Characterization of the common antigenic lipopolysaccharide O-chains produced by Bordetella bronchiseptica and Bordetella parapertussis

FEMS MicrobiologyLetters 97 (1992) 275-282 Federation of European MicrobiologicalSocieties Published by Elsevier

275

FEMSLE 05099

Characterization of the common antigenic lipopolysaccharide O-chains produced by Bordetella bronchiseptica and Bordetella parapertussis J.L. Di F a b i o ~, M. C a r o f f b, D. Karibian b, J.C. R i c h a r d s " and M.B. Perry ~ '* Institute for Biological Sciences. National Research Council of Canada. Ottawa. Ontario. Canada. and h Equipe Endotoxines URA 1116 CNRS. btstitut de Biochimw. Unit'ersit~de Paris-Sud. Orsav Frmwe

Received4 June 1992 Revision received 7 July 1992 Accepted 20 July 1992 Key word~: Lipopolysaccharide; Bordetella bronchiseptica; Bordetella parapertussis; Polysaccharide; Lipopolysaccharide

1. S U M M A R Y

which appeared to be structurally and immunologically similar to a core oligosaccharide of B.

Representative strains of Bordetella bronand B. parapertussis were found to produce smooth lipopolysaccharides (LPS) having identical antigenic O-polysaccharide components composed of linear unbranched polymers of 1,41inked 2,3-diace tamido-2,3-dideoxy-a-L-galactopyranosyluronic acid residues. These LPSs differed from the LPS of B. pertussis which produces only rough-type LPS, devoid of O-polysaccharide. While B. bronchiseptica and B. parapertussis had chemically and immunologically identical O-polysaccharide structures, their core oligosaecharide components differed. The core oligosaccharide of B. parapertussis was chemically distinct from the core of B. bronchiseptica

pertussis.

chiseptica

Correspondence to: M.B. Perry. Institute for Biological Sci-

ences. National Research Council, Ottawa. Ont. KIA OR6. Canada.

2. I N T R O D U C T I O N Bordetella bronchiseptica, a motile aerobic Gram-negative coccoid rod. is a respiratory tract pathogen of mammals [I] while B. parapertussis is a related human pathogen that causes a whooping cough syndrome that is milder than that normally seen with B. pertuss/s [2]. In Bordetella infections, the lipopolysaccharide components of these bacteria have been implicated as virulence factors [3]. In contrast to the detailed information that has been deduced about the structure of the LPS of B. pertussis [4-7] very little information on the chemical nature of the LPS of B. parapertussis or B. bronchispetica is available. With a view to the possibility of using the O-chain polysaccharidcs of the smooth LPS of B.

276

parapermssis and B. bronchiseptica as specific antigenic markers in the development of diagnostic reagents and, in the production of synthetic conjugate vaccines, the analysis of their O-chains was undertaken. This report records the finding that the LPS O-chains of representative strains of B. parapertussis and B. bronchiseptica are identical and are linear unbranched homopolymers of 1,4-1inked 2,3-diacetamido-2,3-dideoxy-a-L-galaetopyrancsyluronic acid residues, a glycose previously not found in nature. Previous analyses of LPS preparations from B. bronchiseptica and B. parapertussis strains by SDS-PAGE, and detection by silver staining and by specific antibody using blotting methods [8-1 l], are consistent with the present conclusions that these LPS preparations have identical O-polysaccharides of approximately the same molecular mass. From the results of these studies, it is likely that the LPS core of B. bronchiseptica and B. parapertussis are chemically distinct from each other and allow the differentiation of the two species.

3. MATERIALS AND METHODS

3.1. Bacterial strains and cell production methods Strains of B. bronchiseptica provided by Dr. .I.L. Bhasin were CIG 16 (NRCC 4175), 4098 (NRCC 4170), PIG 5922, PIG 5783, PIG 5922, DOG 4079D, CAT 5817, and RABBIT CIG 13, from the National Research Council Veterinary Collection. and l l0H Vir + (NRCC 4650)(origin D. Bemis, University of Tennessee). The two strains of B. parapertussis used in chemical structural analyses were ATCC 15989 and ATCC 15311 obtained from the American Type Culture Collection. Cells of B. bronchiseptica were grown in 70-1 fermenters using 3.7% brain heart infusion containing 5% hors~ serum, at 37°C, 200 r.p.m, with aeration, for 18 h. The cells were killed with 2% phenol (final concentration) and were collected by continuous flow centrifugation. Cells of B. parapertussis were grown with rotary agitation in the above media at 37°C, using 4-1 baffled conical flasks, and were killed by 2% phenol (final con-

centration) and ~ollected by low speed ccntrifugation.

3.2 Isolation of LPS and preparation of O.polysac. charide Cells were extracted by the modified enzymephenol water method and the LPS were obtained as precipitated gels by ultracentrifugation (105000×g, 4°C, 12 h) as previously described

I121. Partial hydrolysis of 1.5% solutions of the LPS in 5% acetic acid was carried out by heating at 100°C for 3 h or hydrolysis at pH 4.5 (2 h at 100°C) in the presence of 1% SDS [13]. Insoluble lipid A was removed by centrifugation and the lyophilized aqueous solutions were subjected to Sephadex G-50 gel chromatography. The collected fractions were colorimetrically analyzed for neutral aldose [14], 2-amino-2-deoxyhexose [15], phosphate [16], hexuronic acid [17] and for formaldehyde released on periodate oxidation

[181. The reducing core oligosaccharide terminal ends of the O-polysaccharide fractions were essentially eliminated by: (a) treatment of the fractions (50 mg) with 2 ml anhydrous hydrogen fluoride (HF) at 20°C for 3 h followed by removal of HF in a stream of nitrogen gas and recovery of the polymeric O-chains by Sephadex O-50 gel filtration; or (b) by a sequence involving oxidation of the fractions (50 mg) in water (10 ml) with sodium metaperiodate (100 mg) for 2 days at 20°C, destruction of excess periodate by the addition of ethylene glycol (0.1 ml), dialysis, reduction with NaBH 4 (60 rag, 6 h), acidification with AcOH, Sephadex (3-50 gel chromatography to remove low molecular mass salts, Smith type hydrolysis [19] (2% AcOH, t00°C, 2 h), lyophilization, and collection of polymeric O-chains (approx. 24 mg) by Sephadex G-50 gel filtration

3.3. IH and 13C nuclear magnetic resonance Proton NMR spectra were recorded at 500 MHz on a Bruker AMX-500 spectrometer. Spectra were obtained using a spectral width of 7.3 kHz and a 90° pulse. Broad-band proton decoupied ~3C NMR spectra were obtained at 125 MHz, using the same spectrometer, with a spec-

277 tral width of 33 kHz and a 9(}° pulse employing W A L T Z decoupling. All spectra were recorded on samples in DzO at 37°C. Chemical shifts arc referenced to internal acetone ( 8 . . 2.225; /~c, 31.117). Two-dimensional homonuclear COSY experiments were carried out as previously described [211]. Heteronuclear two-dimensional IH-L~C chemical shift correlations were measured by I H-detected multiple quantum coherence (IH [J3C}HMQC) with a Bruker 5-mm inverse broad band probe using reverse electronics [20].

B. paral~,rtu.~.~i,~ were extracted by a m~lified phenol-water method [12] to yield, by ultracentrifugation of the concentrated, dialyzed, aqueous phases, approx. 6c/c and 1.5~ yields of LPS respectively. The preparations on SDS-PAGE analysis (Fig. I) were all indicated to contain S- an,-: R-type LPS as previously described [8-11]. By reference to standard LPS it was deduced from electrophoretic mobilities that the S-LPS Ochains fell into a narrow molecular m a ~ range and were. from interband spacing, probably homogeneous polymers of a single repeating unit as seen in the homopolymer LPS O-chains of Yersinia entercolitica 0 : 9 [23] and Brucella alu~rtus [241. Fission of the LPS required treatment with 5'% acetic acid at I(I(FC for 3 h to effect removal of the lipid A moiety. The requirement for the unusual forcing condition was probably due to phosphate substitution of a K D O residue [25]• Alternatively, the fission was made at pH 4.5 in the presence of i% SDS [26]. The water ,soluble products on Sephadex G-511 gel chromatography (Fig. 2) showed O-chain fractions (K~, 11.22, approx. 70%), core oligosaccharide (K~, 0.71, ap-

3.4. SDS-PAGE electrophoresis Purified LPS samples (10 /zg) were analyzed by clcctrophorcsis on 1.5 mm thick 14% acrylamide gels with 0.1% SDS, using the system described by Laemmli [21]. Detection of LPS was made by the periodate-silver nitrate staining method [22]. 4. R E S U L T S A N D DISCUSSION Collected saline washed cells of strains of fermenter-grown B. bronchiseptica and flask grown R

1

1'

2

lw':

2'

3

3'

-

4

4'

5

6'

R

.i

..-.

~ .

Fig. I. SDS-PAGE analysisof representative B. brom'hiseptwa LPS preparations with the LPS of Pasteurelhl haemolytwa ~rotype 4 (disaccharide repealing unit in O-chain) as a reference. Lanes: R. P. haemolytica 0:4 reference LPS; I. PIG 5783: 2. RABBITCIG 13; 3, DOG 4079D; 40 CAT 5817; and 5, PIG 5922. Samples were run in duplicate at a 2: I ratio of LPS concentrations (reading left-right) to obtain comprehensiveseparation detail.

278

14

1,2

w a ,~ o.~ ~) o.6 0.4}o.2 ;Io

0

•

=

-

20

30

40

50

60

TUBE No Fig. 2. Sephadt~x G-50 chromatographic clution profile of the water soluble hydrolyzalc of B. bronchi.~'ptica 4()98 (NRCC 4171)) LPS. Symbolsfor colorimetric~nalyses:(o) neutral glycols [411(o) hexuronic acid [17]; ( + ) aminodeoxyglycose[15]: (El) H.CHO. after IO4- oxidation 118];and 11 ) phosphate 1161.

prox. 10%), aud fractions ( K ~ 1.00, approx. 20%) containing K D O and phosphate. The O-chain fractions were purified by D E A E Sephacel ionexchange chromatography. The polysaccharides bound to the column and were subsequently eluted in a sodium chloride gradient (at approx. 0.2 M NaCI) as single sharp homogeneous peaks, in which the ratios of the colorimetrically determined aminodeoxyhexose, hexuronic acid, and

neutral glycose were the same as those determined for the original O-chain fractions. The unusually low molecular mass O-polysaccharide fractions (M r 6000) had similar elemental compositions: C, 40.1-40.6; H, 5.7-5.9; N, 9.39.7, and Ash, 2.5-3.0%. The high nitrogen content, also noted by a previous investigator [24], was not due to contamination by protein or nucleic acid and suggested that an unusual diamino-

CH 3

C=O

160

140

120

100

80

fl0

40

20

ppm

Fig. 3. Proton decoupled t3C-NMRspectrum of the HF treated LPS O-polysaccharideof B. parapertussis (ATCC 15989)

27'1 H

HI

o//

-NHCOCH 8

H4 H6 H2 H3

5.0

4.5

4.0

3.5

3.0

2.5

2.0

ppm Fig. 4. Proton NMR spectrum of the B. puraln'rtu.~.~ix (AT('(" 15989) IIF-treatcd O-polysaccharide. Resonance assignments due to the major repeating 1.4-1inked 2.3-diacetamido-2.3-dideoxy-~-i-galactopyr:mosyluronic acid residues are indicated and the structure is shown in the inset.

dideoxyglycose may be present. T h e positive colo r i m e t r i c reaction for a hexuronic acid c o m p o n e n t (Fig. 2) gave a first indication that the major c o m p o n e n t o f the O - c h a i n might be a diacetamid o d i d e o x y h e x u r o n i c acid residue. A variety o f hydrolysis m e t h o d s , including t r e a t m e n t with anhydrous hydrofluoric acid, failed to yield the major O - c h a i n glycose c o m p o n e n t , a characteristic glycosidic stability o f 2,3-diamino-2,3-dideoxyhexoses [27]. T h e ' H and L~C-NMR spectra o f the O - c h a i n s w e r e c o m p l e x due to the signals arising from the significant p r o p o r t i o n o f core oligosaccharides

p r e s e n t at the reducint, ~-n,ls o f the polymers. T h e spectra o f the respective O - c h a i n s were simplified by essentially removing the core moieties (.see MA'FLRIAI.S ANt) MI-.THODS). A f t e r H F t r c a t m e n t , the purified O-chains from B. parapertussis (K:,,. 0.32. approx. 5(1% y i e l d ) w h i c h had [or],, - 2 3 0 + 10° (c 0.5, water) while the O - c h a i n s from B. bronchiseptica (K:,~ 0.33, approx. 55% yield) had [a],, - 240 + 10° (c 0.5, water). Alternately, the core oligosaccharides were removed by p e r i o d a t e ox;dation a n d Smith type hydrolysis o f the r e d u c e d ( N a B H 4) oxidized products (see MATERIALS A N D METHODS); the O-

Table I zI]- and '~C-NMR resonance as.signments for the 1.4-1inked2.3-diacetamido-2.3-dideox'y-~-t-galacturonic acid residues in the LPS O-polysaccharides of Bordetella parapertus.~'i~ and B. hronchiseptica " Proton

Chemical .shift (p.p.m.)

Coupling conslant

Carbon atom

Chemical .shift (p.p.m.)

C-I C-2 C-3 C-4 C'5 C-6 N HCOC_H~

96.1 46.6 49. I 72.1 71).2 173.5 22.7 22.3 175.1

(Hz)

H-I tt-2 H-3 H-4 H-5

5.17 4.39 4.34 4.65 4.61 (4.86) h

N HCOCH 3

1.q8 1.92

3.1 ~ 10.8 ~3 <3

NHCOCI i.~

174.9 'J Measured at 37°C in D : O (pD ~ 7). h Chemical .shift value for proton resonance shifted due to change in pD (pD ~ 3).

280 chains from B. Imral~'rtussis had [a],, - 165 + 10° (c (I.5, water) and them from B. bronchiseptk'a had [a],, - 1 6 0 + 8(I (c (I.6, water). All purified O-chain samples gave greatly simplified and almost identical N M R spectra that were indicative of a homopolymer composed of a unique linked single glycom residue. This was particularly evident in the u3C N M R spectrum (Fig. 3) which shtca, ed only ten major resonances. The nature of the component glycose residue was determined from a detailed analysis of the N M R data. The ~H N M R spectrum of the O-chain obtamed from B. paralx'rtussis (ATCC 15989) after anhydrous H F treatment (Fig. 4) contained a single major anomeric proton resonance (5.17 ppm, J =- 3 Hz) which served as the initial point of reference for complete assignment of the t H signals by 2D homonuclear chemical shift correlation (COSY). The measured chemical shifts and coupling constants arc presented in Table I. Connectivities observed i~t the COSY spectrum led to the identification of ~he coupled-spin network corresponding to a pyrt*nosyl residue having the a-galacto configuration as evidenced from the magnitude of the vicinal coupling constants [28]. Five I H-resonances could be associated with the spin-network, consistent with the glycose being a uronic acid residue. In agreement with this assertion, the chemical shift of H-5 showed a significant pH dependance (Table 1), and a resonance in the low field region of the n3C N M R spectrum (173.5 ppm) could be attributed to a carboxyl carbon. Assignment of the n3C resonances of the ring carbons was effected by correlation with the °H resonances of the directly attached protons in an H M Q C experiment. In this experiment, the H-2 and H-3 resonances of the glycose ring showed direct one-bond tH.0.~C correlations to °3C resonances at 46.6 and 49.1 ppm respectively, indicating both positions to be amino substituted [29]. The occurrence of signals characteristic of the carbonyl (175.1 and 174.9 ppm) and methyl carbons (22.3 and 22.7 ppm) of two acetamido functions were indicative of the diaminodideoxyglycose residues being present as their acetamido derivatives. From the N M R analysis the major O-chain

glycose component can be identified as a 1,4linkcd 2,3-diacctamido-2,3-dideoxy-a-n.-galactopyranosyl uronic acid where the absolute configuration is indicated from the large negative value of the specific optical rotation. The relatively deshielded value of C-4 resonance (72.1 ppm) compared with data relx)rted [3(I] fi)r methyl 2,3diacctamido-2,3-dideoxy-cr-D-galactopyranosidc (68.3 ppm), is consistent with the glycosyl linkages involving (I-4 in the O-chain [31). The structure of the repeating unit is shown in Fig. 3. The S D S - P A G E analysis of the LPS from all the B. parapertussis strains and B. hronchiseptica strains (Fig. I) isolated from pig, rabbit, dog and cat sources used in the current study, gave results analogous to those reported by other investigators (data not shown). All showed a narrow densely silver staining band of restricted molecular mass range (average M r 8000) corresponding to an O-chain composed of approximately 20 monosaccharide residues. This apparent O-chain size is consistent with the elution point of the fractions on Scphadex G-50 chromatography. From the results it can be concluded that the LPS O-chains of B. bronchiseptica and B. parapermssis are identical, being linear unbranched polymers of i,4-1inked 2,3-diacetamido-2,3-dideoxy-o~-L-galactopyranosyl residues. It is probable that future investigations will reveal that these antigenic polymers are common to all strains of the two species, as already suggested from the use of specific antibodies [8-12]. The O-chains could thus form a specific marker for the identification of B. bronchiseptica and B. parapertussis and differentiation from B. pertussis, as well as forming the basis of a conjugate vaccine. ACKNOWLEDGEMENTS We thank Mr. D.W. Griffith (NRC) for the large-scale production of bacterial cells and Dr. J.L. Bhasin (NRC) for strains of B. bronchisep-

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