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Characterization of the lipopolysaccharide of Yersinia pestis
Microbial Pathogenesis 2001; 30: 49–57
Article available online at http://www.idealibrary.com on
doi:10.1006/mpat.2000.0411
MICROBIAL PATHOGENESIS
Characterization of the lipopolysaccharide of Yersinia pestis Joann L. Prior a∗, Paul G. Hitchenb, E. Diane Williamsona, Andrew J. Reasonc, Howard R. Morrisb, Anne Dellb, Brendan W. Wrend & Richard W. Titballa a
DERA, CBD Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K., bDepartment of Biochemistry, Imperial College of Science, Technology and Medicine, Imperial College Rd, London, SW7 2AY, U.K., c M-Scan Ltd, Silwood Park, Sunninghill, Ascot, SL5 7PZ, U.K., and dDepartment of Infectious and Tropical Disease, London School of Hygiene and Tropical Medicine, Keppel St., London, WC1E 7HT, U.K. (Received April 26, 2000; accepted in revised form October 16, 2000)
Lipopolysaccharide (LPS) extracted from eight strains of Yersinia pestis, which had been cultured at 28 or 37°C, reacted equally well, in Western blots, with four monoclonal antibodies generated against the LPS from a single strain of Y. pestis cultured at 28°C. LPS was extracted and purified from Y. pestis strain GB, which had been cultured at 28°C. When the LPS was analysed by SDSPAGE and MALDI-TOF mass spectrometry it was found to be devoid of an O-antigen. The LPS possessed activity of 2.7 endotoxin units/ng in the Limulus amoebocyte lysate assay. The LPS stimulated the production of TNF and IL-6 from mouse macrophages, but was less active in these assays than LPS isolated from Escherichia coli strain 0111. Y. pestis LPS, either alone or with cholera toxin B subunit, was used to immunize mice. Either immunization schedule resulted in the development of an antibody response to LPS. However, this response did not provide protection against 100 MLD of Y. pestis strain GB. Key words: Yersinia pestis, lipopolysaccharide, LPS, characterization.
Introduction The genus Yersinia contains 11 species of bacteria, three of which (Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica) are pathogenic to humans [1]. Plague, caused by Y. pestis, can occur in the bubonic, septicaemic or pneumonic form. The bubonic form of the disease is a ∗ Author for correspondence. E-mail: prior jo@hotmail. com 0882–4010/01/020049+09 $35.00/0
consequence of the delivery of bacteria into a susceptible host from a flea which has previously fed on an infected host. Y. enterocolitica causes a gastro-enteritis characterized by diarrhoea and invasive involvement of the mesenteric lymphatics. Y. pseudotuberculosis can cause a similar enteric disease in humans, characterized by diarrhoea, mesenteric lymphadenopathy and symptoms of appendicitis. Lipopolysaccharide (LPS) is an integral component of the outer membrane of Gram-negative bacteria and is composed of three domains. The
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lipid-A, which is the major amphipathic molecule in the outer membrane [2], is linked to the core oligosccharide and the O-antigen polysaccharide. The O-antigen is amongst the most polymorphic of known structures [3]. The LPS from Y. pseudotuberculosis or Y. enterocolitica has been shown to contain lipid-A, core oligosaccharide and O-antigen. In contrast, the LPS from Y. pestis contains lipid-A bound to the core oligosaccharide by 3 deoxy-D-mannooctulosonic acid (KDO) [4]. However, purified LPS is not reported to contain an O-antigen [5–7]. Little is known about the properties of Y. pestis LPS. The lethal dose of LPS in mice is reported to be 5–10 mg, and pyrexia was induced in rabbits after the i.v. injection of 7 g/ kg [7]. Like the LPS from other Gram-negative bacteria, the LPS from Y. pestis caused a decrease in liver glycogen, a decrease in plasma glucose and an increase in blood urea nitrogen when injected into mice by the i.p. route [8]. It is also reported that immunization with Y. pestis LPS conjugated to Shigella dysenteriae outer membrane proteins resulted in the development of antibody to LPS, but this was not sufficient to provide protection against Y. pestis infection [7]. In another study, LPS was isolated from Y. pestis as a complex with proteins. When the complex was used to immunize mice it failed to induce protection against a challenge of 1000 LD50 of Y. pestis [9]. The generalized host response to bacterial LPS involves several cytokines, predominantly TNF, IL-6 and IL-1. A previous study has shown that IL-1 is produced in response to Y. pestis LPS [10]. However, the ability of Y. pestis LPS to stimulate the production of TNF and IL-6 is not known. TNF is a proinflammatory cytokine which induces septic shock and fever [11], both of which are responses to bacterial infection. Septic shock induced by LPS can be mimicked by the administration of TNF, indicating the key role of this cytokine in this response. LPS is also known to induce the release of the acute phase proteins [12], which are produced by the liver in response to a bacterial infection or tissue injury. These responses appear to be mediated by IL-6 which is produced by mononuclear phagocytes in response to LPS [13]. The aims of this study were to characterize the LPS isolated from Y. pestis cultured at 28°C with a view towards understanding the role of this molecule in the initial stages of the pathogenesis of bubonic plague.
J. L. Prior et al.
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Figure 1. SDS-PAGE analysis of LPS isolated from Y. pestis strain GB cultured at 28°C (lane 1), S. typhimurium strain L6511 (possessing O-antigen) (lane 2), S. typhimurium strain Ra TV119 (lacking O-antigen) (lane 3), S. minnesota strain Re 595 (lacking O-antigen and core oligosaccharide except the 3-deoxy-Dmanno-2-octulosonic acid residues) (lane 4).
Results LPS purification The phenol chloroform petroleum ether (PCPE) extraction method was used to purify LPS from Y. pestis strain GB cultured at 28°C, producing a yield of 0.6% (35 mg from 6 g dry weight of cells). One mg of this preparation contained less than 7.8 g of protein. When analysed by SDSPAGE and silver stained, one intensely stained band was visible (Fig. 1). This material migrated with a similar mobility to LPS isolated from an Ra mutant of Salmonella typhimurium (Fig. 1). Y. pestis strain GB grown at 28°C was used to produce a proteinase K minipreparation (proK miniprep) of LPS which gave a similar band pattern to that produced by the PCPE extraction.
Monoclonal antibodies against Y. pestis LPS Four monoclonal antibodies were generated, which reacted with purified LPS, from Y. pestis strain GB. These monoclonal antibodies also reacted with LPS isolated from seven other
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Characterization of the lipopolysaccharide of Yersinia pestis
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Figure 2. Reactivity of 2FIB101.3 in an Immuno-blot to proK Minipreps of LPS isolated from eight strains of Y. pestis which had been cultured at 28°C or at 37°C. (a) Y. pestis strain GB grown at 28°C (lane 1), Y. pestis strain GB grown at 37°C (lane 2), Y. pestis strain EV76 grown at 28°C (lane 3), Y. pestis strain EV76 grown at 37°C (lane 4), Y. pestis strain CO92 grown at 28°C (lane 5), Y. pestis strain CO92 grown at 37°C (lane 6), Y. pestis strain 1255 grown at 28°C (lane 7) or Y. pestis strain 1255 grown at 37°C (lane 8). (b) Y. pestis strain TW grown at 28°C (lane 1), Y. pestis strain TW grown at 37°C (lane 2), Y. pestis strain Java9 grown at 28°C (lane 3), Y. pestis strain Java9 grown at 37°C (lane 4), Y. pestis strain 195/P grown at 28°C (lane 5), Y. pestis strain 195/P grown at 37°C (lane 6), Y. pestis strain EVAA grown at 28°C (lane 7) or Y. pestis strain EVAA grown at 37°C (lane 8).
strains of Y. pestis which had been cultured at 28 or 37°C. Fig. 2(a) and (b) show the reactivity of one of the monoclonal antibodies, 3 FIB 1.3, with the LPS extracted from eight strains of Y. pestis grown at 28 or 37°C. There were small differences in the mobility of LPS from bacteria which had been cultured at the different temperatures and this is more pronounced in some of the Y. pestis strains than others. The monoclonal antibodies were further screened in an immuno-blot with proteinase K minipreparations of Y. pestis strain GB, Y. pseudotuberculosis strain 8580, Y. pseudotuberculosis strain 1779, Y. enterocolitica 10598, Y. enterocolitica 10461, E. coli strain 0111, all of these
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Figure 3. MALDI-TOF trace of LPS isolated from Y. pestis strain GB which had been cultured at 28°C.
bacteria were grown at 37 or 28°C. All of the monoclonal antibodies cross reacted with Y. pseudotuberculosis strain 8580 and one of the monoclonal antibodies, 2FIB102.3, cross reacted with Y. pseudotuberculosis strain 1779. When the monoclonal antibodies were screened in an ELISA with whole cells they reacted with Y. pestis strain GB cultured at 28 or 37°C. None of the monoclonal antibodies cross reacted with Y. pseudotuberculosis strain 8580, Y. pseudotuberculosis strain 1779, Y. enterocolitica 10598, Y. enterocolitica 10461 and E. coli strain 0111 all of which had been cultured at 37°C.
Mass spectrometry LPS purified from Y. pestis strain GB was analysed by Matrix-assisted laser desorption timeof-flight (MALDI-TOF) mass spectrometry in the mass range m/z 9000–2000. A major cluster of signals were present near m/z 3000 (Fig. 3). No significant signals were observed above m/z 3400. The observed signals are in the mass range predicted for molecules containing lipid A plus the core oligosaccharide, but lacking an Oantigen. For a complete LPS molecule containing 10–18 repeat units a mass range of m/z 11 000–19 000 would be expected. The mass spectrometry analysis did not show the presence of RNA or DNA.
Biological activity of Y. pestis LPS LPS, purified from Y. pestis strain GB, was tested for activity using the Limulus amoebocyte lysate assay. The assay compared activity of the Y.
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Figure 4. (a) TNF production by mouse macrophages stimulated by 50 g of Y. pestis LPS ( ) or 50 g of E. coli strain O111 LPS (Ε). (b) IL-6 production by mouse macrophages stimulated by 50 g of Y. pestis LPS ( ) or 50 g of E. coli strain O111 LPS (Ε). For each of these data sets three times the SD are included for each time point. In each case this is less than 0.1% of the absorbance value. Each data point was assayed in triplicate.
pestis LPS with the activity of LPS purified from E. coli. The LPS purified from Y. pestis had an activity of 8.85 EU/mol compared to the E. coli LPS which had an activity of 183.6 EU/mol. LPS purified from Y. pestis which had been cultured at 28°C was added to murine macrophage cell cultures and the release of selected
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cytokines in cell culture fluid was measured at intervals up to 32 h using an ELISA. TNF was detected in cell culture supernatant fluid 4 h after the addition of E. coli strain 0111 LPS or Y. pestis strain GB LPS. At all time points after 4 h the level of TNF in the cell culture fluid was higher from macrophages stimulated with E. coli strain 0111 LPS than from macrophages stimulated with Y. pestis strain GB LPS [Fig. 4(a)]. The amount of TNF produced by the cells stimulated by the addition of E. coli LPS rose to a peak at 26 h and then declined. The cells which were exposed to Y. pestis LPS produced increasing amounts of cytokine until a plateau was reached by 26 h. A similar assay was carried out where levels of IL-6 in culture supernatants were measured using an ELISA. IL-6 was detected 7 h after the addition of LPS. At every time point the levels of IL-6 produced by cells stimulated with E. coli LPS were greater than from cells stimulated with Y. pestis LPS [Fig. 4(b)]. The cytokine levels produced by cells stimulated by E. coli LPS increased until a plateau was reached at the end of the experiment. The cytokine levels produced by the cells stimulated with Y. pestis LPS decreased after 30 h.
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Figure 5. Time to death of mice immunized with 10 g of LPS and 10 g of CTB, 10 g of LPS or 10 g of CTB.
Characterization of the lipopolysaccharide of Yersinia pestis
six mice were immunized with purified LPS, LPS + cholera toxin B subunit (CTB) or CTB alone. Serum samples were taken from mice 14 days after the final immunization and the level of antibody against immobilized bacteria determined. Mice immunized with 10 g of LPS and 10 g of CTB had a mean IgM titre of 1:3280 and a mean IgG titre of 1:2360. In each case these titres were higher than those achieved by immunization with LPS alone, which were 1:1688 for IgM and 1:289 for total IgG. The mice were challenged with 100 median lethal doses (MLD) of Y. pestis strain GB which had been cultured at 28°C. All of the challenged mice died within 6 days (Fig. 5). There was no significant difference in the time to death of the mice immunized with LPS and CTB or LPS alone, compared with mice in the control group that had been immunized with CTB alone (Fig. 5).
Discussion LPS plays an important role in the modulation of the host’s response to invading bacteria and is capable of initiating many of the biochemical responses in the host associated with death from Gram-negative bacteremia [14, 15]. The complement cascade is part of the host response and is triggered by the presence of lipid-A-core and O-antigen, generating the membrane attack complexes (MAC). In some bacteria the presence of an O-antigen is thought to stop the integration of the MAC into the cell membrane and therefore stop the formation of pores that lead to cell death. Pathogenic bacteria without an O-antigen are generally sensitive to complement [16] and therefore attenuated. For example, a mutant of Y. enterocolitica 0:3 lacking an O-antigen was 50fold less virulent than the wildtype [17]. Some pathogenic bacteria poses a truncated O-antigen but are still protected from the activity of complement. Others, like some members of the Neisseria species, express polysaccharide components that downregulate complement activation [18]. The LPS of Y. pestis is reported to lack an O-antigen [5–7], but the bacterium is still able to invade its host, and it is reported that despite the lack of an O-antigen the LPS is still able to withstand the action of complement [19]. Most cases of plague are the consequence of a bite from a flea which has previously fed on an infected host. Culture of bacteria at 28°C is considered to result in bacteria which are
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phenotypically similar to those bacteria constituting the infective dose [1]. In this study we have shown by mass spectrometry that the LPS isolated from Y. pestis strain GB cultured at 28°C lacks an O-antigen. We have previously shown that LPS isolated from different strains of Y. pestis which had been cultured at 28 or 37°C appeared identical by SDS-PAGE (Prior et al., unpublished results). The monoclonal antibodies used in this study reacted with a single band on an immuno-blot, confirming the absence of an O-antigen from Y. pestis LPS. The LPS from eight strains of Y. pestis, which had been cultured at 28°C, reacted equally well with the monoclonal antibodies suggesting the LPS from different strains share regions of similar structure. The monoclonal antibodies also reacted with LPS isolated from Y. pestis, which had been cultured at 37°C, suggesting that the LPS isolated from Y. pestis cultured at 28 or 37°C contains an identical binding epitope(s). The activity of LPS is traditionally measured using the LAL assay, which involves the binding of an antimicrobial peptide to the bacterial LPS. Differences in the activity of LPS in this assay may be due to differences in the structure of lipid-A or differences in the solubility of the LPS. In this work we have shown that the LPS extracted from Y. pestis has a significantly lower activity when compared to E. coli LPS. These results agree with the finding that Y. pestis cells and Y. pseudotuberculosis cells cultured at 37°C (i.e. lacking an O-antigen) are more resistant to polymyxin B than Y. enterocolitica [20]. These findings suggest that Y. pestis and Y. pseudotuberculosis may be more resistant to cationic peptides involved in phagocytic killing. IL-1, IL-6 and TNF all play key roles in the host response to LPS [21–23]. The LPS of Y. pestis has previously been shown to be as effective in stimulating IL-1 production from monocytes as the LPS from Salmonella typhimurium or from Shigella flexneri [10]. However, prior to this study the effects of LPS on the production of TNF and IL-6 by immune effector cells were not known. TNF is a proinflammatory cytokine which induces septic shock and fever, both of which are associated with bacterial infection. Traditionally, the pyrogenicity of LPS was measured in the rabbit. However, the discovery that IL-6 is an endogenous pyrogen involved in the stimulation of acute-phase proteins, and induces a monophasic fever in rabbits after i.v. injection [24], enables the use of a cell culture assay in place of the rabbit model. The amount of IL-6
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produced by macrophages activated by LPS is a measure of its ability to induce the release of acute phase proteins [25]. The LPS from Y. pestis can stimulate macrophages to produce TNF and IL-6. However, the level of production was significantly lower than that induced by E. coli LPS. These results suggest that the LPS present on Y. pestis delivered from the flea is likely to induce a lower level of cytokine stimulation in vivo compared to LPS from other Gram-negative bacteria. This may be advantageous to the bacterium as this would promote the avoidance of cellular host defence mechanisms. Immunization with LPS derived from a range of pathogenic bacteria including Salmonella typhimurium and Actinobacilus pleuropneumoniae has been shown to induce protection against Salmonellosis and porcine pleuropneumonia, respectively [26, 27]. In many cases the antibodies recognize the O-side chain, but protective antibodies that recognize the core of LPS have been reported [14, 28]. Previous work has shown that immunization of mice with LPS isolated from bacteria cultured at 37°C resulted in an antibody response which did not protect mice from challenge with Y. pestis [7]. In this study we have immunized mice with LPS isolated from bacteria cultured at 28°C. Although antibody against LPS developed, the immunized animals were not protected against a challenge with 100 MLDs of Y. pestis. In studies with other bacteria, LPS vaccines which have achieved good immune responses and/or protection against challenge are frequently composed of the O-antigen component conjugated to a carrier protein [29–31]. The carrier protein is necessary to convert the typical immune response to LPS from a T-cell-independent to a T-cell-dependent response and thus to establish cellular memory for the LPS. In this study we attempted to achieve this effect by coadministration of LPS and the protein CTB, a strategy previously shown to work for other antigens [32–34]. Although this strategy increased the antibody titres induced to LPS, it did not lead to the development of protective immunity.
Materials and Methods Chemicals and enzymes All chemicals and enzymes used in this study were obtained from the Sigma Chemical Com-
J. L. Prior et al.
pany (Poole, U.K.) or from Roche Diagnostics (Lewes, U.K.). The LPS from S. minnesota and S. typhimurium were obtained from Sigma Chemical Company.
Purification of LPS LPS was extracted using an adapted method based on the PCPE method by Galanos [35]. Instead of using a homogenizer, a pestle and mortar were used to mix the bacteria with the extraction mixture. After washing with ether the extracted LPS was dried in a fume hood rather than under a vacuum. The proK miniprep were produced according to the method of Chart [36].
Mass spectrometry MALDI-TOF MS was performed using a Perseptive Biosystems Voyager Elite mass spectrometer (Foster City, CA, U.S.A.) with Delayed Extraction. Native LPS was dissolved in 90% dimethylformamide in water and aliquots (0.5 l) of the resulting solution were analysed using a matrix of a saturated solution of 2,5dihydrobenzoic acid in 70% acetonitrite, 30%, 0.1% aqueous trifluoroacetic acid. Insulin B chain was employed as an external calibrant.
Culture of bacteria Bacteria used in this study were Y. pestis strains 1255 (CAMR culture collection), 195/p (CBD culture collection), CO92 (human isolate from Colorado, CBD culture collection), EV76 (CBD culture collection), EVAA (Russian vaccine strain, CBD culture collection), GB (human isolate, CBD culture collection), Java9 (F1-negative, Indonesian isolate, CBD culture collection) and Tjiwidej (37) (CBD culture collection). Y. pseudotuberculosis strain NCTC 8580 and NCTC 1779, Y. enterocolitica NCTC 10598 and NCTC 10461 and E. coli 0111. Bacteria were cultured at 28 or 37°C as required. All Y. pestis strains were cultured on blood base II agar (BABII, Oxoid, Basingstoke, U.K., 40 g/l). When agar plates were used, hemin was added (0.25% w/v hemin in 10 mM sodium hydroxide, filter sterilized and 8 ml added/100 ml of agar). Y. pestis colonies appeared pigmented on this growth medium. All other bacteria strains were grown on nutrient agar (Oxoid, Basingstoke, U.K.).
Characterization of the lipopolysaccharide of Yersinia pestis
Production of monoclonal antibodies Four monoclonal antibodies were produced by immunizing CBA×BALB/c mice three times with a crude surface preparation from Y. pestis strain 1255 grown at 37°C. An i.v. boost was given 3 days prior to fusion. Spleen cells were fused with P3-X63-Ag8-clone 6.5.3 cells (American Typed Culture Collection CRL 1580). The cell culture supernatant from clones produced by this fusion was initially screened by a direct ELISA with Y. pestis strain 1255 heat-killed whole cells. Bacteria were bound to the plate in a carbonate/bicarbonate antigen binding buffer overnight at 4°C. Positive wells were expanded and cloned by limiting dilution. The resulting monoclonal antibodies were isotyped using a commercially available kit (Gibco, Uxbridge, U.K.). LPS was purified from Y. pestis strain GB by a phenol, chloroform petroleum ether method [35] and used to screen the monoclonal antibodies.
Immuno-blotting The monoclonal antibodies were also screened in an immuno-blot with LPS proteinase K minipreparations (proK miniprep) [36] from eight different strains of Y. pestis grown at 28 or 37°C. For each proK miniprep a 32 mg/ml suspension of bacteria was digested and 10 l of the suspension was used for each lane of a gel. Electrophoresis was carried out on 12.5% polyacrylamide gels which were produced by a method based on Laemmli [38] and immunoblotted using a semi-dry transfer system. After blocking with 5% BSA overnight and washing three times for 5 min in 0.1% BSA, blots were probed with hybridoma cell culture supernatant diluted to 1:50 in 0.1% BSA for 1–2 h at room temperature. This was followed by three 5 min washes in 0.1% BSA then an anti-species horseradish peroxidase labelled antibody (Harlan Seralab, Loughborough, U.K.) diluted to 1:5000 in 0.1% BSA and applied for 1–2 h. The blot was then washed twice for 5 min in 0.1% BSA and twice for 5 min in PBS. The substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used as suggested by the Sigma Chemical Company until colour developed.
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LPS in phosphate buffered saline was dried under nitrogen and an internal standard added (10 nmole C15:0). Hydrolysis in 1 M methanolic HCl at 80°C for 14–16 h yielded fatty acid methyl esters, the reagent being removed under a stream of nitrogen. Fatty acid methyl esters were dissolved in chloroform, washed several times with water and dried under nitrogen. Analyses were performed by gas chromatography coupled to mass spectrometry (GC-MS) on a Fisons MD800 instrument (Micromass UK Ltd, Manchester, U.K.). Samples were dissolved in hexanes prior to on-column injection on a RTX-5 (30 m×0.25 mm, Restek Corporation, Belleforte, PA, U.S.A.) column at 90°C. The oven was held at 90°C for 1 min before being increased to 130°C at a rate of 50°C/min, held at 130°C for 1 min, then increased to 250°C at a rate of 5°C/min.
Cytokine release assay The J774 mouse macrophage cell line was used for cytokine release assays. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% foetal calf serum, penicillin (100 units/ml), streptomycin (100 g/ml) and glutamine (2 mM). Fifty g of LPS in phosphate buffered saline (PBS; pH 7.2) was added to a well of a 24 well cell culture plate containing 3×105 cells. As a positive control 50 g of E. coli 0111, LPS (Sigma Chemical Company) in PBS was used. As a negative control 50 l of PBS was used. Fifty l samples were removed from the wells at time 0, 4, 7, 23, 26, 30 and 32 h. For the measurement of TNF or IL-6 commercially available kits (R & D Systems, Abingdon, U.K.) were used. Samples were diluted in culture media and PBS (1:1).
Limulus amoebocyte lysate assay Activation of Limulus amoebocyte lysate was determined using the Coatest endotoxin test kit according to the manufacturer’s instructions (Quadratech, Epsom, U.K.). Samples were diluted 10−6 or 10−5 in distilled water prior to analysis.
Quantification of LPS
Immunization with LPS and protection studies
Quantitation of LPS assumed that 1 mol of LPS carries 4 mol of 3-hydroxytetradecanoic acid.
The ability of Y. pestis LPS to protect BALB/c mice from a Y. pestis challenge was determined
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by immunizing groups of six female BALB/c mice (Charles River Laboratorie, Kent, U.K.) by the i.p. route with LPS purified by the PCPE method from Y. pestis strain GB grown at 28°C on BAB II media. On each dosing occasion, mice were given 10 g of purified LPS in PBS alone or LPS in PBS with 10 g of CTB. The mice received six immunizations, each 21 days apart. They were challenged 46 days after the last immunization with 100 cfu of Y. pestis strain GB by the subcutaneous route, delivered in 0.1 ml.
Acknowledgements This work was supported by The Biotechnology and Biological Sciences Research Council (BBSRC) and the Wellcome Trust (grants 030825 and 046294). PGH is a recipient of a BBSRC CASE award. 2001 Crown Copyright
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Characterization of the lipopolysaccharide of Yersinia pestis dysenteriae type 1 (Shiga’s Bacillus) bound to Tetanus toxoid. Infect Immun 1991; 59: 4450–8. 31 Gupta RK, Szu SC, Finkelstein RA, Robbins JB. Synthesis, characterisation and some immunological properties of conjugates composed of the detoxified lipopolysaccharide of Vibrio cholerae 01 serotype Inaba bound to cholera toxin. Infect Immun 1992; 60: 3201–8. 32 Orr N, Arnon R, Rubin G, Cohen D, Bercovier H, Lowell GH. Enhancement of anti-Shigella lipopolysaccharide (LPS) response by addition of cholera toxin B subunit to oral and intranasal proteosome-Shigella flexneri 2a LPS vaccines. Infect Immun 1994; 62: 5198–200. 33 Williamson ED, Sharp GJE, Eley SM, et al. Local and systemic immune response to microencapsulated subunit vaccine for plague. Vaccine 1996; 14: 1613–19.
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34 Wu HY, Russell MW. Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit. Infect Immun 1993; 61: 314–22. 35 Galanos C, Luderitz O, Westphal O. A new method for the extraction of R lipopolysaccharides. Eur J Biochem 1969; 9: 245–9. 36 Chart H. LPS: Isolation and Characterisation. In: Raton B, Arbor A, Eds. Methods in Practical Laboratory Bacteriology. London, Tokyo: CRC Press, 1994: 11–20. 37 Otten L. Immunization against plague with a live vaccine. Ind J Med Res 1936; 24: 73–101. 38 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–5.
Article available online at http://www.idealibrary.com on
doi:10.1006/mpat.2000.0411
MICROBIAL PATHOGENESIS
Characterization of the lipopolysaccharide of Yersinia pestis Joann L. Prior a∗, Paul G. Hitchenb, E. Diane Williamsona, Andrew J. Reasonc, Howard R. Morrisb, Anne Dellb, Brendan W. Wrend & Richard W. Titballa a
DERA, CBD Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K., bDepartment of Biochemistry, Imperial College of Science, Technology and Medicine, Imperial College Rd, London, SW7 2AY, U.K., c M-Scan Ltd, Silwood Park, Sunninghill, Ascot, SL5 7PZ, U.K., and dDepartment of Infectious and Tropical Disease, London School of Hygiene and Tropical Medicine, Keppel St., London, WC1E 7HT, U.K. (Received April 26, 2000; accepted in revised form October 16, 2000)
Lipopolysaccharide (LPS) extracted from eight strains of Yersinia pestis, which had been cultured at 28 or 37°C, reacted equally well, in Western blots, with four monoclonal antibodies generated against the LPS from a single strain of Y. pestis cultured at 28°C. LPS was extracted and purified from Y. pestis strain GB, which had been cultured at 28°C. When the LPS was analysed by SDSPAGE and MALDI-TOF mass spectrometry it was found to be devoid of an O-antigen. The LPS possessed activity of 2.7 endotoxin units/ng in the Limulus amoebocyte lysate assay. The LPS stimulated the production of TNF and IL-6 from mouse macrophages, but was less active in these assays than LPS isolated from Escherichia coli strain 0111. Y. pestis LPS, either alone or with cholera toxin B subunit, was used to immunize mice. Either immunization schedule resulted in the development of an antibody response to LPS. However, this response did not provide protection against 100 MLD of Y. pestis strain GB. Key words: Yersinia pestis, lipopolysaccharide, LPS, characterization.
Introduction The genus Yersinia contains 11 species of bacteria, three of which (Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica) are pathogenic to humans [1]. Plague, caused by Y. pestis, can occur in the bubonic, septicaemic or pneumonic form. The bubonic form of the disease is a ∗ Author for correspondence. E-mail: prior jo@hotmail. com 0882–4010/01/020049+09 $35.00/0
consequence of the delivery of bacteria into a susceptible host from a flea which has previously fed on an infected host. Y. enterocolitica causes a gastro-enteritis characterized by diarrhoea and invasive involvement of the mesenteric lymphatics. Y. pseudotuberculosis can cause a similar enteric disease in humans, characterized by diarrhoea, mesenteric lymphadenopathy and symptoms of appendicitis. Lipopolysaccharide (LPS) is an integral component of the outer membrane of Gram-negative bacteria and is composed of three domains. The
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lipid-A, which is the major amphipathic molecule in the outer membrane [2], is linked to the core oligosccharide and the O-antigen polysaccharide. The O-antigen is amongst the most polymorphic of known structures [3]. The LPS from Y. pseudotuberculosis or Y. enterocolitica has been shown to contain lipid-A, core oligosaccharide and O-antigen. In contrast, the LPS from Y. pestis contains lipid-A bound to the core oligosaccharide by 3 deoxy-D-mannooctulosonic acid (KDO) [4]. However, purified LPS is not reported to contain an O-antigen [5–7]. Little is known about the properties of Y. pestis LPS. The lethal dose of LPS in mice is reported to be 5–10 mg, and pyrexia was induced in rabbits after the i.v. injection of 7 g/ kg [7]. Like the LPS from other Gram-negative bacteria, the LPS from Y. pestis caused a decrease in liver glycogen, a decrease in plasma glucose and an increase in blood urea nitrogen when injected into mice by the i.p. route [8]. It is also reported that immunization with Y. pestis LPS conjugated to Shigella dysenteriae outer membrane proteins resulted in the development of antibody to LPS, but this was not sufficient to provide protection against Y. pestis infection [7]. In another study, LPS was isolated from Y. pestis as a complex with proteins. When the complex was used to immunize mice it failed to induce protection against a challenge of 1000 LD50 of Y. pestis [9]. The generalized host response to bacterial LPS involves several cytokines, predominantly TNF, IL-6 and IL-1. A previous study has shown that IL-1 is produced in response to Y. pestis LPS [10]. However, the ability of Y. pestis LPS to stimulate the production of TNF and IL-6 is not known. TNF is a proinflammatory cytokine which induces septic shock and fever [11], both of which are responses to bacterial infection. Septic shock induced by LPS can be mimicked by the administration of TNF, indicating the key role of this cytokine in this response. LPS is also known to induce the release of the acute phase proteins [12], which are produced by the liver in response to a bacterial infection or tissue injury. These responses appear to be mediated by IL-6 which is produced by mononuclear phagocytes in response to LPS [13]. The aims of this study were to characterize the LPS isolated from Y. pestis cultured at 28°C with a view towards understanding the role of this molecule in the initial stages of the pathogenesis of bubonic plague.
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Figure 1. SDS-PAGE analysis of LPS isolated from Y. pestis strain GB cultured at 28°C (lane 1), S. typhimurium strain L6511 (possessing O-antigen) (lane 2), S. typhimurium strain Ra TV119 (lacking O-antigen) (lane 3), S. minnesota strain Re 595 (lacking O-antigen and core oligosaccharide except the 3-deoxy-Dmanno-2-octulosonic acid residues) (lane 4).
Results LPS purification The phenol chloroform petroleum ether (PCPE) extraction method was used to purify LPS from Y. pestis strain GB cultured at 28°C, producing a yield of 0.6% (35 mg from 6 g dry weight of cells). One mg of this preparation contained less than 7.8 g of protein. When analysed by SDSPAGE and silver stained, one intensely stained band was visible (Fig. 1). This material migrated with a similar mobility to LPS isolated from an Ra mutant of Salmonella typhimurium (Fig. 1). Y. pestis strain GB grown at 28°C was used to produce a proteinase K minipreparation (proK miniprep) of LPS which gave a similar band pattern to that produced by the PCPE extraction.
Monoclonal antibodies against Y. pestis LPS Four monoclonal antibodies were generated, which reacted with purified LPS, from Y. pestis strain GB. These monoclonal antibodies also reacted with LPS isolated from seven other
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Figure 2. Reactivity of 2FIB101.3 in an Immuno-blot to proK Minipreps of LPS isolated from eight strains of Y. pestis which had been cultured at 28°C or at 37°C. (a) Y. pestis strain GB grown at 28°C (lane 1), Y. pestis strain GB grown at 37°C (lane 2), Y. pestis strain EV76 grown at 28°C (lane 3), Y. pestis strain EV76 grown at 37°C (lane 4), Y. pestis strain CO92 grown at 28°C (lane 5), Y. pestis strain CO92 grown at 37°C (lane 6), Y. pestis strain 1255 grown at 28°C (lane 7) or Y. pestis strain 1255 grown at 37°C (lane 8). (b) Y. pestis strain TW grown at 28°C (lane 1), Y. pestis strain TW grown at 37°C (lane 2), Y. pestis strain Java9 grown at 28°C (lane 3), Y. pestis strain Java9 grown at 37°C (lane 4), Y. pestis strain 195/P grown at 28°C (lane 5), Y. pestis strain 195/P grown at 37°C (lane 6), Y. pestis strain EVAA grown at 28°C (lane 7) or Y. pestis strain EVAA grown at 37°C (lane 8).
strains of Y. pestis which had been cultured at 28 or 37°C. Fig. 2(a) and (b) show the reactivity of one of the monoclonal antibodies, 3 FIB 1.3, with the LPS extracted from eight strains of Y. pestis grown at 28 or 37°C. There were small differences in the mobility of LPS from bacteria which had been cultured at the different temperatures and this is more pronounced in some of the Y. pestis strains than others. The monoclonal antibodies were further screened in an immuno-blot with proteinase K minipreparations of Y. pestis strain GB, Y. pseudotuberculosis strain 8580, Y. pseudotuberculosis strain 1779, Y. enterocolitica 10598, Y. enterocolitica 10461, E. coli strain 0111, all of these
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Figure 3. MALDI-TOF trace of LPS isolated from Y. pestis strain GB which had been cultured at 28°C.
bacteria were grown at 37 or 28°C. All of the monoclonal antibodies cross reacted with Y. pseudotuberculosis strain 8580 and one of the monoclonal antibodies, 2FIB102.3, cross reacted with Y. pseudotuberculosis strain 1779. When the monoclonal antibodies were screened in an ELISA with whole cells they reacted with Y. pestis strain GB cultured at 28 or 37°C. None of the monoclonal antibodies cross reacted with Y. pseudotuberculosis strain 8580, Y. pseudotuberculosis strain 1779, Y. enterocolitica 10598, Y. enterocolitica 10461 and E. coli strain 0111 all of which had been cultured at 37°C.
Mass spectrometry LPS purified from Y. pestis strain GB was analysed by Matrix-assisted laser desorption timeof-flight (MALDI-TOF) mass spectrometry in the mass range m/z 9000–2000. A major cluster of signals were present near m/z 3000 (Fig. 3). No significant signals were observed above m/z 3400. The observed signals are in the mass range predicted for molecules containing lipid A plus the core oligosaccharide, but lacking an Oantigen. For a complete LPS molecule containing 10–18 repeat units a mass range of m/z 11 000–19 000 would be expected. The mass spectrometry analysis did not show the presence of RNA or DNA.
Biological activity of Y. pestis LPS LPS, purified from Y. pestis strain GB, was tested for activity using the Limulus amoebocyte lysate assay. The assay compared activity of the Y.
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Figure 4. (a) TNF production by mouse macrophages stimulated by 50 g of Y. pestis LPS ( ) or 50 g of E. coli strain O111 LPS (Ε). (b) IL-6 production by mouse macrophages stimulated by 50 g of Y. pestis LPS ( ) or 50 g of E. coli strain O111 LPS (Ε). For each of these data sets three times the SD are included for each time point. In each case this is less than 0.1% of the absorbance value. Each data point was assayed in triplicate.
pestis LPS with the activity of LPS purified from E. coli. The LPS purified from Y. pestis had an activity of 8.85 EU/mol compared to the E. coli LPS which had an activity of 183.6 EU/mol. LPS purified from Y. pestis which had been cultured at 28°C was added to murine macrophage cell cultures and the release of selected
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cytokines in cell culture fluid was measured at intervals up to 32 h using an ELISA. TNF was detected in cell culture supernatant fluid 4 h after the addition of E. coli strain 0111 LPS or Y. pestis strain GB LPS. At all time points after 4 h the level of TNF in the cell culture fluid was higher from macrophages stimulated with E. coli strain 0111 LPS than from macrophages stimulated with Y. pestis strain GB LPS [Fig. 4(a)]. The amount of TNF produced by the cells stimulated by the addition of E. coli LPS rose to a peak at 26 h and then declined. The cells which were exposed to Y. pestis LPS produced increasing amounts of cytokine until a plateau was reached by 26 h. A similar assay was carried out where levels of IL-6 in culture supernatants were measured using an ELISA. IL-6 was detected 7 h after the addition of LPS. At every time point the levels of IL-6 produced by cells stimulated with E. coli LPS were greater than from cells stimulated with Y. pestis LPS [Fig. 4(b)]. The cytokine levels produced by cells stimulated by E. coli LPS increased until a plateau was reached at the end of the experiment. The cytokine levels produced by the cells stimulated with Y. pestis LPS decreased after 30 h.
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Characterization of the lipopolysaccharide of Yersinia pestis
six mice were immunized with purified LPS, LPS + cholera toxin B subunit (CTB) or CTB alone. Serum samples were taken from mice 14 days after the final immunization and the level of antibody against immobilized bacteria determined. Mice immunized with 10 g of LPS and 10 g of CTB had a mean IgM titre of 1:3280 and a mean IgG titre of 1:2360. In each case these titres were higher than those achieved by immunization with LPS alone, which were 1:1688 for IgM and 1:289 for total IgG. The mice were challenged with 100 median lethal doses (MLD) of Y. pestis strain GB which had been cultured at 28°C. All of the challenged mice died within 6 days (Fig. 5). There was no significant difference in the time to death of the mice immunized with LPS and CTB or LPS alone, compared with mice in the control group that had been immunized with CTB alone (Fig. 5).
Discussion LPS plays an important role in the modulation of the host’s response to invading bacteria and is capable of initiating many of the biochemical responses in the host associated with death from Gram-negative bacteremia [14, 15]. The complement cascade is part of the host response and is triggered by the presence of lipid-A-core and O-antigen, generating the membrane attack complexes (MAC). In some bacteria the presence of an O-antigen is thought to stop the integration of the MAC into the cell membrane and therefore stop the formation of pores that lead to cell death. Pathogenic bacteria without an O-antigen are generally sensitive to complement [16] and therefore attenuated. For example, a mutant of Y. enterocolitica 0:3 lacking an O-antigen was 50fold less virulent than the wildtype [17]. Some pathogenic bacteria poses a truncated O-antigen but are still protected from the activity of complement. Others, like some members of the Neisseria species, express polysaccharide components that downregulate complement activation [18]. The LPS of Y. pestis is reported to lack an O-antigen [5–7], but the bacterium is still able to invade its host, and it is reported that despite the lack of an O-antigen the LPS is still able to withstand the action of complement [19]. Most cases of plague are the consequence of a bite from a flea which has previously fed on an infected host. Culture of bacteria at 28°C is considered to result in bacteria which are
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phenotypically similar to those bacteria constituting the infective dose [1]. In this study we have shown by mass spectrometry that the LPS isolated from Y. pestis strain GB cultured at 28°C lacks an O-antigen. We have previously shown that LPS isolated from different strains of Y. pestis which had been cultured at 28 or 37°C appeared identical by SDS-PAGE (Prior et al., unpublished results). The monoclonal antibodies used in this study reacted with a single band on an immuno-blot, confirming the absence of an O-antigen from Y. pestis LPS. The LPS from eight strains of Y. pestis, which had been cultured at 28°C, reacted equally well with the monoclonal antibodies suggesting the LPS from different strains share regions of similar structure. The monoclonal antibodies also reacted with LPS isolated from Y. pestis, which had been cultured at 37°C, suggesting that the LPS isolated from Y. pestis cultured at 28 or 37°C contains an identical binding epitope(s). The activity of LPS is traditionally measured using the LAL assay, which involves the binding of an antimicrobial peptide to the bacterial LPS. Differences in the activity of LPS in this assay may be due to differences in the structure of lipid-A or differences in the solubility of the LPS. In this work we have shown that the LPS extracted from Y. pestis has a significantly lower activity when compared to E. coli LPS. These results agree with the finding that Y. pestis cells and Y. pseudotuberculosis cells cultured at 37°C (i.e. lacking an O-antigen) are more resistant to polymyxin B than Y. enterocolitica [20]. These findings suggest that Y. pestis and Y. pseudotuberculosis may be more resistant to cationic peptides involved in phagocytic killing. IL-1, IL-6 and TNF all play key roles in the host response to LPS [21–23]. The LPS of Y. pestis has previously been shown to be as effective in stimulating IL-1 production from monocytes as the LPS from Salmonella typhimurium or from Shigella flexneri [10]. However, prior to this study the effects of LPS on the production of TNF and IL-6 by immune effector cells were not known. TNF is a proinflammatory cytokine which induces septic shock and fever, both of which are associated with bacterial infection. Traditionally, the pyrogenicity of LPS was measured in the rabbit. However, the discovery that IL-6 is an endogenous pyrogen involved in the stimulation of acute-phase proteins, and induces a monophasic fever in rabbits after i.v. injection [24], enables the use of a cell culture assay in place of the rabbit model. The amount of IL-6
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produced by macrophages activated by LPS is a measure of its ability to induce the release of acute phase proteins [25]. The LPS from Y. pestis can stimulate macrophages to produce TNF and IL-6. However, the level of production was significantly lower than that induced by E. coli LPS. These results suggest that the LPS present on Y. pestis delivered from the flea is likely to induce a lower level of cytokine stimulation in vivo compared to LPS from other Gram-negative bacteria. This may be advantageous to the bacterium as this would promote the avoidance of cellular host defence mechanisms. Immunization with LPS derived from a range of pathogenic bacteria including Salmonella typhimurium and Actinobacilus pleuropneumoniae has been shown to induce protection against Salmonellosis and porcine pleuropneumonia, respectively [26, 27]. In many cases the antibodies recognize the O-side chain, but protective antibodies that recognize the core of LPS have been reported [14, 28]. Previous work has shown that immunization of mice with LPS isolated from bacteria cultured at 37°C resulted in an antibody response which did not protect mice from challenge with Y. pestis [7]. In this study we have immunized mice with LPS isolated from bacteria cultured at 28°C. Although antibody against LPS developed, the immunized animals were not protected against a challenge with 100 MLDs of Y. pestis. In studies with other bacteria, LPS vaccines which have achieved good immune responses and/or protection against challenge are frequently composed of the O-antigen component conjugated to a carrier protein [29–31]. The carrier protein is necessary to convert the typical immune response to LPS from a T-cell-independent to a T-cell-dependent response and thus to establish cellular memory for the LPS. In this study we attempted to achieve this effect by coadministration of LPS and the protein CTB, a strategy previously shown to work for other antigens [32–34]. Although this strategy increased the antibody titres induced to LPS, it did not lead to the development of protective immunity.
Materials and Methods Chemicals and enzymes All chemicals and enzymes used in this study were obtained from the Sigma Chemical Com-
J. L. Prior et al.
pany (Poole, U.K.) or from Roche Diagnostics (Lewes, U.K.). The LPS from S. minnesota and S. typhimurium were obtained from Sigma Chemical Company.
Purification of LPS LPS was extracted using an adapted method based on the PCPE method by Galanos [35]. Instead of using a homogenizer, a pestle and mortar were used to mix the bacteria with the extraction mixture. After washing with ether the extracted LPS was dried in a fume hood rather than under a vacuum. The proK miniprep were produced according to the method of Chart [36].
Mass spectrometry MALDI-TOF MS was performed using a Perseptive Biosystems Voyager Elite mass spectrometer (Foster City, CA, U.S.A.) with Delayed Extraction. Native LPS was dissolved in 90% dimethylformamide in water and aliquots (0.5 l) of the resulting solution were analysed using a matrix of a saturated solution of 2,5dihydrobenzoic acid in 70% acetonitrite, 30%, 0.1% aqueous trifluoroacetic acid. Insulin B chain was employed as an external calibrant.
Culture of bacteria Bacteria used in this study were Y. pestis strains 1255 (CAMR culture collection), 195/p (CBD culture collection), CO92 (human isolate from Colorado, CBD culture collection), EV76 (CBD culture collection), EVAA (Russian vaccine strain, CBD culture collection), GB (human isolate, CBD culture collection), Java9 (F1-negative, Indonesian isolate, CBD culture collection) and Tjiwidej (37) (CBD culture collection). Y. pseudotuberculosis strain NCTC 8580 and NCTC 1779, Y. enterocolitica NCTC 10598 and NCTC 10461 and E. coli 0111. Bacteria were cultured at 28 or 37°C as required. All Y. pestis strains were cultured on blood base II agar (BABII, Oxoid, Basingstoke, U.K., 40 g/l). When agar plates were used, hemin was added (0.25% w/v hemin in 10 mM sodium hydroxide, filter sterilized and 8 ml added/100 ml of agar). Y. pestis colonies appeared pigmented on this growth medium. All other bacteria strains were grown on nutrient agar (Oxoid, Basingstoke, U.K.).
Characterization of the lipopolysaccharide of Yersinia pestis
Production of monoclonal antibodies Four monoclonal antibodies were produced by immunizing CBA×BALB/c mice three times with a crude surface preparation from Y. pestis strain 1255 grown at 37°C. An i.v. boost was given 3 days prior to fusion. Spleen cells were fused with P3-X63-Ag8-clone 6.5.3 cells (American Typed Culture Collection CRL 1580). The cell culture supernatant from clones produced by this fusion was initially screened by a direct ELISA with Y. pestis strain 1255 heat-killed whole cells. Bacteria were bound to the plate in a carbonate/bicarbonate antigen binding buffer overnight at 4°C. Positive wells were expanded and cloned by limiting dilution. The resulting monoclonal antibodies were isotyped using a commercially available kit (Gibco, Uxbridge, U.K.). LPS was purified from Y. pestis strain GB by a phenol, chloroform petroleum ether method [35] and used to screen the monoclonal antibodies.
Immuno-blotting The monoclonal antibodies were also screened in an immuno-blot with LPS proteinase K minipreparations (proK miniprep) [36] from eight different strains of Y. pestis grown at 28 or 37°C. For each proK miniprep a 32 mg/ml suspension of bacteria was digested and 10 l of the suspension was used for each lane of a gel. Electrophoresis was carried out on 12.5% polyacrylamide gels which were produced by a method based on Laemmli [38] and immunoblotted using a semi-dry transfer system. After blocking with 5% BSA overnight and washing three times for 5 min in 0.1% BSA, blots were probed with hybridoma cell culture supernatant diluted to 1:50 in 0.1% BSA for 1–2 h at room temperature. This was followed by three 5 min washes in 0.1% BSA then an anti-species horseradish peroxidase labelled antibody (Harlan Seralab, Loughborough, U.K.) diluted to 1:5000 in 0.1% BSA and applied for 1–2 h. The blot was then washed twice for 5 min in 0.1% BSA and twice for 5 min in PBS. The substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used as suggested by the Sigma Chemical Company until colour developed.
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LPS in phosphate buffered saline was dried under nitrogen and an internal standard added (10 nmole C15:0). Hydrolysis in 1 M methanolic HCl at 80°C for 14–16 h yielded fatty acid methyl esters, the reagent being removed under a stream of nitrogen. Fatty acid methyl esters were dissolved in chloroform, washed several times with water and dried under nitrogen. Analyses were performed by gas chromatography coupled to mass spectrometry (GC-MS) on a Fisons MD800 instrument (Micromass UK Ltd, Manchester, U.K.). Samples were dissolved in hexanes prior to on-column injection on a RTX-5 (30 m×0.25 mm, Restek Corporation, Belleforte, PA, U.S.A.) column at 90°C. The oven was held at 90°C for 1 min before being increased to 130°C at a rate of 50°C/min, held at 130°C for 1 min, then increased to 250°C at a rate of 5°C/min.
Cytokine release assay The J774 mouse macrophage cell line was used for cytokine release assays. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% foetal calf serum, penicillin (100 units/ml), streptomycin (100 g/ml) and glutamine (2 mM). Fifty g of LPS in phosphate buffered saline (PBS; pH 7.2) was added to a well of a 24 well cell culture plate containing 3×105 cells. As a positive control 50 g of E. coli 0111, LPS (Sigma Chemical Company) in PBS was used. As a negative control 50 l of PBS was used. Fifty l samples were removed from the wells at time 0, 4, 7, 23, 26, 30 and 32 h. For the measurement of TNF or IL-6 commercially available kits (R & D Systems, Abingdon, U.K.) were used. Samples were diluted in culture media and PBS (1:1).
Limulus amoebocyte lysate assay Activation of Limulus amoebocyte lysate was determined using the Coatest endotoxin test kit according to the manufacturer’s instructions (Quadratech, Epsom, U.K.). Samples were diluted 10−6 or 10−5 in distilled water prior to analysis.
Quantification of LPS
Immunization with LPS and protection studies
Quantitation of LPS assumed that 1 mol of LPS carries 4 mol of 3-hydroxytetradecanoic acid.
The ability of Y. pestis LPS to protect BALB/c mice from a Y. pestis challenge was determined
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by immunizing groups of six female BALB/c mice (Charles River Laboratorie, Kent, U.K.) by the i.p. route with LPS purified by the PCPE method from Y. pestis strain GB grown at 28°C on BAB II media. On each dosing occasion, mice were given 10 g of purified LPS in PBS alone or LPS in PBS with 10 g of CTB. The mice received six immunizations, each 21 days apart. They were challenged 46 days after the last immunization with 100 cfu of Y. pestis strain GB by the subcutaneous route, delivered in 0.1 ml.
Acknowledgements This work was supported by The Biotechnology and Biological Sciences Research Council (BBSRC) and the Wellcome Trust (grants 030825 and 046294). PGH is a recipient of a BBSRC CASE award. 2001 Crown Copyright
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