FEMS MicrobiologyImmunology76 (1991) 211-218 © t991 Federation of European MicrobiologicalSocieties 0920-8534/91/$03.50 Published by Elsevier ADONIS 092085349100079A

211

FEMSIM 00167

Relationship of the chemical structure and immunobiological activities of lipoteichoic acid from Streptococcus faecalis ( Enterococcus hirae) ATCC 9790 O s a m u T s u t s u i 1, S u s u m u K o k e g u c h i 2, T o m o h i r o M a t s u m u r a l and Keijiro K a t o 2 1 Department of Oral and Maxillofacial Surgery H and 2 Department of Oral Microbiology, Okayama Unit,ersity Dental School, Okayama City, Japan

Received 20 December 1990 Revision received 6 March 1991 Accepted 9 March 1991 Key words: Streptococcus faecalis (Enterococcus hirae); Lipoteichoic acid; Phosphatidylglycolipid; T u m o u r necrosis factor production; Interleukin-1 production; Interferon production

1. S U M M A R Y Two molecular species of lipoteichoic acid (LTA 1 and LTA 2) were isolated from whole cells of Streptococcus faecalis (Enterococcus hirae) ATCC 9790 by hydrophobic chromatography on Octyl Sepharose CL-4B. Chemical analysis revealed that LTA 1 and LTA 2 contained two and four acyl lipid anchors respectively. ETA 1 was less active than LTA 2 in inducing cytokines, except interleukin-1 (IL-1), but their in vivo antitumour effects were similar. L T A 2 was a potent inducer of tumour necrosis factor (TNF) and interferon (IFN) production and had excellent antitumour activity against Meth A fibrosarcoma established in mice. Deacylation of LTA 2 by alkaline hydrolysis abolished these biological ac-

Correspondence to: K. Kato, Department of Oral Microbiology, Okayama University Dental School, 5-1, Shikata-cho 2 Chome, Okayama City 700, Japan.

tivities. The phosphatidylglycolipid fraction derived from LTA 2 after acid hydrolysis could also induce TNF, IFN, and IL-1 production, as well as having antitumour activity against Meth A fibrosarcoma. Therefore, the lipid anchor portion of S. faecalis LTA may play an important role in the manifestation of these various biological activities.

2. I N T R O D U C T I O N Lipoteichoic acids (LTAs) are membrane-associated amphiphilic macromolecules that are found in many Gram-positive bacteria. They usually consist of a poly(1,3) glycerophosphate backbone (which in some cases is substituted by alanyl esters or glycosidic groups) covalently linked to a glycolipid or phosphatidyl glycolipid [1,2]. Various functions and biological activities have been assigned to LTAs, and they appear to have many

212 activities similar to those of the lipopolysaccharides (LPS) found in Gram-negative bacteria [3,4]. The fact that Lipid A (the hydrophobic portion) is the biologically active center of LPS, which exhibits full endotoxic activity and has various associated immunobiological effects, has been demonstrated by many authors [5,6]. The in vitro and in vivo anti-tumour effects of Streptococcus pyogenes Sv LTA and its low toxicity compared with LPS have been reported [7,8]. However, it has been found difficult to obtain reproducibly an active anti-tumour preparation. Among the commercially available LTA preparations produced by Sigma (St. Louis, MO), S. pyogenes and Streptococcus faecalis LTA induced interleukin-1 (IL-1) and tumour necrosis factor (TNF). In this study, purified LTA was isolated from whole cells of S. faecalis according to the method of Fischer et al. [9]. The chemical structure, its ability to induce cytokine production (e.g., TNF, IL-1 and interferon (IFN)), and its in vivo antitumour effect against Meth A fibrosarcoma were examined.

3.3. Chemical modification of L TA Deacylation of LTA was carried out in concentrated ammonium hydroxide for 18 h at 25 °C, and was followed by chromatography on a Sepharose CL-6B column (1.5 x 90 cm). The LTA was treated with 1 N HCI at 95°C for 20 min. After evaporation of the HC1, the glycolipid fraction (i.e., the acid-hydrolysed LTA) was separated into the chloroform layer by shaking with chloroform-methanol-water (1 : l : 1, v/v/v).

3.1. Bacterial strain S. faecalis (Enterococcus hirae) ATCC 9790 was grown aerobically at 37 °C for 6 h in trypticase-tryptose-yeast, extract medium [10]. Cells were harvested by centrifugation and delipidated with 0.1 M sodium acetate buffer (pH 4.5)choloroform-methanol (1 : 1 : 2, v/v).

3.4. Analytical methods Total hexose, phosphorus, and glycerol were assayed by the colourimetric method described previously [11]. Fatty acid analysis of the LTA was performed by gas-liquid chromatography (GLC), as described by Ikemoto et al. [12]. Amino acids and amino sugars were determined by high-performance liquid chromatography (HPLC; Liquid Chromatograph, CCP & 8000, Toyo Soda Co., Tokyo) with specimens hydrolysed in 6 N HC1 at 100°C for 14 h under N 2. Thin-layer chromatography (TLC) was performed on silicagel G plates (Analtech, Inc., Newark, DE) as previously described [2]. The following solvent systems were employed: A, chloroform-methanol-water (65:25:4; v/v/v), B, chloroformacetone-methanol-acetic acid-water (50 : 20 : 10 :10:5, v / v / v / v / v ) . Two-dimensional TLC was developed in the first dimension with solvent A and in the second dimension with solvent B. The spots on chromatograms were detected with anaphthol/H2SO 4 reagent. The Limuh~s amoebocyte lysate gel test was also used (Limulus SIISingle Test Wako; Wako Pure Chemicals, Osaka, Japan).

3.2. Extraction and purification of L TA LTA was extracted from the delipidated cells with hot aqueous-phenol (80% phenol:0.1 M sodium acetate buffer (pH 4.5)= 1:1, v/v) and was further purified by nuclease digestion (DNase and RNase; Sigma), gel filtration column chromatography (Sepharose CL-6B; Pharmacia, Uppsala, Sweden) and subsequent hydrophobic column chromatography (Octyl-Sepharose CL-4B; Pharmacia) according to the method of Fischer et al. [9].

3.5. Tumour necrosis factor (TNF) assay Serum containing TNF was obtained as follows. Five female ICR mice (8 weeks old; Charles River Japan, Atsugi, Japan) were injected intraperitoneally (i.p.) with 1.5 mg of heat-killed Propionibacterium acnes. After 1 week, the mice were injected intravenously (i.v.) with the indicated doses of the test preparations. Serum specimens obtained from blood collected 1.5 h after injection were pooled and heated at 56 °C for 30 min to reduce their nonspecific cytotoxic activity

3. MATERIALS AND METHODS

213 against tumour cells. After ultracentrifugation at 56000 x g, 30 rain, the supernate was used for the T N F assay. Cytolytic activity of T N F was determined according to the L-929 crystal violet assay method of Ruff and Gifford [13], using actinomycin D as a sensitizing agent. LPS from Salmonella enteritidis (Bacto lipopolysaccharide W.; Difco Laboratories, Detroit, MI) served as the reference TNF-inducing agent.

LPS (Sigma; 0.1 /xg/mouse). The groups without pre-treatment were given an i.v. injection of either 100/xg of various LTAs or 0.1 /xg of E. coli LPS. The anti-tumour effects of the LTAs were determined as follows: tumour size was measured at appropriate intervals according to the formula (length × width t/2 mm), and the treated and untreated groups of mice were compared.

3.6. Interleukin-1 assay IL-1 production was assayed with peritoneal macrophages of C 3 H / H e N mice after induction with 10% ( w / v ) trypticase peptone (BBL; Microbiology Systems, Cockeysville, MD). I L - I production was determined by measuring C 3 H / H e J mouse thymocyte proliferation in the presence of a suboptimal dose of phytohaemagglutinin (PHA; Wellcome Diagnostics, Dartford, U.K.) according to the method of Vacheron et al. [14].

4. R E S U L T S A N D D I S C U S S I O N

3. 7. Interferon-c~ +/3 and IFN-y assays Serum containing I F N - a +/3 and IFN-T was obtained as follows: three female I C R mice (8 weeks old; Charles River, Japan) were injected i.p. with 1.5 mg of heat-killed P. acnes. After 1 week, the mice were injected i.v. with the indicated doses of the test preparations. Blood was collected by cardiac puncture 1.5 h (for I F N - a + /3) or 4 h (for 1FN-y) later, and the serum IFN levels were assayed by measuring inhibition of the cytopathic effect of vesicular stomatitis virus on L-cells according to the method of O k a m u r a et al. [15]. 3.8. Antitumour effects of LTA and LPS against Meth A fibrosarcoma in BALB / c mice Meth A fibrosarcoma (solid type) was inoculated intradermally into B A L B / c mice at 2 × 105 cells/mouse. Groups of five mice bearing Meth A tumours 7 - 8 mm in average diameter were pre-treated by the i.v. injection of N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide, MDP; Wako) at a dose of 1 0 0 / x g / m o u s e (saline was given to groups without pre-treatment). Four h later, the M D P - t r e a t e d mice received an i.v. injection of various L T A preparations (4-100 / z g / m o u s e ) or E. coli ( O l 1 1 : B 4

L T A was isolated from S. faecalis A T C C 9790 according to the method of Fischer et al. [9]. The crude phenol extract was subjected to Sepharose CL-6B gel chromatography after nuclease digestion (Fig. 1), and the first polymer fraction was used as the crude L T A preparation. This was subjected to subsequent chromatography using an Octyl-Sepharose CL-4B column, which separated the L T A preparations into molecular species according to differences in their content of fatty acyl groups (Fig. 2). Two L T A fractions ( L T A 1 and L T A 2) were obtained from the crude S. faecalis E T A preparation. The elution profile of L T A on Octyl-Sepharose CL-4B column was similar to that described by Fischer et al. [9]. The yield of L T A 1 and L T A 2 was 168 mg and 104 mg, respectively from 30 g of delipidated cells. The chemical composition of these L T A preparations is given in Tables t and 2. Chemical analysis

4

I }

0

~

4

i

2

§ 0

40

60

80

IO0

FRACTION NUMBER

Fig. 1. Sepharose CL-6B chromatography of the crude aqueous-phenol extract from S. faecalis ATCC 9790. The crude extract was subjected to chromatography on a Sepharose CL-6B column (2.6×87 cm) in 0.1 M sodium acetate buffer (pH 4.5). Fractions (10 ml) were collected and analysed for their phosphorus content (e) and absorbance at 260 nm (-- - --). The fractions indicated by the bar were pooled and used as the crude LTA fraction for further purification.

214 Table 2 .8O

f'~..

Fatty acid composition of LTA preparations obtained from S.

LTA 1 +

faecalis ATCC 9790

1

O

o

40

Fatty acid

LTA 1

LTA 2

Acidhydrolysed LTA 2

Cl2:0 Ct4:0 CI6:1 C t6:0 C is: t Cz~:o C19:o Unknown

1.66 " 1.59 7.47 29.93 55.9 t 1.84

0.38 2.66 6.39 29.62 54.09 2.46 3.18 1.22

0.10 1.83 5.49 29.94 54.83 2.65 3.17 1.99

I o

~o

0 10

30

50

10

90

110

FRACTION NUMBER

Fig. 2. Chromatography of crude LTA from S. faecalis ATCC 9790 on an Octyl-Sepharose column. The crude LTA preparation was fractionated by hydrophobic interaction chromatography on an Octyl-Sepharose CL-4B column (2.6×60 cm) equilibrated with 0.1 M sodium acetate buffer (pH 4.5) containing 15% propan-l-ol. The column was successivelyeluted with the same eluant and a linear gradient of 15-80% propan-l-ol in the same buffer. Fractions (6 ml) were collected and monitored by their phosphorus content (e) and absorbance at 260 nm ( - - - - - - ) . The fractions indicated by the bar were separately pooled and used as LTA 1 and LTA 2, respectively.

of L T A 1 a n d L T A 2 revealed that the fatty acid c o n t e n t of L T A 2 was a p p r o x i m a t e l y twice as high as that of L T A 1. F i s c h e r et al. [9] described that the two m o l e c u l a r species of L T A isolated from S. faecalis c o n t a i n e d respectively two a n d four acyl groups o n their glycolipid moieties. T h e y p r o p o s e d that the lipid a n c h o r p o r t i o n s of two m o l e c u l a r species of L T A were diglucosyldiacylglycerol a n d phosphatidyldiglucosyldiglycerol, respectively [1,2]. A l k a l i n e - h y d r o l y s e d L T A 2 was almost completely deacylated. A c i d - h y d r o l y s e d L T A 2 consisted m a i n l y of phosphatidylglycolipid, which c o n t a i n e d glucose, glycerol, fatty acid a n d p h o s p h o r u s in a m o l a r ratio of approximately

1.60

Percentage of total fatty acids. 2 : 2 : 4 : 1.5. Its Rf values on T L C in solvent A a n d solvent B were 0.48 a n d 0.35, respectively. T w o - d i m e n s i o n a l T L C c h r o m a t o g r a m of acid-hydrolysed L T A 2 was similar to that of phosphatidylglycolipids described previously [2]. T h e Limulus lysate assay i n d i c a t e d that the ability of 1 mg of L T A 1 a n d L T A 2 to gelate a m o e b o c y t e lysate was e q u i v a l e n t to 1.2 ng a n d 0.12 ng of LPS, respectively. T h e s e L T A p r e p a r a t i o n s were e x a m i n e d for their effects o n T N F a n d I F N p r o d u c t i o n in P. acnes-primed mice a n d IL-1 p r o d u c t i o n by m u r i n e p e r i t o n e a l m a c r o p h a g e s , as well as for their antit u m o u r effect against M e t h A f i b r o s a r c o m a in mice. L T A p r e p a r a t i o n s a n d LPS (positive control) were i n t r a v e n o u s l y injected into I C R mice p r i m e d with heat-killed P. acnes seven days previously, a n d s e r u m samples, o b t a i n e d from blood col-

Table 1 Chemical composition of LTA preparations obtained from S. faecalis ATCC 9790 Component

LTA 1

Phosphorus Glycerol Hexose Alanine Fatty acids

1824.2 a 1804.7 2462.2 434.8 241.8

a nmol/mg. b tool ratio.

LTA 2

1.00 b 0.99 1.35 0.24 0.13

1950.1 1599.0 2237.1 284.8 434.1

Acidhydrolysed LTA 2 1.00 0.82 1.15 0.15 0.22

358.1 451.1 504.9 15.9 948.2

Alkalinehydrolysed ETA 2 1.00 1.26 1.41 0.04 2.65

2210.5 1924.0 2431.8 14.2 21.2

1.00 0.87 1+10 0.01 0.01

215 l e c t e d 90 min a f t e r t h e injection, w e r e u s e d to m e a s u r e t h e c y t o s t a t i c effect on L-929 cells. A l t h o u g h t h e i r T N F - i n d u c i n g a b i l i t y was w e a k e r t h a n t h a t of LPS, as j u d g e d f r o m t h e effective d o s e r e q u i r e d to elicit a c o m p a r a b l e s e r u m level o f T N F , L T A 2 a n d a c i d - h y d r o l y s e d L T A 2 b o t h p r o d u c e d significant i n d u c t i o n o f T N F ( T a b l e 3). L T A 1 was far less active t h a n L T A 2, a n d a l k a l i n e - h y d r o l y s e d L T A 2 d i d n o t elicit significant T N F p r o d u c t i o n . A s shown in T a b l e 4, L T A 2 i n d u c e d b o t h IFN-a +/3 and IFN-y, and acid-hydrolysed LTA 2 also i n d u c e d b o t h I F N s . L T A 1 only i n d u c e d I F N - y a n d its a c t i o n was c o n s i d e r a b l y w e a k e r t h a n t h a t o f L T A 2. A l k a l i n e hydrolysis o f L T A 2 c o m p l e t e l y a b o l i s h e d its effect on I F N . S. faecalis L T A 1, L T A 2, a n d a c i d - h y d r o l y s e d L T A 2 (the glycolipid f r a c t i o n f r o m L T A 2), as well as E. coli O l l l : B 4 LPS (positive control), clearly e n h a n c e d m a c r o p h a g e IL-1 p r o d u c t i o n , which was a s s a y e d by m e a s u r i n g the i n c o r p o r a tion of [ 3 H ] - t h y m i d i n e by C 3 H / H e J mice t h y m o cytes. A l k a l i n e - h y d r o l y s e d L T A i n d u c e d m a r k e d l y less IL-1, b u t the levels w e r e still significant (Table 5).

Table 3 TNF production by LTA preparations obtained from S. faecalis ATCC 9790 Test specimen

Dose (/xg/mouse)

Cytotoxicity a (U/ml)

LTA 1

500 100 20 500 100 20 500 I00 20 500

60 < 20 < 20 8.0 X 106 3.7 × 105 4.0 X 1 0 4 1.3 x 10~ 3.7 x 105 1.9 x 1 0 4 < 20 < 20 NT b

LTA 2

Acid-hydrolysed LTA 2 Alkaline-hydrolysedLTA 2

100

20 Salmonella enteritidis LPS (positive control) Control

1

1.0 × 109 < 20

a Reciprocal of the serum dilution causing 50% lysis of L-929 cells. b NT = not tested.

Table 4 Induction of IFN-a +/3 and IFN-y in mice primed with P. acnes by LTA preparations obtained from S. faecalis ATCC 9790 Test specimen

Dose (ug/mouse)

IFN-a +/3 (IU/ml) a

IFN-y (IU/ml)

LTA 1

500 100 20

< 40 < 40 NT h

80 < 40 NT

LTA 2

500 100 20

320 160 < 40

640 160 80

Acid-hydrolysed LTA 2

500 100 20

80 < 40 < 40

320 80 < 40

500 100 20

< 40 NT NT

< 40 NT NT

Alkaline-hydrolysed LTA 2

E. coli LPS

1

1280

Relationship of the chemical structure and immunobiological activities of lipoteichoic acid from Streptococcus faecalis (Enterococcus hirae) ATCC 9790.

Two molecular species of lipoteichoic acid (LTA 1 and LTA 2) were isolated from whole cells of Streptococcus faecalis (Enterococcus hirae) ATCC 9790 b...
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