An improved medium for preparation of filterable Escherichia coli hemolysin

Journal of Microbiological Methods 8 (1988) 219- 225

219

Elsevier JMM 00266

An improved medium for preparation of filterable Escherichia coli hemolysin Tsutomu Ushijima, Megumi Takahashi and Yoshikatsu Ozaki Department of Microbiology, Shiga University of Medical Science, Otsu, Shiga, 520-21, Japan (Received 14 October 1987) (Revised version received 2 March 1988) (Accepted 24 March 1988)

Summary We found that Tween 80 has potent activity in enhancing passage of E. coli hemolysin through a membrane filter and, on the basis of this finding, we devised a medium, heart infusion broth supplemented with 0.05 o70 Tween 80 and 0.1°70 glucose (HI-TG) for preparation of culture filtrates containing fairly large amounts of hemolysin. When 27 strains of hemolytic E. coli were cultivated to late logarithmic growth phase in the HI-TG broth at 37 °C, the culture filtrates of most strains contained 64 to 128 HU50 (50o70 hemolytic unit) per ml. From these and other results, we established a routine method for partially purifying E. coli hemolysin.

Key words: Escherichia coli; Hemolysin

Introduction E. coli hemolysin is considered as one of the virulent factors in diseases such as sepsis, urinary tract infections or abscess formation [1-4]. Many investigators have found difficulties in obtaining culture filtrates which contained appreciably large amounts of hemolysin, using ordinary laboratory media [5-71. We established a routine procedure of partial purification of E. coli hemolysin by preparing an ordinary laboratory medium, heart infusion (HI) broth, suitable for preparation of culture filtrates containing large amounts of hemolysin, by supplementation of 0.05o70 Tween 80 and 0.1°7o glucose.

Correspondence to: Dr. T. Ushijima, Department of Microbiology, Shiga University of Medical Science, Otsu, Shiga, 520-21, Japan.

0167-7012/88/$ 3.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

220 Materials and Methods

Strain A hemolytic Escherichia coli strain, ATCC 25922, was obtained from the American Type Culture Collection. O f the other hemolytic E. coli strains used, four were isolated from biopsy samples of rectal mucosae of patients with ulcerative colitis and 22 strains were isolated from feces of healthy adults.

Preparation of crude hemolysin One drop of fresh E. coli culture in H I broth was inoculated into 5 ml of the same broth with or without glucose and/or Tween 80 in 15 x 105 m m glass tubes. After incubation at 37 °C for 4 - 5 h, most of the cells were removed by centrifugation at 4500 rpm for 10 min at 5 °C. The supernatant was passed through the membrane filter (Millipore Co., Bedford, MA) of 0.45 tzm pore and effective area of 4 cm 2. In some cases, the membrane filter was pretreated with ca. 1 ml of 0.1% Tween 80 in saline or 0.2% bovine serum albumin (Sigma, St. Louis, MO, USA) in distilled water and then washed with ca. 1 ml of supernatant of the centrifuged culture.

Preparation o f partially purified hemolysin Two milliliters of the fresh E. coil culture in H I broth was inoculated into 200 ml of the same broth, with or without glucose and Tween 80 in 300 ml flasks. After incubation at 37 °C for 4 - 5 h with occasional shaking, sodium azide (0.02o70 w/v) was added and most of the bacterial cells were pelleted by centrifugation at 16000 x g for 30 min at 0 - 2 °C. The residual bacterial cells in the supernatant were removed by filtration through the membrane filter (Gelman Sciences, Inc., MI, USA, Tuffryn membrane filter, HT-450) of 0.45 #m pore and 12 cm 2 effective area. Hemolysin was partially purified by polyethylene glycol (PEG) 4000 precipitation [8] with some modification. The hemolysin preparation was stored at - 2 0 ° C or below to prevent loss of activity.

Measurement of hemolytic activity The crude or partially purified hemolysin were serially diluted two-fold in calcium saline [9]. A 0.5 ml of 1% suspension of sheep erythrocytes in the calcium saline was added to 0.5 ml of the diluted hemolysin preparation and to the control (calcium saline). The tubes were incubated at 37 °C for 2 h with occasional shaking. At the end of incubation, unlysed erythrocytes were pelleted by centrifugation at 3000 rpm for 2 min. The supernatant was transferred to another tube containing 2 ml of distilled water. The optical density at 540 nm (OD540) was read using the control tube as a blank by spectrophotometer (type 124, Hitachi, Co. Ltd., Tokyo). The inverse of the dilution which caused 50% lysis of the erythrocytes was recorded as 50% hemolytic units (HUs0)/ml. Results

Influence of the addition of Tween 80 into culture media on the amount of hemolysins in culture filtrates The hemolysin titer in the filtrate of H I broth culture of E. coli ATCC 25922 was

221 about 2 units/ml, but in the presence of 0.05 - 0 . 1 % Tween 80, hemolysin titers in the culture filtrates were 128 units/ml. In the case of 0.025°7o of Tween 80, the hemolysin titer was slightly lower, i.e., 64 units/ml, than in the cases of 0.05 - 0 . 1 % of Tween 80.

Influence of pretreatment of the membrane filters with Tween 80 or bovine serum albumin solution on the amount of hemolysin in culture filtrates E. coli ATCC 25922 was cultivated in HI broth at 37 °C for 4 h. When the culture was passed through a non-treated membrane filter, the hemolysin titer in the culture filtrate was only 2 units/ml (Fig. 1). When the membrane filter was pretreated with saline containing 0.1% Tween 80, the hemolysin titer in the culture filtrate was as high as 128 units/ml. The same result was obtained when 0.1%0 Tween 80 was added to HI broth. Pretreatment of the membrane filter with 0.2% bovine serum albumin showed the same effect. Influence of addition of glucose into HI broth containing Tween 80 on the amount of hemolysin in culture filtrates Two- to four-times higher amounts of hemolysin were usually found in culture filtrates o f E. coil ATCC 25922 o f HI broth supplemented with 0.1% glucose and 0.05% Tween 80 (HI-TG) as compared with those of HI broth supplemented with only Tween 80. Influence of incubation times on the amount of filterable hemolysin When ca. 6 x 106 cells/ml o f E. coli ATCC 25922 were inoculated into HI-TG broth, the population of E. coliincreased rapidly and reached the maximum level within 5 h (Fig. 2). Amounts of hemolysin in culture filtrates were also increased concomitantly with growth of the bacteria and reached the maximum level at about 4 h, i.e.,

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Dilution of c u l t u r e f i l t r a t e Fig. 1. Influence o f pretreatment of membrane filters with Tween 80 and bovine serum albumin on permeability of the hemolysin. 1. The culture of E. colt ATCC 25922 in HI broth supplemented with 0.1%0 Tween 80 was passed through a native membrane filter. The HI broth culture was passed through a membrane filter. 2. Pretreated with 0.1%0 Tween 80 in saline. 3. Pretreated with 0.2% bovine serum albumin in distilled water. 4. No pretreatment.

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Fig. 2. Influence of incubation times on the amount of hemolysin in the culture filtrates. 1. Population of E. coliATCC 25922 in a culture of HI broth supplemented with 0.05 °70Tween 80 and 0.1°/0 glucose (HITG). 2. Population of 17. coli ATCC 25922 in the HI broth culture. 3. Amount of hemolysin in filtrates of HI-TG broth culture. 4. Amount of hemolysin in filtrates of HI broth culture.

at the late l o g a r i t h m i c g r o w t h phase. T h e m a x i m u m level o f h e m o l y s i n titer in the culture lasted for at least 24 h o f i n c u b a t i o n times. O n the contrary, when the H I b r o t h lacked s u p p l e m e n t s , the a m o u n t o f h e m o l y s i n reached the m a x i m u m level, 8 u n i t s / m l , at 4 h a n d t h e r e a f t e r d e c l i n e d sharply.

Amounts of hemolysin in filtrates of 27 strains of various broth cultures F i g u r e 3 shows the a m o u n t o f filterable h e m o l y s i n p r o d u c e d by 27 strains o f E . coli in three k i n d s o f m e d i a , H I b r o t h (Nissui), H I b r o t h s u p p l e m e n t e d with 0.1070 glucose a n d H I b r o t h s u p p l e m e n t e d with 0.1°70 glucose a n d 0.05°70 Tween 80. H e m o l y s i n titers o f filtrates o f H I b r o t h cultures o f all test strains were as low as 4 u n i t s / m l o r less. In H I b r o t h s u p p l e m e n t e d with glucose a n d Tween 80, h e m o l y s i n titers o f test strains were as high as 32 to 128 u n i t s / m l . In H I b r o t h s u p p l e m e n t e d with o n l y glucose, s o m e strains p r o d u c e d high a m o u n t s o f h e m o l y s i n b u t the h e m o l y s i n titers o f m o s t strains were f r o m 8 to 16 u n i t s / m l .

Optimal concentration of PEG for precipitation of hemolysin T h e h e m o l y s i n titer in the filtrate o f E. coli ATCC 25922 culture in HI-TG b r o t h was 256 u n i t s / m l . T h e h e m o l y s i n titers in P E G precipitates o b t a i n e d by the a d d i t i o n o f 15, 20, 25 a n d 30 g o f the P E G into 100 ml o f culture filtrates were 1 x 103, 2 x 103, 4 x 103 a n d 4 x 103 u n i t s / m l , respectively.

Heat susceptibility of the hemolysin W h e n the P E G p r e c i p i t a t e d h e m o l y s i n p r e p a r a t i o n o f 512 u n i t s / m l was h e a t e d at 56 °C for 60 min in a water b a t h , the h e m o l y s i n titer decreased to 64 u n i t s / m l .

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Fig. 3. Comparison of the amounts of hemolysinin culture filtrates of 27 strains of E. coli in three media; HI broth, HI glucose (0.1%) broth and HI Tween 80 (0.05%)-glucose (0.1070)broth.

Discussion

We prepared a more satisfactory medium, H I broth supplemented with 0.05 % Tween 80 and 0.1°70 glucose (HI-TG) for preparation of culture filtrates o f E. coli containing large amounts of hemolysin. When 27 strains of hemolytic E. coli were cultivated in HI-TG broth, culture filtrates of most of the strains contained as high as 6 4 - 1 2 8 units/ml o f hemolysin. Culture filtrates of 27 strains of hemolytic E. coli cultivated in H I broth contained only 4 units/ml or less of the hemolysin. When a membrane filter was pretreated with 0.1°70 Tween 80 or 0.2°7o bovine serum albumin, the culture filtrate of E. coli ATCC 25922 contained as high as 6 4 - 1 2 8 units/ml of hemolysin. Pretreatment of membrane filters with bovine serum albumin is usually done to prevent the adsorption of proteinous materials. Thus, the low hemolysin contents in the filtrates obtained from H I broth cultures may not be due to a scanty production of the hemolysin but rather to adsorption of the hemolysin to the membrane filters. Most of the ordinary laboratory media are unsuitable for preparation of filterable E. coli hemolysin. Smith [5] devised an alkaline extract broth for preparation of E. coli hemolysin. Jorgensen et al. [7] reported that the presence of a putative hemolysin precursor (HP) in the nutrient broth prepared according the the method of Smith [5] and the amounts of hemolysin found in culture filtrates were proportional to the concentration of the HP. In culture filtrates of the alkaline extract broth, amounts of the hemolysin reached the m a x i m u m levels at the initial [7] or late [10] logarithmic growth phase and thereafter decreased sharply. After incubation for 12-16 h, the hemolysin in the culture filtrates was already reduced to the level no longer detectable by the ordinary hemolysis test. In our studies, an identical result was obtained when E. coli was cultivated in H I broth as shown in Fig. 2. But, when E. coli was cultivated in HI-TG broth, the amounts

224 o f h e m o l y s i n in filtrates reached the m a x i m u m level at the late l o g a r i t h m i c growth p h a s e and, in c o n t r a s t to the cases o f the alkaline extract b r o t h , the high h e m o l y s i n level lasted for at least 24 h o f i n c u b a t i o n . In the P E G p r e c i p i t a t i o n o f the h e m o l y s i n , B h a k d i et al. [8] a d d e d 20 g o f the P E G to 100 ml o f the s u p e r n a t a n t s o f the c e n t r i f u g e d cultures. In o u r experiments, the maxim u m h e m o l y s i n precipitate was o b t a i n e d when 25 - 30 g (ca. 21 - 2407o w/v) o f the P E G was a d d e d to 100 ml o f the culture filtrates. F r o m these results, we established a r o u t i n e m e t h o d o f p a r t i a l p u r i f i c a t i o n o f E. coli h e m o l y s i n as follows. I n o c u l a t e fresh b r o t h culture o f E. coli into HI-TG b r o t h (ca. 1 - 5 x 106 c e l l s / m l ) a n d i n c u b a t e it at 3 7 ° C for 4 - 6 h, i.e., to the late l o g a r i t h mic to early s t a t i o n a r y g r o w t h phase, with o c c a s i o n a l shaking. A d d N a N 3 (0.0207o w/v) to the culture a n d centrifuge the p r e p a r a t i o n at 16000 × g for 30 min at 0 - 2 °C. Pass the s u p e r n a t a n t t h r o u g h a m e m b r a n e filer (0.45 # m pore) a n d a d d P E G 4000 (21-24070 w/v) to the filtrate. Dissolve the P E G a n d i n c u b a t e the p r e p a r a t i o n for 60 m i n in an ice b a t h , with o c c a s i o n a l shaking. C e n t r i f u g e the p r e p a r a t i o n at 16000 × g for 30 m i n at 0 - 2 ° C a n d carefully remove the s u p e r n a t a n t . Dissolve the precipitate in the c a l c i u m - s a l i n e [9] o f 1:100 p o r t i o n o f the v o l u m e o f the o r i g i n a l cultures. C h e c k for the absence o f viable E. coli by s p r e a d i n g 0.1 ml o f the h e m o l y s i n s o l u t i o n o n n u t r i e n t a g a r plates. Store the h e m o l y s i n s o l u t i o n at - 2 0 °C or below. W i t h this procedure, the h e m o l y s i n in the culture filtrates was c o n c e n t r a t e d 2 0 - 4 0 times a n d the p r e p a r a t i o n u s u a l l y c o n t a i n e d 1 - 8 × l03 u n i t s / m l o f hemolysin. T h e h e m o l y s i n p r e p a r a t i o n seems to be c o m p o s e d o f ca. 9007o o f t h e r m o l a b i l e a n d 10% o f t h e r m o s t a b l e h e m o l y s i n [10]. In S D S - P A G E electrophoresis [11], the h e m o l y s i n p r e p a r a t i o n exhibited a m a i n p r o tein b a n d with a m o l e c u l a r weight o f ca. 1.03 x 105 and, in s o m e cases, a faint m i n o r b a n d o f m o l e c u l a r weight o f ca. 5.8 x l04 ( d a t a n o t shown). T h e m a i n b a n d is the native h e m o l y s i n a n d the m i n o r one m a y be a d e g r a d a t i o n p r o d u c t [8].

Acknowledgement We wish to t h a n k M. O h a r a for assistance with the m a n u s c r i p t .

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225 8 Bhakdi, S., Machman, N., Nicaud, J-M. and Holland, I. B. (1986) Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores. Infect. Immun. 52(1), 63-69. 9 Cava•ieri•S.J.andSnyder••.S.(•982)Effect•fEscherichiac••ia•pha-hem••ysin•nhumanperiphera• leukocyte viability in vitro. Infect. Immun. 36(2), 455-461. 10 Snyder, 1. S. and Koch, N.A. (1966) Production and characteristics of hemolysin of Escherichia coli. J. Bacteriol. 91(2), 763-767. 11 Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.