasm, and appearance of minute granules in the cytoplasm were shown with Giemsa stain. One effect of fatty acid was the marked elongation of the cytoplasm. w

u

0

w

10

0

DISCUSSION It is known that phospholipase A contained in habu (Trimeresurus flavoviridis) and bee venom is an indirect hemolysin, which manifests hemolytic activity when combined with lecithin. Lysolecithin, yielded from hydrolyzed lecithin by phospholipase A, affects erythrocytes (6, 15). Such indirect hemolysin was recognized in bacteria. Magnusson and Gulasekharam (10) reported that the hemolytic activity of Vibrio cholerae (El Tor) was related to production of lecithin-hydrolyzing enzyme. Yanagase et al. (19) reported that the hemolytic activity of V. parahaemolyticus was enhanced by lecithin, A 6(7 F

100'

1210C

15

15

B

1

O -2 ! -4 x

6 w

-8 -16

Ii 15

I 30

TIME (MIN) A) HEAT STABILITY

60 TIME (MIN) B) SENSITIBITY TO TRYPSIN

FIG. 2. (A) Heat stability and (B) sensitivity to trypsin of CIH. Symbols: _ *, CIH; =, 0, phospholipase A;z, lipase. ®,

0.5

¶19?

0 Q32

0/1

32

B

0

4 5 1 3 2 FIG. 4. Detection of hemolytic substances by TLC. Number in figure are hemolytic titers. 1, Lecithin; 2, lysolecithin; 3, CIH + lecithin; 4, phospholipase A + lecithin; 5, lipase + lecithin.

and the causal factor of this phenomenon is phospholipase A. The hemolytic activity of fatty acid, however, was known previously (12). Orcutt and Howe (13) reported that some nonhemolytic staphylococci have hemolytic activity in the presence of milk fat. This activity is due to fatty acids produced by decomposition of the fat by lipase derived from the organism. Although a few reports have appeared in the literature regarding the hemolytic activity of Y. enterocolitica (4, 7, 14), hemolytic activity observed in the present experiment was essentially different from hemolysis shown on ordinary blood agar. Lecithinase reaction is essential in biotyping of Y. enterocolitica (G. Wauters, Ph.D. thesis,

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University of Louvain, Louvain, Belgium, 1970). Because all strains of biovar 1 (lecithinase positive) have hemolytic activity in the presence of lecithin, we considered that the hemolytic activity of Y. enterocolitica was possibly related to lecithinase (phospholipase A) derived from biovar 1 strains. Because separation of pure substances with high enzymatic activity from CIH was difficult, physical and chemical properties of CIH are shown as a mixture of those of the enzymes phospholipase A and lipase. Although further study is necessary, it is evident that the lecithinase-positive strain of Y. enterocolitica produced phospholipase A, an indirect hemolysin. Vries et al. (17) reported that when phospholipase A from snake venom was inoculated into a rabbit intravenously, hemolysis may have resulted from lysolecithin produced by decomposed lecithin contained in plasma. The strains of biovar 1 Y. enterocolitica have recently been identified as different strains from typical Y. enterocolitica (5, 9, 11, 16). However, they have frequently been isolated from a variety of clinical sources in humans (3). Thus, it is necessary to have a complete understanding of their potential pathogenicity. Lysolecithin and fatty acid produced with enzymes originating from Y. enterocolitica have cell toxicity. It is unknown, however, whether or not these hemolytic substances were produced in vivo since the hemolytic activity in vitro was never observed at 370C. Further study is necessary to verify the pathogenicity, including the hemolytic activity in vivo of biovar 1 Y. enterocolitica. LITERATURE CITED 1. Aoyama, I., S. Kimura, T. Eda, and K. Iwata. 1968. Studies on the extracellular enzymes of staphylococci. 2. Examination of lipase test for identification of pathogenic staphylococci with special reference to the medium ingredients. Jpn. J. Bacteriol. 23:401-409. 2. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 3. Bottone, E. J. 1978. Atypical Yersinia enterocolitica:

INFECT. IMMUN. clinical and epidemiological parameters. J. Clin. Microbiol. 7:562-567. 4. Bottone, E. J., B. Chester, M. S. Malowany, and J. Allerhand. 1974. Unusual Yersinia enterocolitica isolates not associated with mesenteric lymphadenitis. Appl. Microbiol. 27:858-861. 5. Brenner, D. J., A. G. Steigerwalt, D. P. Falcao, R. E. Weaver, and G. R. Fanning. 1976. Characterization of Yersinia enterocolitica and Yersinia pseudotuberculosis by deoxyribonucleic acid hybridization and by biochemical reactions. Int. J. Bacteriol. 26:180-194. 6. Collier, H. B. 1952. Factors affecting the hemolytic action of lysolecithin upon rabbit erythrocytes. J. Gen. Physiol. 35:617-628. 7. Farstad, L., T. Landsverk, and J. Lassen. 1976. Isolation of Yersinia enterocolitica from a dog with chronic enteritis. Acta Vet. Scand. 17:261-263. 8. Gillespie, W. A., and V. G. Alder. 1952. Production of opacity in egg-yolk media by coagulase-positive staphylococci. J. Pathol. Bacteriol. 64:187-200. 9. Knapp, W., and E. Thal. 1973. Differentiation of Yersinia enterocolitica by biochemical reactions. Contrib. Microbiol. Immunol. 2:10-16. 10. Magnusson, B., and J. Gulasekharam. 1965. A lecithin-hydrolysing enzyme which correlates with haemolytic activity in El Tor Vibrio supernates. Nature (London) 206:728. 11. Maruyama, T., Y. Yanagawa, S. Sakai, and H. ZenYoji. 1975. A proposal on the classification of Yersinia enterocolitica. Annu. Rep. Tokyo Metr. Res. Lab. Public Health 26-1:7-13. 12. McPhedran, W. F. 1913. On the hemolytic properties of fatty acid and the reaction to the pernicious anemia. J. Exp. Med. 18:527-542. 13. Orcutt, M. L., and P. E. Howe. 1922. Hemolytic action of a Staphylococcus due to a fat-splitting enzyme. J. Exp. Med. 35:409-420. 14. Pederson, K. B. 1976. Isolation of Yersinia enterocolitica from Danish swine and dogs. Acta Pathol. Microbiol. Scand. Sect. B 84:317-318. 15. Roy, A. C. 1945. Lecithin and venom haemolysis. Nature (London) 155:696-697. 16. Sakazaki, R., K. Tamura, and T. Shimada. 1977. Numerical classification of Yersinia enterocolitica and their enteropathogenicity. In Proceedings of an International Symposium on Yersinia and Pasteurella, Mont Gabriel. 17. Vries, A. D., C. Kirshmann, C. Klibansky, E. Condrea, and S. Gitter. 1962. Hemolytic action of indirect lytic snake venom in vivo. Toxicon 1:19-23. 18. Wakui, K., and S. Kawachi. 1954. New determination of lecithinase A activity. Yakugaku Zasshi 79:867-871. 19. Yanagase, Y., K. Inoue, M. Ozaki, T. Ochi, T. Amano, and M. Chazono. 1970. Hemolysins and related enzymes of Vibrio parahaemolyticus. I. Identification and partial purification of enzymes. Biken J. 13:77-92.

Production of indirect hemolysin by Yersinia enterocolitica and its properties.

Vol. 25, No. 3 INFECTION AND IMMUNITY, Sept. 1979, p. 939-942 0019-9567/79/09-0939/04$02.00/0 Production of Indirect Hemolysin by Yersinia enterocol...
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