Novel Type of Endotoxin Structure Present in Bordetella pertussis. Isolation of Two Different Polysaccharides Bound to Lipid A

Eur. J. Biochem. 84, 579 -589 (1978)

A Novel Type of Endotoxin Structure Present in Bo rdetella pertussis Isolation of Two Different Polysaccharides Bound to Lipid A

Annick LE DUR, Martine CAROFF, Richard CHABY, and L.adislas SZARO

Lquipc No. 55 du Centre National de la Recherche Scientifiquc, lnstitut de Biochimie, Univcrsite de Paris-Sud, Orsay

(Received October 20, 1Y77)

The endotoxin of Bort~etc~llapertus.sis was cleaved by mild acidic hydrolysis to yield a polysaccharide (polysaccharidc I, 15 %), a glycolipid (63 x ) and lipid X (2 %). Further treatment of the glycolipid with stronger acid released a second polysaccharide (polysaccharide 11,9 Yi,) and material similar to lipid A prcscnt in entcrobactcrial cndotoxins. Both polysaccharides possess a single molecule of 3-deoxy-2- octulosonic acid as the reducing, terminal sugar. In polysaccharide I1 the octulosonic acid is phosphorylated in position 5 and presumably substituted in position 4; in polysaccharide I the octulosonic acid is not phosphorylated, but is substituted in position 5. Following treatment of the endotoxin with strong basc, a fragment was isolated that contained bound, non-phosphorylated 3- deoxy-2-octulosonic acid, glucosamine phosphate and fatty acids. This indicated that polysaccharide I , like polysaccharide 11, was bound to the lipid region of the endotoxin. The endotoxin structure thus defined is different from that proposed for the lipopolysaccharides of enterobacteria.

Endotoxins of gram-negative bacteria are macro- molecules of the outer membrane of the cell envelope and account for more than half of the mass of this membrane [ I , 21. It has been established [3] that for many enterobacteria their gross structure is similar: a species-specific polysaccharide chain, made up of re- peating units is bound to a single sequence of sugars, called the ‘core’ which, in turn, is bound to a complex lipid usually referred to as ‘lipid A’. In the vast majority of cases investigated hitherto 3-deoxy-D-manno-2- octulosonic acid appears to serve as the link between the core and the complex lipid. Cleavage of the very acid-labile deoxyketosidic bond releases the water- soluble polysaccharide-core part, while the complex lipid which is devoid of neutral sugars forms a water- insoluble precipitate. Detailed chemical structures have been established for many bacteria which mostly belong to the family Enterobacteriaceae [3]. The structure of the core region has been less often investigatcd. Since in the wild-type (‘smooth’) bacteria it represents only a small part of the macromolecule, detailed chemical structures for this region of Salmonella endotoxins were obtained [3] by use of mutants which arc cither unable to attach the side chain to the core (Ra

Dedicated to Professor Fdgar Lederer on the occasion of his 70th birthday.

mutants) or are deficient in the biosynthesis of the core itself (Rb-Re mutants). An ‘epimeraseless’ strain [4] of Escherichia coli which produces an endotoxin devoid of the specific polysaccharide chain was used to establish [5] that three different core structures were present in the incomplete endotoxin. Fractional molar concentration values for amounts of extracatenary core sub- stituents were also found in Salmonellae [6] and heterogeneity of the core region was demonstrated for R forms of Shigellae [7] as well as for E. c d i [8] by use of serological methods. There is thus ample evidence that different structures may be present in the endotoxin of a given microorganism and this has been hitherto referred to as ‘microheterogeneity’ [9]. Another typc of heterogeneity within endotoxin preparations appears to be the incomplete substitution of the core regions by the specific polysaccharide chains [lo]. It is not established whether such types of heterogeneity are of general occurrence in endotoxins, nor is it known if they are due to different types of endotoxin molecules present in any given batch of material. The purity [ I 11 and homogeneity [12,13] of endotoxin preparations have been questioned by several authors.

Endotoxin possessing a structure differcnt from that elaborated by enterobacteria appears to be present in Bordetella pertussis (Brucellaceae) ; as reported in this paper, comparable amounts of two different poly-

580 Novel Endotoxin Structure in Bordetella pertussis

saccharides can be isolated from this endotoxin. Both represent entire specific polysaccharide-core regions, both possess as reducing, terminal sugar a single molecule of 3-deoxy-2-octulosonic acid and both are bound to the complex lipid moiety of the endotoxin.

MATERIALS AND METHODS

Bordetellapertussis strains L84 phase 1 and IV were obtained from Dr Jean M. Dolby (The Lister Institute of Preventive Medicine, Elstree, United Kingdom); strain 1414, phase I, is a vaccine strain of the Institut Merieux (Lyon). All strains were grown in the liquid medium of Cohen and Wheeler [14] in 40-1 fermenters. The large-scale cultures of phase IV bacteria contained about 20 % of phase I cells (reversion). The endotoxins were extracted from the wet, sedimented cells with phenol/water [ 151 and purified by repeated high-speed centrifugation at 150000 x g for 2 h.

Analytical methods used to estimate aldohexoses, aldoheptoses, hexosamines, phosphorus and fatty acids were the same as employed previously [16, 171. 3- Deoxy-2-octulosonic acid was estimated by the mo- dified [16] thiobarbiturate reaction [18], with semicarbazide [ 191 and with diphenylamine [20]. Conditions for gas-liquid chromatography (in the present study only the SE-30 column was used) and mass spectrometry were described previously [17]. 0.2 M buffer of pH 7.2 was obtained by dissolving N-methylmor- pholine (200 ml) and acetic acid (76 ml) in water (final volume: 10 I).

Analytical data refer to lyophilised material equili- brated with ambient humidity. The water content of the material, determined by keeping it at 100 C in vucuo until constant weight was reached, was found to be 8 - 10 % (w/w).

RESULTS

Release of Polysaccharide I fr-om the Endotoxin

The endotoxin (5.5 g) from strain 1414 was sequen- tially extracted with chloroform, toluene/methanol ( I / ] ) and methanol at room temperature, The dried residue (5.445 g) was suspended in aqueous trifluoroacetic acid of pH 3 (1360 ml, 4mg/ml) and stirred at 50 “C until an homogeneous, opalescent dispersion was obtained. If necessary the pH was readjusted to 3 and the stirring continued at the same temperature until the thiobarbiturate reaction of a sample reached a constant value (Fig. I ) (60 - 90 h). The precipitated crude glycolipid was removed by centrifugation ( I 6000 x g, 30 min), washed with trifluoroacetic acid of pH 3 and lyophilised (3.4 g). The clear supernatant was dialysed and the retained material freeze-dried (polysaccharide I, 940mg, 17% w/w). This material was purified by acrylamide gel chromatography (Biogel P10, 100-

04. E

% 0 3 -

c 0

c 5

g 02. 5 ? =I 01- n Q /.’

I 10 20 30 40 50 60

Time ( h ) Fig. 1. Kinetiu of the ridease of pol.vsucchuride I ,from B. pertussis endotoxin treated with trifluoroacetic acid at p H 3 and 50 C, us measurcd ?y the uppeurance of thiohurbiturate-positive material

1 2 3 big 2 Sedimentation puttern (Jynthetic f ionlre l ) ofpolvrnctharide I 14ing/ml in 0 1 M KCI at 360000 x z (mean value), 8 C Migration from left to right Pictures were taken at 8 ( l ) , 24 (2) and 56 ( 3 ) min after reaching full speed (1 5 min) Phase pldtc anglc 60

200mesh, 95 x 2.5 cm; charge: 30mg in 2ml of water; eluent: water); a very small amount of material appeared in the void volume and was discarded; the main fraction emerged as a single, symmetrical peak and represented 90 % of the material ; it was recovered by lyophilis’ation. Polysaccharide I thus obtained appeared to be homogeneous during sedimentation (Fig. 2). It was free of fatty acids and contained heptose (20.0 %), aldohexose (8 y;), glucosamine (19.0 %) but no phosphorus. The content of 3-deoxy-2-octulosonate was 7.8 % as measured by the diphenylamine test [20], 8.8 by the semicarbazide test [I91 and 1.1 by the thiobarbiturate test [ 161, using crystalline ammonium 3-deoxy-~-manno-octulosonate as standard. The terminal reducing sugar was the octulosonate, as upon treatment with sodium borohydride the positive diphenylamine and thiobarbiturate reactions disappeared.

Suh~titution Pattern of the Terminal 3-Deouj- 2-octulosonate Molecule in Polysaccharide I

Polysaccharide I (2mg) was dissolved in water (1 ml) and to thc cooled (0 C) stirred solution 0.1 M

A. Le Dur, M Caroff, R. Chaby, and L. Szab6 581

C H 2 0 A c 7OOH C OONa COOEe

7HOH CHOH CHOH CHOAc CH20H I I I

I 1 s

CH 1 2 y 2 - R ~ ~ - O C H __+ H+H - A c O h

cn 1 2

3 HOCH 11,l HOYH 111 HOCH 1 , V l AcOCH 7 H 2

I

I - lV,V ROCH

OH, COOH H y OH CH20H CH20H C H 2 0 A c

HCOH I R-o CH20H

R e a g e n t s : 1, N a B H 4 ; ii, N a I 0 4 ; 111, H'; i v , OH-; v, M e O H / I R 1 2 0 ( H + ) f o l l o w e d by CH2N2; v i , A c 2 0 / p y r i d i n e

Scheme

NaBH,(l ml) was added; 30 min later glacial acetic acid (20 pl) was added followed by 70 mM NalO, solution (0.4 ml). The stirred, cooled (0 C) mixture (pH 3 - 4) was kept for 12 h. Its pH was then adjusted to about 7 (5 M NaOH, 50 pl) and 0.5 M NaBH, solution (0.3 ml) was added at 0 C to the stirred solution, followed 30 min later by glacial acetic acid (20 pl). After removal of the solvents, 1 M HCI (1 ml) was added to the dry residue and the mixture was kept at 100 C for 5 h. The solvent was then evaporated and the residue kept in a desiccator (KOH) overnight. The solid was dissolved in a small amount of water, the solution passed through a small column of Amberlite 1R 120 H + resin ( I - 2 ml) and the effluent taken to dryness. Methanol was evaporatcd several times from the residue which was then taken up in water. The solution was warmed to 50 - 60 C for 15 min and its pH continuously adjusted to 9 with dilute barium hydroxide solution. The solvent was removed and dry ethanol was evaporated several tiines from the residual solid which was then kept overnight in vacuo over phosphoric oxide; anhydrous methanol and some dry Amberlite IR 120 H + resin were then added and the suspension was thoroughly mixed. Solids were filtered off and a slight excess of ethereal diazomethane solution was added to the filtrate which was then taken to dryness. Ice-cold 0 5 M NaBH, solution (0.3 ml) was added to the residue and the mixture was allowed to stand (2 h, 0 C). Excess acetic acid was then added and the cations removed (Amberlite IR 120 H') before the solution was taken to dryness. Methanol was evaporated several times from the dry residue which was then treated with a mixture of acetic anhydride/pyridine (1 / l ) for 1 h at 100 C and at 40 C overnight. Solvents were then removed and the residue was extracted with ethyl acetate. The soluble material was analysed by gas chromatography (3 xSE30 column, 155 "C, isothermal) combined with mass spectrometry. Peaks likely to represent deoxy- alditol acetates were selected by monitoring fragments characteristic for this class of compounds. Mass spectra of all of these peaks were taken; their analysis indicated that the only deoxy-alditol acetate present in the mixture corresponded to a 3-deoxy-hexitol peracetate. It had the same retention time (alone and with authentic

markers added to the sample) and the same mass spectrum as the pair of epimeric hexitol acetates obtained from glucometasaccharinic acid after re- duction of the carboxyl group (3-deoxy-~-ribo-hexi- to1 acetate and 3-deoxy-~-arabino-hexitol acetate, Scheme). It has been shown previously that the reaction sequence described above when applied to 5-0-benzyl- 3-deoxy-2-octulosonic acid 8-phosphate [21] and to 3-deoxy-5-O-(~-~-glucopyranosyl)-2-octu~osonic acid [22] gave 3-deoxy-hexitol peracetates (3-deoxy-L-rilm hexitol peracetate and L-auabino-hexitol peracetate) which had the same retention time and identical mass spectra.

Purification of the Crude Glycolipid; Isolation of Lipid X

The lyophilised, crude glycolipid (3.4g) was ex- tractcd with a mixture ( l / l , v/v) of toluene/methanol (5 x 300ml). 'Lipid X' [23] was recovered from this extract. The residual glycolipid (3.2 g) contained hep- Lose (5.473, aldohexose (5.1 x), glucosaminc (1470, phosphorus (2.3 %) and fatty acids (37.8 y<, as tetradecanoic acid). Although it gave a negative thiobarbiturate reaction, the presence of a 3-deoxy-2-octulosonic acid as one of its components could be demonstratcd: following mild acide hydrolysis (0.25 M HC1, 100"C, 30 min) an apparent 3-deoxy-octulosonatc content of 0.24% was measured with thiobarbiturate; upon treatment with strong acid a phosphorylated 3-deoxy-2- octulosonic acid was released from the glycolipid. A sample of glycolipid (1 mg) was treated with 2 M HCI (2h, 100 .C). The hydrolysate, freed from HCI by evaporation to dryness irz i~acuo, was submitted to paper elcctrophoresis (pH 5, 53 V/cm, 75 min) and the paper was sprayed with the pel-iodatclthiobarbiturate reagent [24]. Two compounds were detected. The minor one (traces) migrated like authentic 3-deoxy-~- manno-2-octulosonic acid, while the major one had the same mobility as authentic 3-deoxy-~-munno-2-octulosonic acid %phosphate [21] or the corresponding 5-phosphate [25]. It was indistinguishable from the 3- deoxy-2-octulosonic acid phosphate released upon acidic hydrolysis (2 M HC1, 100 C , 2 h) of the whole

582 Novel Endotoxin Structure in Bordetella pertussis

endotoxin. It has been established previously [16] that this was a 3-deoxy-2-octulosonic acid 5-phosphate. Only free, non-phosphorylated 3-deoxy-2-octulosonic acid was released from polysaccharide I upon similar treatment.

Releuse of Polysucchuride II from the Glycolbid

Kinetics. As the phosphorylated, terminal, reducing 3-deoxy-2-octulosonic acid of polysaccharide I1 responds only very weakly in the thiobarbiturate test (see below) the kinetics of the cleavage of the glycosidic bond by which polysaccharide I1 was attached to the complex lipid could not be followed directly. Conditions for the 'selective' cleavage of this bond were established as follows. Accurately weighed samples (1 - 2 mg) of the glycolipid were treated sep- arately with 0.25 M HCI (0.5 ml) at 100°C for variable lengths of time (0 - 90 min) ; the samples were taken to dryness in vucuo at room temperature. Any residual HCI was removed by keeping the samples over KOH pellets overnight in vucuo and then NaBH, solution (1 ml, 1 mg/ml, 0 C, 30min) was added: terminal octulosonic acid molecules with free glycosidic carbon atoms were thereby reduced to thiobarbiturate- negative 3-deoxy-octonic acids. After addition of acetic acid (1 00 PI) the samples were taken to dryness and then treated with 2 M HCI (1 ml, 100 C, 90 min) as it has been established previously that the glycolipid gave a maximal response in the thiobarbiturate test following such acidic hydrolysis. The content of 3- deoxy-octulosonate was then estimated with thiobarbiturate. The progress of the cleavage of the glycosidic bond of 3-deoxy-octulosonic acid was calculated from the difference between the apparent octulosonate content of the glycolipid and the apparent octulosonate content of the treated samples. The results, expressed as percentage of the octulosonate set free as a function of time of hydrolysis with 0.25 M HCl, are represented in Fig. 3. To minimise the presumed, concommitant hydrolysis of other bonds present in the glycolipid, for the isolation of polysaccharide I1 an hydrolysis time of 30min was chosen; at this point 75-80';/, of the octulosonate present had its glycosidic bond cleaved.

Isolution. Purified glycolipid (3.3 g) was treated with 0.25 M HCI (1 600 ml) for 30 min at 100 "C under constant stirring. The cooled, heterogeneous mixture was centrifuged (1 50000 x g , 2 h). The sedimented solid was washed once with 0.25 M HCl, once with water and then lyophilised and gave the crude lipid A fraction (1.23 g). The volume of the pooled supernatant and washing was reduced by concentration in vucuo after its pH had been adjusted to 7 (NaOH). The material was then dialysed and freeze-dried to yield crude polysaccharide I1 (960 mg). A sample (50 mg) was purified by acrylamide gel chromatography in exactly the same way as described for polysaccharide I . Pure polysac-

02 i

I 10 20 30 LO 50 60 70 80

Time lm in )

Fig. 3 . Kinetics of'the release oJ'poly.saccharide 11 from the glycolipid upon Ireatment with 0.25M HC1 at 100 C us measured hy the thiobarhituratr test

1 2 3 Fig. 4. Sedimentuiion pattern is~nthetic.f loni ier) ofpolysaccharidt~ II . 4mg/ml in 0.1 M KC1 at 360000 x g (mean value): 7 C. Migration from left to right. Pictures were taken at I8 ( I ) , 26 (2) and 42 ( 3 ) min after reaching full speed (16min). Phase plate angle: 60

charide I1 (25 mg, 9 of the endotoxin, 14.5 % of the glycolipid w/w) had a retention time very similar to that of polysaccharide I and was eluted as a symmetrical peak. It was preceded by some high-molecular-weight material appearing in the exclusion volume and was followed by material of lower molecular weight. It appeared homogeneous upon sedimentation (Fig. 4). It was free of fatty acids and contained heptose (17.9 "/;I, hexose (9.9 %,), glucosamine (17.0 %) and phosphorus (1.8 %). Its content of 3-deoxy-octulosonic acid was 0.26% as measured by the thiobarbiturate test and 6.6 as estimated directly with semicarbazide. The terminal reducing sugar was the octulosonate as both the semicarbazide and the thiobarbiturate reactions disappeared after treatment of the material with borohydride.

Release o j 3-Deoxy-2-octulosonic acid 5-phosphutc. jiom Polysucchuride 11

When a sample (1 mg) of polysaccharide 11 was treated with 2 M HCI in the same conditions as the glycolipid (see above) it released, besides traces of free

A. Le Dur, M. Caroff, R. Chaby, and L. Szabo 583

3-deoxy-2-octulosonic acid, 3-deoxy-2-octulosonic acid 5-phosphate as the sole thiobarbiturate-positive compound. This confirmed that the phosphorylated 3- deoxy-2-octulosonic acid was part of polysaccharide 11.

Fragmentation of the B. pertussis Endotoxin by Base

Endotoxin (l00mg) was treated with 2 M KOH (20 ml, 100 ’C, 8 h) in a teflon flask. The cooled (0 -C) mixture was acidified (HCI, pH2) and the free fatty acids were removed by extraction with chloroform (3 x 20 ml). The pH of the solution was brought to 6.5 (1 M KOH); it was then dialysed. All the 3-deoxy- octulosonic acid and 3-deoxy-octulosonic acid 5-phosphate was retained (Table 1). The solution was concentrated in vacuo to a volume of 4ml ; 2 m l of this solution were submitted to polyacrylamide gel column chromatography (Biogel P60, 87 x 1.5 cm in water; eluant :water; flow rate: 0.16 ml/min; I-ml fractions). Elution was followed by (a) measuring the absorbance at 220 nm and (b) estimating neutral sugar content. The elution patterns observed are represented in Fig. 5 . The 3-deoxy-octulosonate, 3-deoxy-octulosonate 5-phosphate and total phosphate contents of fractions con-

Table I. Anulyticul duta ohtuined,for noii-dialy,mhle material reunwed from the B. pertussis mdotoxiii treated nith 2 M KOH-fhr 8 h at 100 C Values for 3-deoxy-2-octulosonate (dOclA) and its 5-phosphate (dOclA-5-P) do not represent the true 3-deoxy-octulosonate and 3- deoxy-octulosonate 5-phosphate content of the material (c$ [ I 61)

Component Amount found in

l00mg recovered, non- B. pertussis dialysable material endotoxin obtained from

100 mg of endotoxin

mg

dOclA, by the thiobarbiturate method [I61 0.35 0.35

dOcl A-5-P 0.25 0.25

Phosphorus 2.3 1.6 Neutral sugars I I 9.4

taining the highest concentrations of neutral sugars in each peak were estimated. Results are given in Table 2. Four independent experiments gave identical results.

Analysis of Peak 2

Pooled fractions of peak 2 (Fig. 5 ) from a single experiment were brought to dryness by lyophilisation and the residue was dissolved in water (1 ml); this solution will be referred to as ‘stock solution 2’.

Cleavage of the Glycosidic Bond of 3-De0.x.v- octulosonate. Dilute acetic acid (pH 3.4, 500~1) was added to a sample (100 pl) of stock solution 2 and the

50 100 150 Eluate iml)

6

Table 2. Analytical data obtained,fiw the main fractions of peaks 1-12 (Fig. 5 )

Fig. 5. Fra~mentation OJ the B. pertussis endoraxin with alkali. The endotoxin (100 mg) was kept with base (2 M KOH, 20 mi) at 100 C for 8 h ; the pH of the cooled (0 C) solution was brought to 2 (4M HCI), the mixture extracted with CHCI,, neutrdlised (1 M KOH), dialysed, concentrated in vacuo (= 2 ml) and applied to a Biogcl 1’60 column (87x 1.5cm); elution (9.6ml/h) was carricd out with water and fractious (1 ml) were collected. Neutral sugars were estimated in each fraction (c f . (161)

-

Component Amount found in peak (fraction)

1 2 3 4 5 6 7 8 9 10 I I 12 ~ ~___-___ ________

(104) (107) (110) (112) (114) (117) (120) (122) (129) (131) (134) (136)

nmol/ml

P 270 280 380 700 700 690 720 540 370 450 410 350 dOclA 30 80 90 80 90 70 80 90 20 30 20 28 dOcl A-P 12 9 16 26 40 50 30 20 10 20 20 0 Neutral sugars 311 400 822 755 844 1022 844 666 444 377 420 577

584 Novel Endotoxin Structure in Borderelkt pertussLs

mixture was kept at l00"C for 1 h. Solvents were removed in vacua at room temperature and toluene was evaporated from the residue several times to remove residual acetic acid. Ice-cold NaBH, solution (100 pl, 1 mg/ml) was added to NaB3H, (5 mCi; specific activity 20 Ci/mmol) and the radioactive reagent poured on the dry residue obtained above. The mixture was kept in ice (1 h), acetic acid ( 5 drops) was added and the solvents were removed. The dry residue was dissolved in water (1 ml) and a sample (10pl) was submitted to paper electrophoresis (pH 1.9, 53 V/cm, 2 h). A sample (IOOpl) of stock solution 2 which had not been submitted to hydrolysis with acetic acid was similarly treated with radioactive borohydride and served as the blank. A single radioactive fragment was detected in the hydrolysed material; it migrated as a cation (20cm) while no radioactive material was detected in the blank. From the unused part of the hydrolysed, reduced material, this radioactive substance was recovered by preparative paper electrophoresis and hydrolysed with 2 M HCi (1 ml, 2 h, 100 'C). After removal of the acid in vacua at room temperature, the residue was taken up in water. The pH was adjusted to 9 with NaOH and the solution kept at 100' C for 5min to hydrolyse any lactones present. The pH of the cooled solution was lowered to 6 - 7 with IR 120 (H') resin and a sample of the material was submitted to paper electrophoresis (pH5, 53V/cm, 90min); a single radioactive compound was detected. It migrated as an anion and had the same mobility as reduced 3-deoxy-D-munno- octulosonic acid. This proved that the hydrolysis with acetic acid had cleaved the glycosidic bond of 3-deoxy- octulosonic acid and that treatment with 2 M HCI released the reduced 3-deoxy-octulosonic acid.

Release of Two Fragments Possessing Opposite Charges upon Hydrolysis with Acetic Acid of pH3.4. A sample (200pl) of stock solution 2 was treated with acetic acid as described above and the solvents were removed in vucuo. The material was then submitted to paper electrophoresis (pH 7.2, 53 V/cm, 2 h) along with an unhydrolysed sample (200 pl) of the same solution. When the pherogram was revealed first with ninhydrin and then with alkaline silver nitrate, two substances were detected in the hydrolysed material: one moved (2cm) as a negatively charged entity and could be detected with both the ninhydrin and the silver nitrate reagents; the other moved (4 cm) as a positively charged fragment and reacted with silver nitrate only. It was shown in a separate experiment that the latter had the same mobility as the radioactive fragment described above. The unhydrolysed sample remained on the base line. I t was concluded that cleavage of the glycosidic bond of 3-deoxy 2-octulosonic acid released from the material contained in peak 2 two fragments which, at pH 7.2, had opposite charges; the positively charged fragment had a terminal 3-deoxy-octulosonate molecule.

Analysis of Peak 12

Pooled fractions of peak 12 (Fig.5) were freeze- dried and the residue was dissolved in water (1 ml) ; this solution will be referred to as 'stock solution 12'.

Release of a Phosphoryluted Fragment upon Hydrolysis at pH3.4. The phosphorus content of a sample (50pl) of stock solution 12 was estimated (found: 1.55 pg). Another sample (50pl) was treated with acetic acid (pH 3.4) and the mixture was brought to dryness as described above. The hydrolysate was submitted to paper electrophoresis (pH 7.2, 53 V/cm, 2 h) along with an unhydrolysed sample (50 pl) of stock solution 12. Strips (3-cm long) were cut from the dried pherogram for both samples as shown in Fig. 6; the strips were eluted with water and the phosphorus content of the eluate estimated after concentration. The total amount of phosphorus applied was recovered from strips 1 A (found : 1.5 pg) and 2 B (found : 1.5 pg) ; none was found in the other strips. This proved that cleavage of the glycosidic bond of 3-deoxy- octulosonate released, besides the octulosonate-containing phosphorus-free fragment, a second, negatively charged fragment that had retained all the phosphorus content of the sample.

Release and Identification of Glucosamine Phosphate. A sample (200 pl) of stock solution 12 was taken to dryness, treated with 4 M HCI (0.5 ml, 100 C , 2 h), and then taken to dryness again. NaB3H, solution (200 pl) was prepared by dissolving NaB3H, (total activity: 5 mCi; specific activity: 18 Ci/mmol) in aqueous NaBH, solution (200 pl, 1 mg/ml); this was added to the residue and the mixture was kept in ice for 1 h. It was then acidified (acetic acid, 5 drops) taken to dryness and boric acid was removed by repeated addition and evaporation of methanol. The borate-free residue was dissolved in water (I ml) and percolated through a column (20 x 1 cm) of Dowex G 50 (H') resin and the column was washed with water (20 ml/h). Fractions (2 ml) were collected; those containing radioactive material were pooled and concentrated. A sample of this material was submitted to paper electrophoresis (pH 7.2, 53 V/cm, 90 min); authentic radioactive glucosaminitol 6-phosphate, alone and admixed with the sample to be analysed, were used as controls. It was found that the radioactive compound present in the sample to be analysed had the same mobility as authentic glucosaminitol 6-phosphate both alone and when mixed with the control. Identical results were obtained when the radioactive compound was submitted to ascending paper chromatography in two solvents: isobutyric acid/ammonium hydroxide (513, v/v) 40 h, R F = 0.86; 2-butanone/formic acid/ methanol/water (4/2/2/1.5, v/v) 5 h, R, = 0.28. Finally, a sample (60 pl) of the same material was treated with 6 M HCI (100 C, 10h) in order to hydrolyse the phosphate ester. After removal of the solvent and

A. Le Dur, M. Caroff, R. Chaby. and L. Szabb 585

3 ’ 2 ’ 1 2 3 ! I

! I

3 ’ 2 ’ 1 2 3

Fig. 6. Sqiurution hy pupw e/ectrophoresi.r (pH 7.2, 5.3 V/cin, 2 h ) of u p~io.~~’l’hor,)?/utedfi.a~ment re/ru.ved,frorn rnalrrial o/peuX 1-7 (Fix. 5 ) rrpon hydro1y~i.s with acetic trcid o f p H 3 . 4 at 100‘ C for 1 h. (A) Unhydrolyscd, (B) hydrolysed material

I x X ” U

L x

a! a 0 c 0 ” W U

+

: c W c

10 20 Time I min I

Fig. 7. Fultj, ucid melhd esters ,fionz maleria/ of’ pcuh 12 /Fix. 5) id iwt i f id by gti.~-liyuid c/zronirrto~rcrpliy fhllonwl by ma.w ,spwtroiii- crry. Gas-liquid chromatography details: 3 ”<) SC 30, column: 1.5 m x 3.2mm: tcmpcraturc program: 100-250 C, 8 Cimin

residual free acid, the residue was analysed by paper electrophoresis (pH 7.2, 53 V/cm, 1 h); a single, radioactive compound, co-migrating with authentic glucosaminitol, was detected. It was concluded that a phosphorylated derivative of glucosamine was present in the negatively charged fragment which was released upon treatment of the material in peak 12 with acetic acid of pH 3.4.

R&wrr crnd Idmtification of Futty Acids. The dry residue of a sample (200~1) of stock solution 12 was treated with 4 M HC1 (100 C, 2h) and the cooled, acidic mixture was extracted with chloroform. A slight excess of cthcrcal diazomethane solution was added to the dried (Na,SO,), filtered extract. The chloroform was evaporated, the residue was dissolved in ethyl acetate (l0pl) and a sample ( 1 pl) analyscd by gas- liquid chromatography/mass spectrometry. Two main

1 2 3 Fig. 8. Srdlnirrittrtiotz pattcwz of the R. pertussis ordoto\iir. 4.8 mg/ml in 0.1 M KCI at 360000 x R (mean value); 20 C. Migration from left to right. Pictures were taken at 16 ( I ) , 32 (2) and 48 (3) min after reaching full speed (lhmin). Phase plate angle: 45

peaks (Fig. 7) were observed; thcse had the retention times and the mass spectra of methyl tetradecanoate and methyl 3-hydroxy-tetradecanoate.

DISCUSSION

The phenol/water procedure [ 141 followed by high- speed centrifugation, which has been successfully employed by previous authors [26- 281 to obtain endotoxin from B.pertz.r.rsis, was also used in the present work. The material isolated had biological properties similar to those described [26 - 281 and gave a single line of precipitate when tested by immunodiffusion against sera of mice immunized with whole B. licjrtussis cells. Upon ultracentrifugation the preparation appeared homogeneous (Fig. 8).

The parameters of the hydrolysis with trilluoro- acetic acid were chosen to minimise cleavage of glycosidic bonds other than those of 3-deoxy-2-octulosonate: it has been shown that no 3,6-dideoxyhexose was released from the endotoxin of Salmonrllu typlzi- murium in these conditions [22]. It is noteworthy that in spite of the very mild conditions used to cleave polysaccharide I, inorganic phosphate was released simultaneously from the B.pertussi.7 endotoxin (Fig. 9). However, release of polysaccharide I and inorganic phosphate are probably unrelated : kinetics of the

586 Novel Endotoxin Structure in Bordetellu pertussis

Time ( h ) Fig. 9. Ralease ofinorganicphosphure,fiorn the B. pertussis endotoxin during freutnwnt bcith trifluoroucetic acid of p H 3 nl 50°C

release of phosphate and of polysaccharide I (as measured by the thiobarbiturate test) do not parallel each other, inorganic phosphate continues to be formed when release of polysaccharide I is complete and the amounts of inorganic phosphate and reducing 3-deoxy-2-octulosonate released are not equimolar.

Contrary to that which was observed when Salmonella typhimurium endotoxin was treated with trifluoroacetic acid of pH 3 at 50 C. no free, dialysable 3-deoxy-2-octulosonic acid was released from the B. pertussis endotoxin. It was concluded that polysaccharide I was bound through a single 3-deoxy-2- octulosonate molecule within the endotoxic complex. It is known that three molecules of 3-deoxy-2-octulosonate which form a branched trisaccharide serve as a link between the core and lipid A in the Salmonella typhimurium endotoxin [29].

Three large fragments were formed from the B. pertussis endotoxin upon treatment with trifluoroacetic acid: polysaccharide I (15%), glycolipid (63 %) and IipidX (273; about 18% of the material was transform- ed into small dialysable fragments during this step. The diffusate contained all of the characteristic components (neutral sugars, phosphate, hexosamine) of the endotoxin, but no free 3-deoxy-2-octulosonate. As the starting endotoxin was devoid of dialysable material, the latter must have been formed during the hydrolytic step, but the nature of the bonds broken has, as yet, escaped identification.

Polysaccharide I was obtained as an homogeneous substance as judged both by acrylamide gel chromatography and by ultracentrifugation. Its molecular weight, as calculated from its terminal 3-deoxy-2-octulosonate content, appears to be approximately 2700. It has a remarkablyhigh nitrogen content (5%); besides glucosamine which accounts for one third of it, two other nitrogen-containing compounds have been observed to be present. The structures of these are under in- vestigation. The branched trisaccharide 7-0-(E-D-

glucosaminyl)-2-O-(~-~-glucur0nyl)-~-glycero-~- manno-heptose described previously [17] is part of polysaccharide I.

Both the semicarbazide [19] and the diphenylamine [20] methods indicated for polysaccharide I an apparent 3-deoxy-2-octulosonate content about 7 times higher than the value obtained by the thiobarbiturate method. It has been established previously that in the thiobarbiturate test the molar absorption coefficient of 5-0- methyl 3-deoxy-2-octulosonic acid is about one seventh of that of the unsubstituted acid [30]. The preliminary conclusion was drawn that in polysaccharide I the terminal 3-deoxy-2-octulosonate was substituted in position 5 . This was confirmed by applying the chemical degradation previously shown to transform 5-0- benzyl-3-deoxy-2-octulosonic acid [21] and ~-O-(P-D- glucopyranosyl)-3-deoxy-2-octulosonic acid [22] first into a mixture of epimeric 3-deoxy-hexonic acids and thence to the corresponding 3-deoxy-hexitols (Scheme).

The fact that 3-deoxy-hexitol acetates were formed during this degradation is unequivocal proof for the presence of a terminal 5-0-substituted 3-deoxy-2-octulosonic acid in polysaccharide I. The same degradation procedure would transform a 4-0-substituted 3- deoxy-2-octulosonic acid into peracetylated, epimeric 3-deoxy-pentitols, while a 7-0-substituted or 8-0-substituted 3-deoxy-2-octulosonic acid would yield 3- deoxy-tetritol acetates. In spite of a thorough search of the chromatogram by selected ion monitoring no such compounds were detected.

In the above reaction sequence, a 5-0-substituted 3- deoxy-u-munno-2-octulosonic acid (hydroxyl groups at C-4 and C-5 cis) yields a mixture of 3-deoxy-~-riho- hexitol peracetate, and 3-deoxy-~-arahino-hexitol peracetate which in the chromatographic system used appears as a single peak ; 5-0-substituted 3-deoxy-~- gluco-2-octulosonic acid (hydroxyl groups on C-4 and C-5 trans) is degraded to a mixture of 3-deoxy-~-xylo- hexitol peracetate and 3-deoxy-~-lyxo-hexitol peracetate; this mixture also emerges as a single peak and is well separated from the former [21]. The fact that the 3- deoxy-hexitol acetate(s) formed upon degradation of polysaccharide I had the same retention time as the mixed 3-deoxy-~-riho-hexitol and L-arahirio-hexitol peracetates established that the hydroxyl groups on C-4 and C-5 of the terminal 3-deoxy-2-octulosonic acid of polysaccharide I had a ci5 relationship as in 3-deoxy-u- manno-2-octulosonic acid, but is not unequivocal proof that this 3-deoxy-2-octulosonic acid has, indeed, the D-

manno configuration. Treatment of enterobacterial endotoxins with di-

lute, aqueous acetic acid gives precipitates (lipid A) which contain glucosamine, phosphate and fatty acids but none of the neutral sugars originally present in the endotoxin. While in similar conditions a water- insoluble fragment was also formed from the B. pertussis endotoxin, this material was not comparable

A. Le Dur, M. Caroff, R. Chaby, and L. Szabo 587

to the lipid A fragment of enterobacterial endotoxins. The portion (6.6%) of the insoluble material formed upon treatment of the B. pertussis endotoxin with trifluoroacetic acid of pH 3 at 50 C which was soluble in a mixture of toluene/methanol; lipid X (3.1 %), and which contained 2-methyl-3-hydroxydecanoic and 2- methyl-3-hydroxytetradecanoic acids, has been recovered from this material [23]. The insoluble part (over 90 %) of the material was a glycolipid; it contained a high proportion (10 %,; endotoxin: 14%; in both cases as D-glycrro-L-manno-heptose) of covalently bound neutral sugars, and also 3-deoxy-2-octulosonic acid. It is noteworthy that the presence of the latter could be demonstrated only following relatively strong acidic hydrolysis. The behaviour of this 3-deoxy-2-octulosonic acid was most unusual: its glycosidic bond was cleaved neither by the usual treatment with acetic acid of pH 3.4 at 100°C nor by trifluoroacetic acid of pH 3 at 50 "C as described above, but can be achieved by using 0.25 M HC1 at 100 "C for 30-40 min. The reducing 3-deoxy-octulosonate thus formed, which is the terminal reducing sugar of polysaccharide 11, responds only very weakly in the thiobarbiturate test: the 3-deoxy-octulosonate content of polysaccharide I1 as measured by this method (0.26 yo) is only 1/25th of that given by the semicarbazide test (6.6%). The reasons for this unexpected behaviour, in particular the strongly increased stability of its glycosidic bond and its negligible reaction in the thiobarbiturate test, are not well understood. It is clear, however, that a 'negative' thiobarbiturate reaction should not be taken to indicate the absence of 3-deoxy-2-octulosonic acid in endotoxin preparations.

Treatment of the glycolipid with 0.25M HCI at 100 C for 30min released a water-soluble polysaccharide (polysaccharide 11) and left an insoluble residue (lipid A). Thc reaction mixture remained heterogeneous throughout and, because of the relatively harsh conditions that had to be used, release of polysaccharide I1 from the glycolipid was not as selective as that of polysaccharide I from the intact endotoxin. Acrylamide gel chromatography revealed that, besides polysaccharide 11, several other water- soluble fragments were formed; moreover about 30 % of the material (calculated for the initial amount of glycolipid) was lost during dialysis. Although the amount of pure polysaccharide I1 actually recovered (9 % of the endotoxin, 14.5 % of the glycolipid, w/w) was only half of the amount of polysaccharide I (1 5 of the endotoxin) that was isolated from the endotoxin, polysaccharide I and I1 are very probably present in equal amounts within the intact endotoxin molecule. Indeed, as the lipid A portion of the endotoxin is devoid of neutral sugars, the polysaccharide I1 content of the endotoxin can be calculated from the glycolipid content (60 %) of the endotoxin on the one hand and from the heptose content of the glycolipid (5.4 76) and of that of

polysaccharide I1 (18 %) on the other hand, to be approximately 17 %.

As in polysaccharide I, the terminal reducing sugar of polysaccharide I1 is a single molecule of 3-deoxy-2- octulosonic acid. However, while in polysaccharide I the 3-deoxy-2-octulosonic acid bears a polysaccharide chain in position 5 , a phosphate group esterifies position 5 of the terminal 3-deoxy-2-octulosonic acid of polysaccharide 11. Upon treatment of polysaccharide 11 (or of the intact B. pertussis endotoxin) with 2 M HC1 at 100 "C for 2 h, this phosphorylated 3-deoxy- 2-octulosonic acid is released; its structure has been rigorously established [16]. Although it is well known that phosphate groups attached to polyhydroxy compounds can undergo migration [31- 331, in the present case the formation of a 3-deoxy-2-octulosonate 5- phosphate as a consequence of transesterification can be excluded for the following reasons. Upon sequential treatment of the intact B. pertussis endotoxin with periodate, borohydride and mild acid a 3-deoxy-2- heptulosonic acid 5-phosphate was released [ 161; it follows that in the intact endotoxin positions 7 and 8 of the phosphorylated 3-deoxy-2-octulosonic acid are free. Because of its stability to acid-catalyscd hydrolysis it is most unlikely that within the endotoxin the phosphorylated 3-deoxy-2-octulosonate should be present in the acyclic or furanose forms. Position 6 being engaged in the pyranose ring, the only positions which the phosphate group can esterify are 4 and 5 and the direction of the transesterification must have been from 4 to 5. 3-Deoxy-~-arahino-2-heptulosonic acid 4-phosphate has been synthesized and it has been demonstrated that, in the conditions used to set free 3-deoxy- 2-octulosonic acid 5-phosphate from the B. pertussis endotoxin, 84% of the phosphate content of the heptulosonate 4-phosphate was released as inorganic phosphate [22]. No phosphate migration was observed upon acidic treatment of methyl 4, 6-0-benzylidene- 2-deoxy-a-~-lyxo-hexopyranoside 3-phosphate, a compound which has the same steric arrangement of its phosphorylated and free hydroxyl groups as 3-deoxy- n-nzanno-2-octulopyranonic acid 4-phosphate. Phos- phate migrations lead to equilibrium mixtures [31- 331 ; it would, therefore, be expected that the reaction sequence used to establish the position of the phosphate group would yield both phosphorylated 3- deoxy-pentonic and 3-deoxy-hexonic acids; only the latter could be identified. The fact that only 3-deoxy-2- aldulosonic acid 5-phosphates were isolated from the B. pertussis endotoxin whether it was hydrolysed with strong acid (2M HCI, 2h , 100 C) or under the conditions of the Smith degradation (0.25M HCI, 20min, 100 C) also excludes all likelihood of the 5- phosphate arising from the 4-phosphate.

It has been shown previously that in the thiobarbiturate test 3-deoxy-~-manno-2-octulosonic acid 5- phosphate reacts 9 - 10 times [25] and 4-O-methyl-n-

588 Novel Endotoxin Structure in Bovdeklla pertussis

arahino-2-heptulosonic acid reacts 28 times [34] less than unsubstituted 3-deoxy-~-manno-2-octulosonic acid. If the figure obtained with semicarbazide is considered to represent the real value, the terminal 3- deoxy-2-octulosonic acid 5-phosphate of polysaccharide I1 appears to react about 29 times less in the thiobarbiturate test than unsubstituted 3-deoxy- -~-manno-2-octulosonic acid. Since in the intact endotoxin the 3-deoxy-2-octulosonic acid is most probably in the pyranose form (as it carries a phosphate group in position 5 and as it is unsubstituted in positions 7 and 8), the discrepancy between the 3- deoxy-2-octulosonate content as measured with semicarbazide and with thiobarbiturate strongly suggests that in polysaccharide I1 the terminal 3-deoxy-2- octulosonate-5-phosphate is substituted in position 4.

The precipitate (40 % of the glycolipid, 23 % of the endotoxin) remaining after the release of polysaccharide I1 contains only negligible amounts of neutral sugars; its main components are esterified phosphate, glucosamine, tetradecanoic acid, 3-hydroxytetradecanoic acid and 3-hydroxy-decanoic acid. This material was thus analogous to the lipid A fragment released by mild acidic treatment (acetic acid of pH 3.4, 1 h, 100 .’C) from enterobacterial endotoxins.

The presence of two different polysaccharide chains in the endotoxin of B. pertussis is not restricted to the vaccine strain 14 14; stepwise hydrolysis with trifluoroacetic acid and 0.25 M HCl of endotoxin extracted from B. pertussis L84 phase I and phase IV strains also released two different polysaccharides apparently identical with those isolated from strain 1414. As a corollary, release of phosphorylated and non- phosphorylated 3-deoxy-2-octulosonic acid in comparable amounts was also observed upon treatment of these endotoxin samples with 2 M HCI at 100 ,C for 2 h followed by paper electrophoresis (pH 5 , 53 V/cm, 90 min, thiobarbiturate spray).

The isolation of two different polysaccharides with terminal 3-deoxy-2-octulosonic acids could be due to the existence of two types of endotoxin molecules in these preparations. In this connection it must be remembered that endotoxins are notoriously difficult to purify [l l] ; their homogeneity is usually assessed on the insufficient basis of their sedimentation pattern and the demonstrable absence of nucleic acids. By these criteria, the endotoxin preparations used in this study had properties analogous to endotoxins previously isolated from Bordetella pertussis [26,27], from other members of the genus Bor&~c.tella [26] and to those extracted from enterobacteria. Its apparent homogeneity as revealed by ultracentrifugation does not exclude the possibility that it contained more than one molecular species. On the other hand, as the same phenomenon was observed not only for two unrelated strains (1414 and L84, phase I) but also for the two phases of strain L84 and as all of these preparations

0 c a 1 2 3 4 5 6 7 8 9

Time ( h ) Fig. 10. Rdeu,w qf’ino,.gunicphosphatr from the B. pertussis eiidotoxin during treatment with 2 M KOH at 100 C

gave single lines upon immunodiffusion, it is unlikely that they should represent rough mixtures.

The existence of two polysaccharides in comparable amounts is, therefore, more likely to indicate that the general structure of this endotoxin is not the same as that assigned to the endotoxin obtainable from Enterobacteriaceae. This conclusion was corroborated by the results obtained by fragmentation of the endotoxin by alkali.

As acid-catalysed selective removal of polysaccharide I from the intact endotoxin left a glycolipid containing polysaccharide 11, clearly polysaccharide I1 was bound to a constituent of the lipid moiety of the endotoxin. On the other hand polysaccharide I could be linked either to polysaccharide TI to give the serial arrangement I - I1 - lipid, or, like polysaccharide 11, be linked to a constituent of the lipid. Proof for the latter type of structure was obtained by the alkaline degradation of the endotoxin.

Surprisingly harsh conditions were required to fragment the endotoxin with base: treatment with 2 M KOH at 100 C for only 2 h instead of 8 h gave mainly breakdown products that were still excluded from the polyacrylamide gel (Biogel P60) used for the chromatographic separation of the fragments. Release of inorganic phosphate (kinetics shown in Fig. 10) occurs during the alkaline treatment; it represents about 30 ;i of the total phosphate present in the endotoxin. I t can be concluded from these observations that the complex lipid moiety of the B. pertussis endotoxin which cor- responds to the lipid A fragment of enterobacterial endotoxins is unlikely to have the structure assigned [35] to the latler, or to that of Ch/nrnuhac/eriiii1?? violaceurn [36].

The isolation, after alkaline treatment, of material containing simultaneously non-phosphorylated 3-deoxy-2-octulosonic acid, neutral sugars and elements characteristic of the complcx lipid moiety of the endotoxin, but no phosphorylated 3-deoxy-2-octulo-

A. Lc Dur, M. Caroff, R. Chaby. and L. Szab6 589

sonic acid, strongly suggests that in the intact endotoxin both polysaccharide I and polysaccharide 11 arc bound to the complex lipid moiety of the macromolecule. It is most unlikely that significant amounts of unphosphorylated 3-deoxy-2-octulosonic acid were formed by dephosphorylation of the phosphorylated 3- deoxy-2-octulosonic acid ; indeed while large amounts of phosphorylated 3-deoxy-octulosonic acid are present in the separated fragmcnts, the release of measur- able amounts of inorganic phosphate ceases (3 - 4 h) well before the end of the alkaline treatment (8 h). The stability of phosphomonoesters to alkaline hydrolysis is well known [37]. On the other hand, the simultaneous presence of phosphorylated and non-phosphorylated 3-deoxy-2-octulosonic acids, i .e. the characteristic components of polysaccharides I and 11, in most fragments isolated following the alkaline treatment suggests that both polysaccharide chains arc attached to a single entity of the complex lipid.

As regards the minor complex lipid, lipid X, its position within the endotoxic complex is, as yet, unknown. As it represents only a very small part (about 2 x) of the endotoxin it appears highly unlikely that it should be the complex lipid counterpart of polysaccharide I which accounts for about 15% of the endotoxin's mass, although these two fragments are released simultaneously.

The existence of two non-identical polysaccharide chains within the B. pertu endotoxin was discovered because of the very different rates of acid-catalysed hydrolysis of the glycosidic bond through which each of them is joined to the complex lipid moiety of the endotoxin. It is conceivable that similar structural features are also present in endotoxin preparations from other gram-negative bacteria but have remained undetected because of similar rates of hydrolysis of the glycosidic bonds concerned. Should this be the case thcy could serve as the structural basis for the microheterogeneity of endotoxins commonly observed.

The authors are indebted to the Directors of the Institut Mltrieux (Lyon), to Drs G.Ayme, R.Donikian and M. C. Mynard who prepared and supplied the R. pw/us.sis extracts used, and to Dr

The Lister Institute, Elstrcc, England) lor supplying ' LX4 strains. This work was supported by the ircilc u lo Rivhwchr Scicvztifiquc r t Technique.,

Coniniissiorz: Menibrunes Biologiyuc,.~ (grants 72.7.0678 and 74.7.0204). the Iiixiiiur Natiotiul dc Iu Sanli; et (16, la Rcdzerchc Midicul(~ iind the F~~ntlcrtioii pour la Rcch~wlrc~ M&ka/c Frun~uisa.

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Documents

Novel Type of Endotoxin Structure Present in Bordetella pertussis. Isolation of Two Different Polysaccharides Bound to Lipid A