during Bordetella pertussis growth - ScienceDirect

(~) INSTITUTPASrEuR/ELSEVlER Paris 1993

Res. MicrobioL

1993, 144, 201-209

Release of lipopolysaccharide

during Bordetella pertussis growth D. Hozbor (ll(*) M.E. Rodriguez {I), A. Saran 01, A. Lagares (21 and O. Yantorno I , ¢o Centro de lnvestigacidn y Desarrollo en Fermentaciones lndustriales (CINDEFI), and r2~ Inslituto de Bioquhnica y Biolog/a Molecular, Facultad de Ciencias Exactas UNLP, Calla 47~y-115 (1900) La Plata (Argemiila)

SUMMARY The effect of the addition of (2,6-O-dimethyll-~-cyclodextrin (Me~CD) during growth of Bordetelfa pertussis in synthetic Stainer-Scholte liquid medium ISSl on lipopolysneoharide (LPS; endotoxinl release was investigated. The Me[~CD concentration used (3 mg/ml) was chosen according to the optimal level found in previous studies to enhance major soluble antigen ~oduction. The profiles in SDS-PAGE (sodium dodecyl sulphete/polyacrylamida gel electrophoresia) of LPS extracted from calls grown in SS and S$ + Me~CD media revealed similar patterns. Although the LPS content of whole cells decreased during cell growth, yields obtained at different growth periods in cycl;.',deatrin medium were lower than those corresponding to SS medium elune. Consequently. the level of LPS released in supematants of both media increased during cegular growth. This amount of free LPS was higher in the cyolodextriu liquid medium and became significant at the beginning ,)f the stationary growth phase. Binding of cyclodextrin to pottussle ceils could account for the data obtained. Similar results were obtained with all species of the genus Bordatella. Key-words: Cell wall, LPS, Cyclodextrin, Bordere//a pertussis, Vaccine; LPS release.

INTRODUCTION Pertussis vaccines currently used in most countries are suspensions of killed whole cells of the causative organism, Bordetella pertussis. Those vaccines include some components which are unnecessary for promoting a protective response. Instead, such components could be responsible for the adverse reactions observed after vaccination (Robinson et al., 1985).

"['he consensus is that the lipopolysaccharide (I.PS) (Le Dur et al., 1980) in the traditional whole cell vaccine is non-protective and might instead contribute to the vaccine's toxicity and reactogenicqy (Bergmazl and M anoz, 1977; Stdnman et al., 1982). Moreover, in a new ace!lular vaccine consisting of characterized constituents of the organism, the LPS is regarded as an undesirable one. LPS should therefore be reduced or excluded from all vaccine formulations.

Submitted November 19, 1992, accepted February 1, 1993. (*) Correspondingauthor.

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With regard to vaccine production, the Stainer-Scholte (SS) synthetic medium (Stainer and Scholte, 19-/I) has been recommended for B. pertussi$. However, when this medium was used, levels of protective antigens were low, in spite of good cell growth under shaking conditions (Imaizumi et aL, 1983b; Bellalou and Relyveld, 1984). In large-scale production, major progress has been achieved by introducing "Hept a k i s " ((2,6-O-dimethyl)-~,-cyclodextrin ; Me~CD) to enhance ceil growth and synthesis of the main antigens. Several authors have reported that the optimal level of cyclodextrin for increasing the yields of such immunogens ranged from 2 to 3 mg/ml (Suzuki etal., 1985; Imaizumi etat., 1983a,b; H o z h o r e t a L , 1991). However, whether this concentration of cyclodextrin affects the LPS level remains to be studied. A better knowledge of LPS release must be taken into account for the production of a new non-toxic acellular vaccine. Bearing these considerations in mind, we studied the release of LPS during the growth of the bacteria in liquid synthetic medium, either in the presence (3 mg/ml, SS-Me[3CD) or in the absence of cyclodextrin. We analysed whether this concentration of cyclodextrin affects the SDS-PAGE {sodium dodecyl sulphate/polyacrylamide gel electrophoresis) profiles of free and cell-bound LPS at different periods of growth. With regard to the effect of cyciodextrin, comparisons with two other Bordetella species (B. bronchiseptica and B. parapertussis) were also made, Furthermore, data on the binding of eyctodextrin on B. pertussis cdls are reported.

CIP 55110T(Institut Pa~tcur Collection) were maintained in 15 % glycerol 1% casaminoaeids at - 70°C. Cultures were grown for 48 h (B. bronchiseptica and B. parapertussis) or for ?2 h (B. pertussis) and subcuhured for 24 h at 36°C on Bordet Gengou agar (Difeo Laboratories. Detroit, MI) supplemented with 10g peptone/I and 15 ~a (v/v) dcflbrinated sheep blood. Cells harvested from these plates were transferred to I-litre Erlenmeyer flasks containing 250 ml of SS medium, and incubated for 24 h at 36"C with rotary shaking at 160 rev/min. The seed suspension (25 ml) was inoculated into SS medium alone or with 3 mg of MeI~CD per ml (SS + Me[3CD). Under these conditions, the initial optical density at 650 nrn (OD6m) ranged from 0.1 to 0.2. Ceils were grown for 17-48 h in the same conditions described above. LPSextraclion Ceils grown for 24, 30 and 48 h at 36°C in SS and SS + Me~CD media were ecmrifugated at tO,000 g for 15 rain at 40C and washed twJce in distilled water. The bacterial concentrations were adjusted and LPS was extracted by the hot phenol-water method of Westphal and Jann (19655, with minor modifications. The volume of phenol used to suspend the organisms was 55 % (v/vS. The extracts were dialysed overnight at 4°C against 4 litres of water and then c.,'ntrifuged at 100,000 g for 2 h. The sediment (LPS) was resuspended in distilled water and stored at -20°C until used. LPS release LPS was determined in supernatams from SS and SS + Me[3CD media by means of electrophoresis and 3-deoxy-D-manno-2-octulosonie acid I.KDO). Sampies taken at different periods of cell ,~rowth were centrifuged at I0,000 g for 15 rain at 4°C. Under these conditions, cell-free supernatants were obtained as shown by microscopic examination.

MATERIALSAND METHODS Strains and growth conditions

~DS-PAGE of Upopolysaccharides

B. pertussis strain 8132 phase 1, B. parapertussis strain CIP 64.11T and B. bronchiseplica strain

The Laemmli discontinuous buffer system was used (Laemmi'. 19705.The total acrylamide concert-

B.b. = Bordet¢llobronchiseptic,. B.pp. - Bordetellaporoperlussis. HEPES = N-2-h~droxyethylpperazine-N'-2-ethanesu phonic acid. KDO = ]-deoxy-D-manno-2-oclulosonicacid. LPS - lipopolysaccharid~.

Me~CD = OD6~, = PAGE : SDS = SS =

(2,6-O-dimc~hyl]-~-cTdodcxtdn. opticaldensityat 650 am. polya¢~lamideg~l~i~:~rophot~sis. sodiumdodecy sulphae. Stain=r-Schohe.

R E L E A S E O F L P S DURING B. PERTUSSIS G R O W T H tration used in the separating gel ranged from 12.5 to 15 070 as indicated in the figure legends. LPS preparations of whole ceils and supernatams were solubilized in sample buffer aod were heated at 100°C for 10 mln. Twenty-five ttg of proteinase K in 10 pd of buffer were added per 50 td of LPS suspension. The mixtures were incubated in a water bath at 60°C for 1 h with occasional vortexing. Proteinase-g-trented samples were applied to gels. Determlnatlun of KDO LPS (Le Dur et el., 1978) from B. pertussis contain lipid A and an oligosaceharide core having KDO (Moreau et aL, 1984), hut not the long-chain polysaceharide characteristic of the enterobacterial LPS (Hitchcock et aL, 1986). KDO was determined by the thiobarbiturie acid method of Karkhanis et al. (19 ~ ) with modifications (0.1 M H2SO4 hydrolysis for 60 rain instead of 0.05 M H2SO4 for 30 rain) using KDO (Sigma) as a reference. Preparation of outer membrane Envelope fractions (outer membrane plus peptidogly,mn) were prepared using the method of Schnaitman et aL (1971) with minor modifications. Ceils harvested by centrifugation (10,000g for 15 rain at 4°C), were washed and resuspended is 10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid), pH 7.4. After ultrasonic disruption for 10 rain with cooling, unbroken cells and large fragments were removed by centrifugarish (3,000 g for l0 mio at 4oC). The crude envelopes were pelleted by centrifugation (16,000 g for 60 min at 4°C), resuspended in l0 ram HEPES buffer containing 7.5 mM MgCI_, and 2 % TritonXI00 (Sigma) and left at room temperature for 30 rain. The Triton-insoluble material was pelleted by centrifugation m 16,000 g for 60 min. The pellet was resaspended in distilled water and the protein coucemration was determined. Protein assay Protein content was estimated by the Lowry method, usiog bovine serum albumin as the standard (Lowry et al., 1952). SDS-PAGE of outer membrane pmteius Separating and stacking gels ~,ere 12.5 % (w/v) and 4 % (w/v), respectively. Envelope proteins were solubilized by heating at 100°C for 5 min in 0.0625 M Tris/HCl buffer pH 6,8, containing SDS

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(2 %, w/v), glycerol (10 %, w/v), bromophenol blue (0.001%, w/v) and mercaptoethanol (5 %, w/v). SolubiUzed proteins were applied to the gels. Electraphoresls Whenever electrophoresis had to be applied, it was perforn.ed at room temperature and constant voltage. Polypeptides were stained overnight in a solution of Coomassie blue R250 (0.2 %, w/v) in methanol 40 % (v/v) and acetic acid l0 % (v/v). Molecular weights were estimated by means of a Pharmacia Calibration Kit. Hitchcock and Brown (1983) silver stain technique was used to spot LPS. Fluorescence assay for cyclodextrin The content of cyc!cdegtrlu h~ solulion was determined by the method of Gorriuge et aL (1988), which is based on the ability of cyclodextdn to greatly enhance the fluorescence of the dye 2-p-toluidinylnaphtalene-6-sulphate. Cyelodextrin distribution in the solution bearing SS + MeI3CD medium was analysed by performing its determination in the supernatant, in the supernatant filtered with au ultrafiltration micropartition system (Millipore, "Ultrafree-CL", Iow-blnding cellulose 30,000 MNWL) and in the cells. Ceils from the SS medium having the same optical density and the same culture time were used as controls. The concentration of Me[3CD was determined by comparison with a standard curve for Me[3CD(0-0.133 rag/ral) obtained for each assay. Statistical analysis Data from the studies described above were subjected tO analysis of variance, with the Fisher proteeted lesst-signifieant-dif ference procedure (Stell and Torrie, 1980) used for comparisons of treatment means. RESULTS AND DISCUSSION The LPS preparations extracted from B. tiertussis cells grown in SS and in SS + Mel3CD media exhibited similar patterns in polyacrylaraide gels (fig. la). These profiles consist of two bands (rough-type LPS), an upper, dominant (LPSa) and a lower, small band (LPSb), which closely resemble those reported by Peppier etal. (1984). These results indicate that when cyclodextrin is added to the SS medium, the LPS cellular pro-

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D. H O Z B O R E T A L .

files remain unchanged. Figure Ib shows that the total LPS content of whole cells (estimated by densitometry) decreased during cell growth in both media. However, this decrease was greater in cells grown in SS + Mei3CD. These results seem to be important for the production of tt less reactogentc cellular vaccine.

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According to these rest!Its, it seems that the LPS is released into the surrounding medium during cell growth. Therefore, the time course of LPS release in both media was analysed by determining KDO, which has been found to be an ubiquitous component of the endotoxins (LPS) of G r a m - bacteria, Figure 2b shows that the amount of KDO in supernatants of both SS and SS + Me~CD media increased during cellular growth. In SS medium, the rate of LPS secretion remained constant throughout the growth curve. Although the secretion rate in SS + Mei3CD medium is similar for the first 24 h, between 24 and 32 h, a marked increase in LPS release is observed. Then, the rate is maintained as in the initial stage. The enhanced release daring the stationary phase in both media, on a per cell basis, may he due to partial cell death and lysis as proposed by Cadieux et al. (1983) or merely due to partial disintegration of the outer membrane. As can also be seen in figure 2b, the KDO level was higher in S S + Me~CD medium. This difference becomes significant at the deeelerating growth phase (p < 0.05). By comparing the results obtained with both media, maximal cell growth in SS + Me}CD was about 24 % greater than that in SS medium (fig. 2a), whereas the level of KDO was about 37 070higher than that corresponding to the cyclodextrin-free medium (fig, 2b). These results correlate directly with SDSPAGE profiles of free LPS, shown in figure 3. Again, this pattern was similar for both media and similar to those described above for cellbound LPS. These findings are in agreement with those reported by Perera et al. (1987), who showed that the addition o f cyclodextrin (I mg/ml) during growth caused a small, but consistent increase in the release of LPS in four wild-type strains of B. pertussis.

Fig. I L SDS-PAGE {I 2.5 % w/v) comparing LPS prepar~lions from ce|is of B. ~rlus$is grown ir~ both SS medium (lanes [, 2 and 3) and ~S + Me~CD medium (lanes 4,

5 and 6). Cells were harvested a! differentgrowth periods and adjuslcd to identicalOD~, before LPS isolation.LPSa and LPSb are indicated. Fig. l b . Densitometric tracing of figure la.

On the other hand, other authors (Ibsen et eL, 1989) have found that the amount of LPS in the supernatants of cyclodextfin cultures was either somewhat reduced or unaffected, in comparison with the amounts in cultures grown in S$ medium alone. The difference with our results could be explained by considering that in the

R E L E A S E O F LPS DURING B. PERTUSSIS G R O W T H

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Fig, 2. Time-course of bacterial gro~vth and KDO level in liquid cultures of 8. pertussis. a) Cell growth in SS medium ((31 and in SS + Me[3CD medium ( I l L b) KDO level in supernatant cullures of SS medium ((3) and 5 S + Me)CD medium ( o ) . All the results are mean values of six experiments. Verllcal bars indicate standard error of the means.

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Fig. 3. SDS-PAGE(15 °70w/v) comparing free LPS from culture supernatants of B. pertussis grown in SS llledium (lanes I and 2) and in SS+ Me[3CDmedium(lanes3 and 4).

work of Ibsen et aL (1989), the concentration of cyclodextr!n employed (500 ~g/ml) was six times lower than the one used by us. As the ratio Erlenmeyer flask volume:liquid medium volume used was also lower, the aeration conditions as well as the kinetics of cell growth might have been different ; the detection technique was also different. The higher amounts of LPS in the presence of cyelodcxtrin were probably due to an increase in the extraction of LPS from the cerls mediated by cyclodextrin, as is suggested by the decrease in cell LPS in figure I, rather than to an increase in LPS production, in order to investigate this possibility, cells harvested from SS medium were incubated in three different conditions: with an agent which is known to increase LF3 release (TrJs I00 m M + E D T A 20 raM) (Vaara, 1992), with cyclodextrin (Tris 100 mM + Me[3CD 3 g/I) and with buffer Tris alone (100 mM). The amount of LPS released was compared. Figure 4 shows that the presence of Me~CD in the sample caused an increase in the level of

D. HOZBOR ET AL.

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Fig. 4. SDS-PAGE (!5 % w/v) of free LPS from supernatants o f three different incubation mixtures : B. perlllssis cells plus 100 mM buffer Tris {lane 1), 8. pertussis cells plus 100 mM buffer Tris and 20 mM EDTA (lane 2) and B. pertussis cells plus 100 mM buffer Tris and 3 g/l of Me~CD (lane 3). Baeletial cell preparations were adjusted, incubated for 2 b at room temperature and centrifugated at 10,000 g for 15 rain at 4°C before supernatant LPS determination.

LPS released, similar to that observed in the TrisE D T A supernatant. In order to determine if the effect of cyclodextrin is a general p h e n o m e n o n a m o n g Bordetella species, the released L P S was also analysed in cultures of B. parapertussis (B.pp) and B. bronchiseptica (B.b) (fig. 5). T h e pattern o f free L P S from B.pp showed a ladder-type arrangement o f the bands o f high molecular mass (typical o f smooth type L P S ) and a single fast migrating band o f low molecular mass (fig. 5) lanes 7, 8, 9, 10) (Ray et al., 1991). The latter band was similar in molec~;lar mass to the I-PSb band from B. pertussis. L P S preparations f r o m B.b, however, showed two bands (fig. 5, lanes 3, 4,

Fig. 5. SDS-PAGE 05 ~0 w/v) of free LPS from culture supernatams of three different strains of Bordetella grown in SS medium (lanes 1,2.3.4, 7 and 8) or in SS + Me[~CD medium (lanes 5, 6, 9 and 10l. Lanes 1 and 2: B. pertussis 8132; lanes 3, 4, 5 and 6: B. bronchiseptica CIP 55110T; lanes 7, 8, 9 and 10: B. paraperlussis CIP 6400T. KDO (gg/ml)/OD~50): lane 1=0.75; lane2-2.94; lane 3=0.00; lane 4=0.22; lane 5=0.00; lane 6=0.54; lane 7 = 0.00; lane 8 = 0.98; lane 9 = 0.00; lane 10 = 1.35.

5, 6). T h e slow migrating band corresponded to a slightly higher molecular mass than that f r o m B.p. ( L P S a ) , and the other b a n d was similar to L P S b o f B. pertus$is. These results concerning the different profiles o f B.b~, and B. b L P S confirm a recent study p e r f o r m e d by M a r t i n et aL (1992). In all the supernatants from cyclodextrinstimulated cultures tested, the L P S patterns were found to be the same as those in SS m e d i u m , but the a m o u n t per OD650 at 48 h o f culture was higher. T h e addition o f this agent (3 g / l ) produced a 2.43-fold increase in extracellular K D O for B. bronchiseptica and a 1.37-fold increase for B. parapertussis. However, for both species, the levels o f K D O / O D 6 s 0 at each g r o w t h period assayed were lower than those corresponding to B. pertussis. Spontaneous release o f L P S from cultures o f other G r a m -

207

R E L E A S E O F LRS DURING B. PERTUSSIS G R O W T H

bacteria into the supernatant during the exponential growth phase has been demonstrated by other authors. Even during the stationary phase of growth, release of LPS is observed (Devoe et al., 1973; Cadieux et aL, 1983). Despite the apparent widespread occurrence of endotoxin release in G r a m - bacteria, up to the present, there is little conclusive data explaining this generalized release phenomenon.

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The increase of LPS release by the addition of cyclodextrin to the culture medium could be accounted for by a destabilization of the outer membrane, as has been suggested by Perera et aL (1987). in accordance with this hypothesis, eyclodextrin may form inclusion compounds with hydrophobic residues, such as fatty acid (Imaizumi et aL, 1983a,b). Since the endotoxin (LPS) of B. pertussis contains several fatty acids (Starkoff and Szabo, 1986), it is possible that Met3CD interacts in some way with this component of the outer membrane. To gain further insight into this subject, the cyelodextrin distribution in the liquid culture was determined by a fluorescence technique (Gortinge et al., 1988). Under our experimental conditions (as described in "Materials and Methods"), the highest cyelodextrin level was found in the supernatant. By using "Centrifree'" filters, it was possible to determine that 5 % o f this agent was associated with the soluble fraction of supernatant with molecular mass 30 kDa, and cyelodextrin-bound whole cells were estimated as 2 °70 of the total. This binding to the cell might disorganize the outer membrane, favouring the release of LPS into the surrounding medium. Bearing these results in mind. we also analysed the polypeptides of the outer membrane from cells grown in SS and SS + Mei3CD media. SDS-PAGE revealed variations in the patterns of polypeptidas of cells harvested from both media after 48 h of growth, as shown in figure 6. At present, we are studying the possible relationship between the modifications of the outer membrane described abcwe (LPS amount and polypeptide profile) and the secretion of the main soluble antigens (filamentous haemagglutinin, pertussis toxin and adenylate cyclase), especial-

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FiR. 6. SDS-PAGE (12.5 % w/v) of outer membraneproteins of B. pertussis from SS+Me~CD (lane II and SS (lane 2l media. Cells were harvested at 48 h. Molecular sizes in kDa are indicated. ly under conditions where the yields of such antigens were enhanced (SS medium plus cyclodextrin). In this study, we have demonstrated and quantified the release of LPS during exponential and stationary growth phases of B. pertussis in SS and S S + Mei3CD media, and what is more important, the enhancement of the release of LPS in the presence of cyelodextrin. These results should be taken into account during the purification of the main soluble antigens for the production of a non-toxic acellular pertussis vaccine. Acknowledgements We thank R. Erlola for critical reading of the manuscript.

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This work was supported by tile Programme of Secretaria de Ciencia y Tecnologia, Comitd ArgentinoBrasik'fio de BioteL.nologia (Argentina), D.H., M.E.R. and A.S. are members of Scienlific Career of CIC BA. A.L. and O.Y. are members of Scientific Career of CONICET.

Liberation de lipopolyosld~ au cours de la croissance de Bordetelta pertussis

Nous averts ~tudi~ les eons6quences de raddition de (2,6-O-dimethyl)-[Leyclodextriue (Me[~CD), darts le milieu liquide de Slainer-Seholte (SS), sur la [ib~ration du lipopolyoside (LPS) ap coats de la troissance de Bordetella oertussis. Nous avons theist )a coucenlralion de 3 m g / m l pour la Me~CD en fonclion d'observations prealnbles ofJ cette concentration ~'tait le taux optimal pour augmenter [a production des antig~nes solubles majeurs. Les profils SDSPAGE de LPS extraits de cellutes d6velopp~es en milieux SS et SS+Me[~CD, r6v~::eut des r6actions semhlables. Quoique los contenus de LPS des cellules, dans leur totallt~, diminuem an cours de la croissance cellulaire, los quantit~'s obtenues/t cliff,rents moments de la croissance dans le milieu SS + Me~CD sent inf~rieures aux quautit~s obtenues duns le milieu SS soul. Par cons6x]uent le niveau de LPS diffus~ darts les surnageants des 2 milieux augmente au tours de la eroissance cellulaire. La quantit6 de LPS lib6r6 est plus ,~levd,e dans le milieu S S + M e ~ C D et elle devient sigulficative au d6but de la phase stationnaire de croissance. La liaison de la cyclodextrine aux eellules de B. pertussis pourrait ~tre en relation avec les donndes obteuues. Des r~suhats semblables oat ~t6 ohtcnus avee tomes los esp~:es du genre Bordetelta. Mots-cl(s: Pardi cellulaire, Cyclodextrine, LPS, Bordetella pertussis, Vaccin ; Elimination du LPS.

References BellaIou, J. & Relyveld, E. (i984L Studies on cuhure conditions of Bordetella pertussis and relationship to immunogenicity of vaccines. Ann. Inst. Pasteur/ MicrobioL, 135B, 101-110. Bergman. R.K. & Manoz. f.]. (1977), Increased histamine sensitivity in mice after adminislration of endotoxins, Infect. Immun,, 15, 72-77. Cadieux, L.E.. Kuzio, J., Milaz~o, F.IL & Kropinski, A.M. (19831. Spontaneous release of lipopolysaccharide by Pseudornonas aeriuginosa, J. Bact., 155. gl7-825. Devoe, I, & Gi~christ, J. (19731, Release of endotoxin in form of cell wall blebs during i~ vi,'ro growth of Neisscrip meningitidis. J. exper. Med., 138. 1156-1167.

Gorringe. A.R., irons, L.I. & Robinson. A. (198g), The effect of mcthyl-]~,cyclodextdn on the stability of Bordetelta pertussis filamentous haemagglutinin. FEMS Mierobiol. Letters, 55, 315-320. Hitchcock, P. & Brown, T.M. (1983), MorphologieM heterogeneity among Sahnonella tipopolysaceharide chemotypes in silver-stained polyacrylnmide gels. J, Beet., 154. 269-277. Hitchcock, P., Loire, L., Makela, P., Rietshel. E., Slrittmatter, W. & Morrison, D, ll986), l,ipopolysaccharide nomenclalme -- past, present, and future. ,L Bact.. 166. 699-705. Hozbor, D., Same, A, & Ynnlorno, O. (1991). Effect of metbyl-cyclodext rin on adenYlale cyclase of Bordetella perlu~is. World J. MicrobioL BioteehnoL, 7, 309-315. Ibsen, P.. Schon, C.. An-Jonson, M. & Heron, I. (198gL The effec~ of cyclo~xtrin on lipopob'saccharide production in cultures of Borffetellapertussis. J. BioL Stand., 17, 321-330. lmaizumi, A,, Suzuki, Y., One. S., Sate, H. & Sate, Y. (1983a), Heplakis-(2,6-dimethyl)-13-cyclodextrln: a novel growth stimulant for Bordelefla permssis. J. Clin. MicrobioL, 17, 78t-786. Imalzumi, A., Suzuki, Y.. One. S.. Sate. H. & Sate, Y. (1983b), Effect of Heplnkis-(2.6-dimethyl)-[L cytflodextrin on the production of pertussis toxin by Bordetella perntssis, infect, hnmun., 41, l [38-1143. Karkhanis, Y., Zelmer, J., Jackson, J. & Carlo, D. (1978), A new and improved microassay to determine 2-keto-3-deoxyoetonate in lipopolysaccharid¢ of Gram-negative bacteria. Analyt. Bio£.hem., 85. 595-601. Laemmli, U .K. (1970). Cleavage of!~ructural proteins during the assembly of the head of bacteriophage T4. Nature (Lend.), 227, 680-685. Le Dur, A., Caroff. M., Chaby, R. & Szabo, L. (1978), A novel type of endotoxin structure present in Bor. detella pertussis. Europ. J. Biochem., 84, 579-589. Le Dur, A., Chaby, R. & Szabo, L. (1980). Isolation of two protein-free and chemically different llpoolysaccharide of BordewBa perlussis phenol exlracted endotoxin. J. Bat't, 143, 77-88. Lowry, O., Rosebrough, N.. Farr, A. & Randall, ]. (1952). Protein measurement with folin phenol reagent. J. BioL Chem.. 193, 265-275. Martin, D., Peppier, M. & Brodeur, B. (1992), ImmunOlogical characterization of the lipooligosaccharide B band of Bordetella l~ertussi& Infect. Immun., 60. 2718-2725. Moreau. M.. Chaby, R. & Szabo, L. 0984). Structure terminal reducing heptasaceharide of polysaccbaride 1 isolated from Bordetella perntssis endotoxin. J. Bacl., 159, 611-617. Peppier. M. (1984), Two physically and serologically disline1 lipopolysaccharid¢ profiles in strains of Bordetella pertusMs and their phenotype variants, infect. hntnun., 43, 224-232. Perera, V., Wardlaw, A. & Freer, J. (1987), Release of pertussis toxin and its interaction with outer-membrane antigens. J. gen. MicrobioL, 133, 2427-2435. Ray, A., Redhead, K., Selkirk, S. & Peele. S. (1991), Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis. FEMS Microbiol. Letters, 79. 211-218.

RELEASE

OF LPS DURING

Robinson. A., Irons, L. & Ashworth, L. (1985), Pcrtussis vaccine: presciiI status and future prospccls. Vaccine, 3, I 1-21. Schainlman, C. (1971), Solubilizalion of the cyloplasmic membrane of Pschericbia ('oh by Triton X-I00. J. Bat'L, 108, 545-552. Slainer, D. & Scholte, M. (1971), A simple chemically defined medium for the production of phase I Gordetella per/ussis. J. gen. Microbiol., 63, 211-220. Slarkloff, A. & Szaho, L. (1986), The I~ltty acid content o f Bordetelfa pertussis e ndoloxin. J. gen. MicrobioL. 132, 97-102. S[einman, L., Sriraln, S., Adelman, N., Zanvil, S., McDevitt, H. & Urich, S. (1982), Murine model for pertussis vaccine encephalopathy linkage to H-2. Nature (Lond.), 299, 738, Slell, R. & Torrie, J. (1980), Principles and procedures of

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