Structural variability and originality of the Bordetella endotoxins

Proceedings Structural variability and originality of the Bordetella endotoxins Martine Caroff 1, Laurent Aussel 1, Hassan Zarrouk 1, Adèle Martin 2, James C. Richards 2, Hélène Thérisod 1, Malcolm B. Perry 2, Doris Karibian 1 1 Equipe Endotoxines, UMR 8619, Centre National de la Recherche Scientifique, Biochimie, Université de Paris-Sud, Orsay, France 2 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Structural studies of Bordetella endotoxins (LPSs) have revealed remarkable differences: (i) between their LPSs and those of other bacterial pathogens; (ii) among the LPSs of the seven identified Bordetella species; and (iii) among the LPSs of some Bordetella strains. The lipid As have the classical bisphosphorylated diglucosamine backbone but tend to have fewer and speciesspecific fatty acid components compared to those of other genera. Nevertheless, three strains of B. bronchiseptica have at least three different fatty acid distributions; however, the recently identified B. hinzii and B. trematum LPSs had identical lipid A structures. The B. pertussis core is a dodecasaccharide multi-branched structure bearing amino and carboxylic groups. Another unusual feature is the presence of free amino sugars in the central core region and a complex distal trisaccharide unit containing five amino groups of which four are acetylated and one is methylated. The B. pertussis LPS does not have O-chains and that of B. trematum had only a single O-unit, unlike the LPSs of all the other species of the smooth-type. The O-chain-free cores of non-b. pertussis LPSs were always built on the B. pertussis core model but most were species-specifically incomplete. The LPS structures of three B. bronchiseptica strains were found to be different from each other. The O-chains of B. bronchiseptica and B. parapertussis were almost identical and had some features in common with B. hinzii O-chain. Serological analyses are consistent with the determined LPS structures. INTRODUCTION At the present time, the Bordetella genus consists of seven species. B. pertussis, the agent of whooping cough, infects only humans. B. parapertussis causes a less severe human disease, but also affects sheep. Other respiratory diseases are caused by B. bronchiseptica (in mammals), B. hinzii and B. avium (in poultry). 1 B. bronchiseptica has recently been isolated from immunodepressed patients. 2 Recent isolates of B. holmesii 3 and B. hinzii 4 are opportunistic bacteria that have been found in cases of septicemia, and B. Received 27 August 2000 Revised 22 February 2001 Accepted 28 February 2001 Correspondence to: Dr Martine Caroff, Biochimie, Bâtiment 432, Université de Paris Sud, 91405 Orsay cedex, France Tel: +33 1 69 15 71 91; Fax: +33 1 69 85 37 15; E-mail: martine.caroff@bbmpc.u-psud.fr trematum 5 has been found in human ear and blood infections; all three are associated with low immune status. LPSs have been implicated as virulence factors in enterobacterial infections 6 and, more recently, in whooping cough by a process resulting in damage to ciliated trachea cells, involving production and release of NO. 7 Thus, the need to explore the structure/activity relationship amongst all the LPSs of Bordetella pathogens has become apparent. The B. pertussis endotoxin has been serologically characterized 8 and its complete structure recently refined. 9 The polysaccharide region is a complex, branched dodecasaccharide structure containing uncommon sugars such as 2- acetamido-4-methylamino-2,4,6-trideoxy-β-l-galactose (Fuc2NAc4NMe), and 2,3-diacetamido-2,3-dideoxy-β-Dmannuronic acid (Mann2,3NAcA). B. bronchiseptica and B. parapertussis, unlike B. pertussis, produce smooth-type LPS structures. 10 All three have different and characteristic lipid A structures. 11 Journal of Endotoxin Research, Vol. 7, No. 1, 2001 W. S. Maney & Son Ltd

64 Caroff, Aussel, Zarrouk, Martin, Richards, Thérisod, Perry, Karibian Bacterial strains MATERIALS AND METHODS B. pertussis 1414 and A100 virulent strains 12 as well as virulent and non-virulent L84 strains were obtained and grown at the Institut Mérieux, Lyon, France. All other Bordetella species and strains (B. bronchiseptica strains NRCC 4170, 4175 and 4650; B. parapertussis strains ATCC 15989, 15311; B. hinzii strain ATCC 51730; B. trematum strain NRCC 4948 and B. holmesii strain ATCC 51541) were obtained from the Canadian National Research Council collection and grown as previously described. 10 LPS The cells were killed in 2% phenol before harvesting. The LPSs were extracted by the modified enzyme-phenol-water method. 13 Lipid A and polysaccharides were separated after hydrolyzing LPS preparations in 20 mm Na-acetate/acetic acid ph 4.5 + 1% Na-dodecylsulfate at 100 C for 1 h as described. 14 Spectrometry Plasma desorption mass spectrometry (PDMS) was performed on a home-made Depil 21 15 Time-of-Flight (TOF) instrument as described. 9 Matrix-assisted laser desorption/ ionisation mass spectrometry (MALDI/MS) was performed using a Perseptive Voyager STR model (PE Biosystem, France) mass spectrometer as described. 9 1 H- and 13 C-NMR were performed as described. 9 SDS-polyacrylamide gel electrophoresis of LPS preparations O-chain-free LPS molecules. These were the first signs of differences from other LPSs. The distance between consecutive bands of B. hinzii and B. holmesii LPS molecules showed that the repeating unit was composed of more than one sugar. The B. parapertussis and B. bronchiseptica O-chain-linked LPSs did not give distinct bands because their O-chains are homopolymers of 2,3- diacetamido-2,3-dideoxy-α-l-galactopyranosyluronic acid. 10 Recent complementary structural investigations 18 indicated that the terminal sugar of B. bronchiseptica O- chains is tri-aminated, and the amines had variable substitutions, depending on the strain. B. hinzii O-chain was found to be relatively homogeneous with a major highmolecular species containing five trisaccharide subunits of which one sugar is common to the B. parapertussis and B. bronchiseptica repetitive unit. 19 In the electrophoresis region of O-chain-free LPS molecules, there was the slow migrating A-band of B. pertussis 20 corresponding to the LPS containing a dodecasaccharide 9 which included a complex distal trisaccharide, and a smaller faster moving B-band which lacked the distal trisaccharide. B. bronchiseptica behaved similarly, and the B band is the major band of strain A100 B. pertussis LPS. B. trematum LPS, which is a semi-rough type that migrated more slowly than the pertussis A- band, as expected. The O-chain-less LPSs of B. parapertussis and B. hinzii migrated faster than the B. pertussis B-band. B. avium O-chain-free LPS gave multiple bands and the main B. holmesii band migrated like the B. pertussis B-band. These observations were found to be consistent with the core structures presented in Figure 2 which were determined in this work by chemical analyses, NMR, and mass spectrometry and compared to the B. pertussis core. 9 Monoclonal antibodies raised against B. bronchiseptica and B. parapertussis O-chain-linked LPSs 21 did not show serological reaction with B. hinzii O-chain-linked LPSs. 19 Some antibodies raised specifically against B. pertussis Aliquots (0.2 0.5 µg) of the native LPS preparations were electrophoresed as previously described 16 and stained. 17 RESULTS AND DISCUSSION SDS-gel electrophoresis (Fig. 1) of the LPS preparations of the seven Bordetella species revealed one rough-type LPS (B. pertussis), one semi-rough (B. trematum), and five smooth-type LPSs. The latter were mixtures of O- chain-linked and O-chain-free LPSs (smooth and roughtypes, respectively). When compared to Escherichia and Salmonella LPSs, the O-chain-linked Bordetella LPSs appeared to cluster at relatively low masses and had no intermediate bands between the clusters and the band of Fig. 1. Electrophoretic profiles of the LPSs from: (a) Escherichia coli O119; (b) Bordetella pertussis 1414; (c) B. trematum; (d) B. bronchiseptica; (e) B. parapertussis; (f) B. avium; (g) B. hinzii; (h) B. holmesii; and (i) Salmonella typhimurium.

Structural variability and originality of the Bordetella endotoxins 65 Fig. 2. Schematic representation of the O-chain-free core structures of the Bordetella LPSs. Variable sugars are indicated by shaded areas. core structures showed cross-reactivities with the cores of the O-chain-free LPSs of other species, consistent with the presence of common core epitopes in all members of the genus tested so far. The B. pertussis distal trisaccharide was the immunodominent part of the structure. Antibodies against it cross-reacted with the O-chain-free LPSs of B. bronchiseptica isolated by chromatography, but not with those of B. parapertussis and B. hinzii. 8,19

66 Caroff, Aussel, Zarrouk, Martin, Richards, Thérisod, Perry, Karibian Another way of comparing the LPS sizes and heterogeneity was by thin-layer chromatography (TLC) in the solvent system isobutyric acid/ammonium hydroxide (1 M). 22 The O-chain-free LPSs migrated, leaving the O- chain-linked molecular species at the origin. This solvent system was used to separate preparative quantities of the two groups on silica-gel columns. 23 In some cases, the lipid A and the cores of the two LPS groups were shown to have structural differences. 16,19,24 A further use of the solvent has been developed which involves TLC chromatography of bacterial cells, extracting and separating the LPS molecular species which are then scraped off the plate for direct analysis by mass spectrometry (unpublished results). This use gives rapid results with very little material. The analysis of native endotoxins by PDMS 25 was considerably improved when the samples were first decationized and their micelles disrupted to give spectra of LPS up to 4 kda. With MALDI mass spectrometry, the higher masses of smooth-type Bordetella LPSs could also be determined. 19 Mass spectrometry has also been indispensable for ensuring that all the LPS components have been accounted for in other types of analysis. The first lipid A to be studied, that of B. pertussis, had only five fatty acids one of which was the short-chained Table 1. Comparison of the fatty acid distribution in the main molecular species of different strains and species of Bordetella R1 R2 R3 R4 B. pertussis 4 strains C 14 C 10 B. parapertussis 3 strains C 10 C 16 C 14 B. bronchiseptica strain 4175 C 14 C 12 C 14 B. bronchiseptica strain 4650 C 14 OC 16 C 14 C 12 B. hinzii C 14 (2-) C 12 B. trematum C 14 (2-) C 12 Fatty acids are presented in the table in a simplified way. The UPAC abbreviations are: 14:0 (2-) and 14:0 (3- ) for C 14 (2-) and C 14 respectively. They are 14:0 [3-O (14:0)] or 14:0 [3-O (14:0 (2-)] for the acyloxyacyl residues. C 10. This might explain its low in vitro toxicity. The structural analysis of all the other Bordetella lipid A species 11 revealed that they varied from one species to another although the lipid A of B. hinzii 16 and B. trematum proved to be identical. Preliminary evidence indicates that B. holmesii lipid A resembles one found in B. bronchiseptica strains. All Bordetella lipid A species share a β-1,6 bisphosphorylated glucosamine disaccharide backbone structure but differ considerably in fatty acid substitution as seen in Table 1. The structural variability of the Bordetella lipid A suggests species 11 (and strain 26 ) variability of the enzymes involved in their biosynthesis compared with the well-documented Escherichia coli system. In the latter, a symmetrical distribution of C 14 at the C-2 (R4 in Table 1), C-3 (R3), C-2 (R2), and C-3 (R1) positions of the glucosamine residues (GlcNs) is observed, whereas in Bordetella species the C-3 and C-3 positions, as well as the secondary acylation at C-2, have different species- or strain-specific fatty acids. This kind of distribution implies a dual specificity of the enzyme acting at the C- 3 and C-3 level or the existence of two different GlcN esterifying enzymes. These unusual differences involve hydroxylation and chain length of the fatty acids and, in two cases, the presence of a non-hydroxylated fatty acid in direct linkage to the sugar as indicated in Table 1. Only the amide-linked C 14 were invariable. These differences could not be attributed to growth or extraction conditions, since they were constant. The core structures are presented in Figure 2. That of B. pertussis strain 1414 9 is a highly branched structure with only one partially phosphorylated 3-deoxy-Dmanno-octulosonic acid residue (Kdo), three free amino and two uronic acid groups in addition to the GlcNAc Man2,3NAcA Fuc2NAc4NMe terminal trisaccharide, an extreme case of complexity. B. pertussis strain A100 is missing the distal trisaccharide. The LPS of the virulent and non-virulent strains L84 gave MALDI negative-ion spectra identical to that of virulent strain 1414 (not shown). Figure 3 shows the different masses obtained by PDM spectra for free cores isolated from three different strains of B. bronchiseptica. These cores are the most similar to that of B. pertussis (Fig. 2), but differ in carrying an additional phosphate group on the second heptose (Hep) residue. One strain (4170) was only partially phosphorylated, as shown by a peak appearing at m/z 2293 (80u less than m/z 2373). M/z 2293 is also the main peak of the dehydrated B. pertussis core, 9 following β-elimination of the Kdo substituent at position 4 during the acid hydrolysis used to split the LPS. Another strain (4650) lacked the terminal Hep (m/z 2293 minus 192), and still another (4175) had the 4-N of the fucosamine derivative only partially methylated as shown by a peak appearing at m/z 2359 (m/z 2373 minus 14) and by NMR. The

Structural variability and originality of the Bordetella endotoxins 67 which is most probably linked to the latter glycose residue. If the terminal trisaccharide has a biosynthesis pathway different from that of the non-acetylated core, the above disaccharide would be an intermediate product in that pathway. Structural variability was observed in the LPSs of all Bordetella species. The exceptional example of the B. bronchiseptica strains and the species-wide host range lends credence to the idea that evolutionary pressure was exerted toward changes that may have helped the bacteria to evade the host immune systems. Some of the unusual structural characteristics of the Bordetella endotoxins have been confirmed in molecular biology studies. 27 Some major points still remain to be explored. It may be that Bordetellae are not the exceptions in variability, as suggested by recent data obtained on the lipid A structures of Yersinia. 28 ACKNOWLEDGEMENTS We thank Dr Y. LeBeyec (IN2P3, Orsay) for access to the Depil mass spectrometer. NATO gave financial support for travel expenses between laboratories in 1999 and MRC/CNRS in 2000. REFERENCES Fig. 3. Negative-ion PDM spectra of O-chain-free cores from B. bronchiseptica strains: (a) 4650; (b) 4170; and (c) 4175. spectra of the three cores also showed peaks corresponding to the core minus the distal trisaccharide at m/z 1710 and 1630. The B. hinzii free core lacked the terminal Hep as well as the distal trisaccharide. 19 The core of B. parapertussis lacked in addition the terminal galactosaminuronic acid residue (GalNA). The B. avium O-chain-free core was a mixture of 4 molecular species of which the two longest ones were missing only the terminal GlcNAc residues but retained the two other sugars of the distal trisaccharide; one of these also lacked the terminal Hep. Usually, the distal trisaccharide is present or absent as a complete unit. This and the fact that its amino groups, unlike those of the rest of the core, are acetylated or methylated, suggested that it had a separate biosynthetic pathway, like the O-chain units. The O-chain-linked cores of B. parapertussis and B. hinzii, on the other hand, contained additional glycosidic residues, 19,24 i.e. Fuc2NAc4NMe and Man2,3NAcA. They were more complete relative to the B. pertussis core, than their O-chain-free cores obtained from the same LPS preparation. The latter two sugars seem to be necessary for the addition of the O-chain 1. Hinz KH, Glunder G, Luders H. Acute respiratory disease in turkey poults caused by Bordetella bronchiseptica-like bacteria. Vet Rec 1978; 103: 262 263. 2. Meiss JFGM, van Griethuijsen AJA, Muytjens HL. Bordetella bronchiseptica bronchitis in an immunosuppressed patient. Eur J Clin Microbiol Infect Dis 1990; 9: 366 367. 3. Weyant RS, Hollis DG, Weaver RE et al. Bordetella holmesii sp. Nov., a new Gram-negative species associated with septicemia. J Clin Microbiol 1995; 33: 1 8. 4. Cookson BT, Vandamme P, Carlson LC et al. Bacteremia caused by a novel species, B. hinzii. J Clin Microbiol 1994; 32: 2569 2571. 5. Vandamme P, Heyndrickx M, Vancanneyt M et al. Bordetella trematum sp. Nov., isolated from wounds and ear infections in humans, and reassessment of Alcaligenes denitrificans Rüger and Tan 1983. Int J Syst Bacteriol 1996; 46: 849 858. 6. Rietschel ET, Schade U, Jensen M, Wollenweber HW, Lüderitz O, Greisman SG. Bacterial endotoxins: chemical structure, biological activity and role in septicaemia. Scand J Infect Dis 1982; 31: 8 21. 7. Flak TA, Goldman WE. Signaling and cellular specificity of airway nitric oxide production in pertussis. Cell Microbiol 1999; 1: 51 60. 8. Le Blay K, Caroff M, Blanchard F, Perry MB, Chaby R. Epitopes of Bordetella pertussis lipopolysaccharides as potential markers for typing of isolates with monoclonal antibodies. Microbiology 1996; 142: 971 978. 9. Caroff M, Brisson JR, Martin A, Karibian D. Structure of the Bordetella pertussis 1414 endotoxin. FEBS Lett 2000; 477: 8 14. 10. Di Fabio JL, Caroff M, Karibian D, Richards JC, Perry MB. Characterization of the common antigenic lipopolysaccharide O-

68 Caroff, Aussel, Zarrouk, Martin, Richards, Thérisod, Perry, Karibian chains produced by Bordetella bronchiseptica and Bordetella parapertussis. FEMS Microbiol Lett 1992; 76: 275 281. 11. Caroff M, Aussel L, Zarrouk H, Perry MB, Karibian D. Contribution of 252 Cf-plasma desorption mass spectrometry to structural analysis of lipid A: examples of non-conservatism in lipid A structure. J Endotoxin Res 1999; 5: 86 89. 12. Caroff M, Chaby R, Karibian D, Perry J, Deprun C, Szabo L. Variations in the carbohydrate regions of Bordetella pertussis lipopolysaccharides: electrophoretic, serological, and structural features. J Bacteriol 1990; 172: 1121 1128. 13. Johnson KG, Perry MB. Improved techniques for the preparation of bacterial lipopolysaccharides. Can J Microbiol 1976; 22: 29 34. 14. Caroff M, Tacken A, Szabo L. Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the isolated lipid A fragment of the Bordetella pertussis endotoxin. Carbohydr Res 1988; 175: 273 282. 15. Della Negra S, Deprun C, Le Beyec Y. Le Spectromètre de masse à temps de vol Depil et son utilisation pour la mesure de masse de protéines de masse élevée dépassant 5000. Rev Phys Appl 1986; 21: 401 406. 16. Aussel L, Brisson JR, Perry MB, Caroff M. Structure of the lipid A of Bordetella hinzii ATCC 51730. Rapid Commun Mass Spectrom 1999; 13: 2252 2259. 17. Tsai CM, Frasch CE. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 1982; 119: 115 119. 18. Vinogradov E, Peppler MS, Perry MB. The structure of the nonreducing terminal groups of the O-specific polysaccharides from two strains of Bordetella bronchiseptica. Eur J Biochem 2000; 267: 7230 7237. 19. Aussel L, Chaby R, Le Blay K et al. Chemical and serological characterization of the Bordetella hinzii lipopolysaccharides. FEBS Lett 2000; 485: 40 46. 20. Peppler MS. Isolation and characterization of isogenic pairs of domed hemolytic and flat nonhemolytic colony types of Bordetella pertussis and their phenotype variants. Infect Immun 1982; 43: 224 232. 21. Le Blay K, Caroff M, Richards JC, Perry MB, Chaby R. Specific and cross-reacting monoclonal antibodies to Bordetella parapertussis and Bordetella bronchiseptica lipopolysaccharides. Microbiology 1994; 140: 2459 2465. 22. Caroff M, Karibian D. Several uses for isobutyric acidammonium hydroxide solvent in endotoxin analysis. Appl Environ Microbiol 1990; 56: 1957 1959. 23. Lebbar S, Karibian D, Deprun C, Caroff M. Distribution of lipid A species between long and short chain lipopolysaccharides isolated from Salmonella, Yersinia, and Escherichia as seen by 252 Cf plasma desorption mass spectrometry. J Biol Chem 1994; 269: 31881 31884. 24. Zarrouk H, Karibian D, Godard I, Perry MB, Caroff M. Use of mass spectrometry to compare three O-chain-linked and free lipopolysaccharide cores: differences found in Bordetella parapertussis. J Endotoxin Res 1997; 4: 453 458. 25. Caroff M, Deprun C, Karibian D. 252 Cf plasma desorption mass spectrometry applied to the analysis of underivatized rough-type endotoxin preparations. J Biol Chem 1993; 268: 12321 12324. 26. Zarrouk H, Karibian D, Bodie S, Perry MB, Richards JC, Caroff M. Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution. J Bacteriol 1997; 179: 3756 3760. 27. Preston A, Allen AG, Cadisch J et al. Genetic basis for lipopolysaccharide O-antigen biosynthesis in Bordetellae. Infect Immun 1999; 67: 3763 3767. 28. Aussel L, Thérisod H, Karibian D, Perry MB, Bruneteau M, Caroff M. Novel variation of lipid A structures in strains of different Yersinia species. FEBS Lett 2000; 465: 87 92.