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Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization
FEMS Immunology and Medical Microbiology 45 (2005) 221–230 www.fems-microbiology.org
Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization Danuta Witkowska a, Małgorzata Mieszała a, Andrzej Gamian a,*, Magdalena Staniszewska a, Anna Czarny a, Anna Przondo-Mordarska b, Michel Jaquinod c, Eric Forest d a
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wrocław, Weigla 12, Poland b Department of Microbiology, Medical University, 50-368 Wrocław, Chałubin´skiego 4, Poland c Laboratoire de Chimie des Prote´ines, CEA, 17, avenue des Martyrs, 38054 Grenoble, France d Institut de Biologie Structurale, CEA-CNRS, LSMP, F-38027 Grenoble, France Received 14 January 2005; received in revised form 22 March 2005; accepted 5 April 2005 First published online 26 May 2005
Abstract Fimbriae are filamentous structures present on the cell surface of many bacteria, including genus Klebsiella. The use of fimbriae as protein carriers in conjugates may allow to formulate effective multivalent vaccines and suitable diagnostics. However, the evidences have been reported that fimbriae may enhance the inflammatory response. This prompted us to examine the degree of cytokine induction by the type 1 and type 3 Klebsiella fimbriae and their conjugates. Fimbriae were assessed as carrier proteins for Escherichia coli K12 endotoxin core oligosaccharide. MALDI-MS revealed the molecular mass of fimbrial monomer major protein, which was 15,847 Da for type 1 and 18,574 Da for type 3 fimbriae of Klebsiella. These two types of fimbriae were moderate inductors of IL-6 and interferon and almost inactive with regard to the stimulation of TNF when tested in human whole blood assay. Coupling of fimbriae with E. coli K12 core oligosaccharide gave immunogenic conjugates with respect to a saccharide ligand and protein carrier, although only 10% of the pilin monomers possessed the attached oligosaccharide. Rabbit antiserum reacted with a broad spectrum of lipopolysaccharides, as measured by ELISA and immunoblotting assays. The antibodies against glycoconjugates were bactericidal for the wild, S-type bacteria of some species. Regarding the induction of cytokines by conjugates only the TNF level was noticeably elevated. These results prompt for the practical use of fimbriae, as effective protein carriers for conjugates to obtain broad-spectrum antisera for diagnostic applications. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Klebsiella; Fimbriae; Conjugate; Carrier protein; Fimbrillin; Endotoxin; Diagnostics; MALDI mass spectrometry; Cytokine; Pilin
1. Introduction Fimbriae are bacterial surface structures which mediate the attachment of many pathogenic bacteria to the *
Corresponding author. Tel.: +48 71 337 11 72; fax: +48 71 337 13
82. E-mail address: [email protected] (A. Gamian).
host cells. They are thin appendages exposed outside the lipopolysaccharide and capsular polysaccharide layers of the cell envelope. Fimbriae are built mainly of the monomers of major structural protein of average molecular mass of 20 kDa, and adhesin subunits usually of lectin properties [1]. The type 1 mannose-specific fimbriae are expressed in many enterobacterial species, whereas the type 3 fimbriae specific for collagen IV
0928-8244/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2005.04.005
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and V, are present in the majority of Klebsiella strains. Type 1 fimbriae, which are present in Klebsiella are closely related to the type 1 fimbriae of Escherichia coli [2,3]. Fimbrial proteins, with major structural monomer FimA or MrkA in types 1 and 3, respectively, and adhesin subunits FimH or MrkD in these fimbriae of Klebsiella, may be encoded by chromosomal and plasmid genes [4]. Klebsiella pneumoniae and K. oxytoca are opportunistic bacteria, which may cause urinary tract and respiratory tract infections. They are often involved in hospital outbreaks of nosocomial infections, which may be the cause of septicemia [5]. Klebsiella are also considered to participate in an intestinal translocation to the blood stream. In these bacteria, fimbriae are important pathogenic factors by the mechanism of adhesion to host tissue [1,4,6]. Prompt and specific diagnostic identification of Klebsiella antigens is important to control the disease. For this purpose, the search for common epitopes suitable for the construction of immunogen to obtain diagnostic antisera with a broad specificity for a genus, seems to be the proper approach. Fimbriae induce production of cytokines in a differential manner in various host cells, though the proinflammatory molecules are induced most efficiently, in that case they may contribute to the pathogenic effects [7–10]. Fimbriae mediate also phagocytosis of bacteria by phagocytic cells and this process is complemented by opsonophagocytosis in the presence of serum opsonins. It was shown that the interaction between Klebsiella fimbriae and human polymorphonuclear leukocytes depends on the type of fimbriation [11]. Type 3 fimbriae coated on latex particles stimulated leukocytes to a higher extent than did type 1 fimbriae, while in the presence of human serum opsonins the response of leukocytes was significantly higher with type 1 than with type 3 fimbriae [11]. The induction of antibodies may be important when opsonic activity is impaired at the initial stages of inflammation or in the neonatal period. In spite to the numerous capsular K- and lipopolysaccharide O-specific antigens in Klebsiella, these bacteria have only a few types of fimbrial structures, therefore, fimbriae are more conservative as compare to K and O antigens and as protein carriers might be as well the common antigens of a broad specificity. The aim of the present study was to prepare and characterize the major structural fimbrial proteins in view of their use as carriers and immunogens of broad specificity. Another aim was to answer the question whether Klebsiella fimbriae are involved in cytokine induction and therefore contribute to the inflammation. Studies into the stimulation of immune mediators require immunologically pure fimbrial preparation, which criterion might be relevant for possible vaccine construction. Finally, our present study aimed to prepare immunogenic conju-
gates of fimbriae with an oligosaccharide hapten from E. coli K12 endotoxin. This core oligosaccharide contains glycine which has been found in several bacterial lipopolysaccharides [12].
2. Materials and methods 2.1. Bacterial strains Two non-capsular strains were used for the preparation of fimbriae: K. pneumoniae 304 (K3 , fimbriae of type 1) and K. oxytoca 666 (K58 , fimbriae of type 3) [11]. Bacteria were cultivated on nutrient agar plates, washed and centrifuged for the preparation of fimbriae. In order to cultivate fimbriae-enriched bacteria, use was made of serial passages [11]. The degree of fimbriation was checked in the hemagglutination test and by electron microscopy. For the preparation of lipopolysaccharides, E. coli C600 K12 and some strains of Shigella, Hafnia, Salmonella and E. coli were derived from the Polish Collection of Microorganisms (PCM), Institute of Immunology and Experimental Therapy, Wrocław. Bacterial growth in liquid medium, as well as isolation and purification of the lipopolysaccharide (LPS), was carried out as detailed previously [12]. 2.2. Preparation and purification of fimbriae The procedure is based mainly on the methods developed by Fader et al. [3], Gerlach and Clegg [13] and Honda et al. [14]. Bacterial wet mass (20 g) was homogenized with 5 mM Tris–HCl buffer pH 7.5, containing 1 M NaCl (100 ml) in a blender homogenizer, in an ice-cold bath for 15 min. The cells were centrifuged and homogenized for the second time. Combined supernatants were salted out with 5% ammonium sulfate at room temperature for 30 min. The pellet containing fragments of bacteria was discarded and the supernatant was adjusted to 30% of concentration with ammonium sulfate and kept at 4 C for 20 h. Pelleted fimbriae were dialyzed against water, concentrated by ultrafiltration and purified on a Sepharose 4B column (1 cm · 27 cm), with 50 mM Tris–HCl buffer pH 7.0 containing 6 M urea as eluant. Fimbriae were eluted in a void volume, dialyzed against water, concentrated by ultrafiltration and subjected to preparative electrophoresis or hydrophobic chromatography on a Phenyl-Sepharose CL-4B column (0.5 cm · 20 cm) in 5 mM Tris–HCl buffer pH 7.0 with an NaCl gradient from 4 to 0 M. Protein was determined by the method described by Lowry et al. [15]. Fractions were monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with Coomassie Brilliant Blue staining for protein and with a silver reagent specific for both protein and LPS [16].
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2.3. Preparative SDS–PAGE Preparative separation of the proteins was performed at the conditions of Laemmli [17], using 80 ml of 10% or 12.5% resolving gel and 20 ml of 5% stacking gel prepared in 37 mm ID tube. The Model 491 Prep Cell (Bio-Rad) was set up for electrophoresis using an electrode buffer containing 25 mM Tris, 0.192 M glycine, 1% SDS pH 8.3 in cathode and anode reservoir and the same buffer as an elution buffer. After application of the 30–40 mg sample of fimbriae to the top of the stacking gel, electrophoresis was run at maximum setting 260 V and 109 mA current. Elution was started when the bromophenol blue indicator band reached the base of the separating gel. Fractions of 1.4 ml were collected according to the continuously monitored protein with UV detector set at 280 nm and checked in SDS–PAGE. Fractions with fimbrial proteins were dialysed against water, pooled and concentrated using vacuum centrifugation. 2.4. Analytical methods Sugar analysis was carried out as previously described [12,16,18]. Sugar derivatives in the form of alditol acetates were analyzed by gas–liquid chromatography–mass spectrometry (GLC–MS), using a Hewlett– Packard MSD 5971A with an HP-1 column (0.2 mm · 12 m) and programmed temperature (150–270 C, 8 C min 1).
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were dispensed to 24-well cell culture polystyrene plates (Linbro). Fimbrial, conjugate or LPS preparations were added (0.1 ml) in doses of 5, 10 and 50 lg. The samples were cultured in a cell culture incubator (at 37 C in the presence of 5% CO2). Supernatants were collected after 20 h and stored at 70 C. Samples of human peripheral blood treated with PBS served as controls. The titers of IFN, TNF and IL-6 were determined in the supernatants by biological microtests, using cell lines A549, L929 and 7TD1 as previously [22–24], where the specificity and sensitivity of these bioassays were also determined. 2.7. Determination of TNF activity Monolayer cultures of murine fibroblasts L929 were prepared on 96-well plastic plates (Linbro) by adding 4 · 104 cells/well in 100 ll Eagle medium. The plates were incubated in a cell culture incubator for 24 h. On separate plates, sequential twofold dilutions of the tested supernatants were prepared in the culture medium containing 5 lg ml 1 of actinomycin D (Sigma) and then 100 ll of each dilution were transferred to the wells with L929 cell monolayers. The cells in the medium with and without actinomycin D served as controls. After 24 h of incubation in a cell culture incubator, cell death was estimated using the colorimetric method described by Hansen et al. [23]. TNF-a activity in the supernatants was calculated by comparing the OD of the samples under test with those of the different concentrations of the standard TNF-a (Genentech).
2.5. MALDI mass spectrometry 2.8. Determination of IFN level Matrix-assisted laser-desorption ionization (MALDI) mass spectra were recorded on a time-of-flight (TOF) instrument from Perseptive Biosystems, equipped with a delayed extraction ion source [19,20]. Spectra were recorded from 256 laser shots (nitrogen laser, 337 nm) with an accelerating voltage of 20 kV in a linear mode. As a matrix, 2,5-dihydroxybenzoic acid was dissolved in aqueous acetonitrile solution (70%) containing 0.1% TFA. Then 1 ll of a 2,5-dihydroxybenzoic acid matrix was added to 1 ll of the sample and mixed together. The sample was then simply placed on top of the matrix surface and allowed to dry either by itself or in a gentle stream of nitrogen. The spectra were calibrated using 1 pmol ll 1 solution of myoglobin (m/z = 16,952), under the same conditions. 2.6. Induction of cytokines A whole-blood model was used in principle as described by Wang et al. [21]. Samples of whole peripheral blood from healthy donors were collected to a 0.015% solution of EDTA and diluted fivefold with an antibiotic-enriched RPMI 1640 medium. Volumes of 1 ml
Interferon was determined with a microtest, using the human lung carcinoma cell line A549 and the strain Col MM of the EMC virus. Dilutions of the tested supernatants (100 ll/well) were prepared on 96-well plates (Nunc), and then transferred to 100 ll cultures of A549 cells (2 · 104 cells/well) in the Dulbeco culture medium supplemented with 10% calf serum and antibiotics. The control culture was with or without supernatant over human blood cells not treated with fimbrial preparations. The cultures were incubated in a cell culture incubator (37 C, 5% CO2) for 24 h and then 50 ll of virus ColMM was added at a concentration of 100 TCI D50/ml. The results were read after 48 h of incubation, using the colorimetric method [23]. The preparation B69/19 of HuIFN-Med.Res.Council was used as a standard. 2.9. Determination of IL-6 activity The activity of IL-6 in the tested supernatants was estimated by measuring the growth of the IL-6 dependent cell line 7TD1 [24]. Cell suspensions (5 · 103 cells
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in 100 ll of Iscove culture medium supplemented with 10% fetal calf serum, antibiotics, hypoxantine and thymidine) were distributed into 96-well cell culture plates (Nunc) and then 100 ll of the tested supernatants were added to each well. The control culture was with or without supernatant over human blood cells not treated with fimbrial preparations. After incubation for 72 h (37 C, 5% CO2), the proliferation of the cells was determined with the colorimetric method [23]. IL-6 activity in the tested supernatants was expressed by comparing the OD of the samples and those of the different standard IL-6 concentrations. 2.10. Isolation of LPS core oligosaccharide and preparation of conjugates Lipopolysaccharide of E. coli K12 C600 (strain PCM 2184) was subjected to mild acid hydrolysis (1% CH3COOH, 100 C, 1 h). The carbohydrate material present in the supernatant was separated by gel filtration on a Bio-Gel P-4 column (1.6 cm · 100 cm) in order to isolate the core oligosaccharide fraction containing glycine, as detailed previously [12]. Core oligosaccharide was coupled to fimbrial proteins by a high-temperature method of conjugation as described elsewhere [25]. The oligosaccharide (0.5 mg) and fimbriae (0.5 mg) were dissolved in distilled water (0.5 ml). The solution was frozen in an acetone-dry ice bath in a glass test tube and lyophilized. Dry lyophilizate was then heated in an air oven at 105 C for 15 min. The tube was allowed to cool down to room temperature and the powder was dissolved in PBS and dialyzed against PBS. The conjugates were analyzed for sugars [16] and protein [15] and then subjected to SDS–PAGE and serological assays [18] as well as to MALDI-MS analysis. 2.11. Preparation of rabbit antisera and immunoblotting analysis Immune sera were obtained after multipoint intradermal threefold immunization of rabbits with the conjugates suspended in Freund complete adjuvant (1:1, v/v) [26]. Prior to the experiments, samples of rabbit sera were checked by immunoblotting assay for the presence of antibodies against fimbriae and lipopolysaccharides. SDS–PAGE and immunoblotting experiments were performed as previously described [18]. SDS–PAGE was carried out in 15% acrylamide gel. LPS suspension (1 mg ml 1) in sample buffer was boiled for 5 min and 2 ll samples were applied to the gel. After electrophoresis, the gel was stained with a silver reagent. For immunoblotting, after SDS–PAGE, the gel was electrophoretically transferred to a nitrocellulose membrane (Schleicher-Schuell, 0.45 lm). The blot was blocked with 20 mM Tris–HCl buffer with 50 mM NaCl (TBS) containing 3% (w/v) of gelatin at 36 C for 1 h, followed
by incubation at 36 C overnight with rabbit antiserum diluted 1:200 with TBS-1% (w/v) gelatin. The nitrocellulose membrane was then incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (diluted 1:5000 in TBS-1% (w/v) gelatin), at 36 C for 1 h and stained with a 4-chloro-1-naphthol substrate solution in the presence of H2O2. 2.12. ELISA The experiments were performed with lipopolysaccharides as described previously [18]. The wells of the microtiter plates (Nunc) were coated at 37 C for 3 h with solutions of various lipopolysaccharides (2 lg/100 ll) in 0.05 M carbonate buffer pH 9.6. The plates were then blocked at room temperature for 10 min. with 0.2% casein in TBS buffer containing 0.05% Tween 20, pH 7.5 (T-TBS). Thereafter, the wells were filled with 100 ll of serial dilutions of antiserum in PBS and the plates were incubated at room temperature for 2.5 h. After washing, 50 ll of a peroxidase-labeled goat antirabbit IgG (ICN, USA) diluted 1:10000 with PBS were added to each well. Following incubation at room temperature for 1 h, the plates were washed three times with T-TBS and the o-phenylenediamine substrate (50 ll well 1) was added. The reaction was stopped with 50 ll of H2SO4 and the optical densities were read at 492 nm, using a Behring EL311 Microplate Reader. 2.13. Bactericidal assay The experiment was essentially performed as detailed previously [27]. The bactericidal assay was carried out on tissue culture 96-well polystyrene plates (Costar, No. 3595). The E. coli C600 K12 (PCM 2184), E. coli O104 (PCM 270), Shigella sonnei PhI (PCM 2336), Sh. sonnei PhII (PCM 1984), K. oxytoca 666, Hafnia alvei PCM 1196, H. alvei PCM 537 and Salmonella enterica ser. Toucra O48 (PCM 2515) strains used in this assay were from the PCM, Institute of Immunology and Experimental Therapy, Wrocław. Bacteria were grown on nutrient agar plates at 37 C for 19 h. Suspensions of bacterial strains in HanksÕ balanced salt solution (HBSS) containing 1% of casein hydrolysate were prepared to give an OD600 = 0.26–0.29. A final working dilution of bacteria was obtained by further 1:400,000 or 1:800,000 dilutions. Twofold dilutions of rabbit polyclonal sera were performed directly on the plate to a final volume of 50 ll well 1, using HBSS-1% casein hydrolysate. Freshly thawed guinea pig complement was added to each well (20 ll) followed by 30 ll of the working dilution of bacteria (100 CFUs/well). Controls included 70 ll HBSS-1% casein hydrolysate and 30 ll of bacteria or 50 ll HBSS-1% casein hydrolysate and 20 ll of complement and 30 ll of bacteria. The tissue culture plates were incubated at 37 C for 30 min,
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then the contents of each well were mixed and 30 ll was plated onto the nutrient agar (2 plates/well). The plates were incubated at 37 C overnight and the number of colonies (CFU) was counted. The bactericidal activity of the serum expressed as the percent of the bacteria killed by the serum was calculated with respect to the mean value of the HBSS/bacteria/complement control well in the following manner: percentage of killing = [(CFUcontrol CFUserum)/CFUcontrol] · 100%. 2.14. Statistical evaluation The results were analyzed as medians and absolute ranges (Min–Max). The significance of measurements was compared using the WilcoxonÕs matched-pair signed-ranks test (Statistica software).
3. Results 3.1. Preparation of fimbriae and their immunochemical characterization To isolate pure fimbriae, use was made of a procedure which involved homogenization, ammonium sulfate precipitation, gel filtration in the presence of 6 M urea and hydrophobic chromatography. The latter step might be replaced by preparative electrophoresis. In every step, purity was checked by SDS–PAGE, with Coomassie Brilliant Blue for protein and silver staining specific for proteins and lipopolysaccharides. The yield of fimbrial protein varied between 0.5 and 2.5 mg of the obtained 20 g of wet bacterial mass. Fragments of bacterial membranes were removed via precipitation with 5% ammonium sulfate. After gel filtration on Sepharose 4B in the presence of urea, the void volume peak contained fimbriae with some proteins and LPS. The purification steps are exemplified for type 1 fimbriae (Fig. 1, lanes 1 and 2). Fimbriae could be separated from lipopolysaccharide and nonfimbrial proteins on a hydrophobic column (Fig. 1, lanes 3–6), where – with a linear gradient of NaCl – LPS and fimbriae were eluted with 3.8 and 0.2 M of sodium chloride, respectively. Nonfimbrial proteins were then eluted with ethanol. The staining of the gels with a silver reagent allowed a sensitive detection of the lipopolysaccharide. Fractions containing fimbriae were rechromatographed when necessary. Alternative to hydrophobic chromatography was preparative electrophoresis, the efficient way of one step separation of major structural fimbrial protein as a single component (Fig. 1, lines 7 and 8). Purified fimbriae were analyzed with MALDI-TOF mass spectrometry to determine the molecular mass of pilin monomers. The positive ion MALDI-TOF spectra of type 1 pilin monomers of K. pneumoniae and of type 3 pilin monomers of K. oxytoca generally revealed one
Fig. 1. SDS–PAGE analysis of fimbrial preparations of Klebsiella pneumoniae 304 (type 1) after gel filtration on Sepharose 4B column in the presence of 6 M urea (lanes 1 and 2), after hydrophobic chromatography on phenyl-Sepharose CL-4B of LPS-contaminated fractions (lanes 3 and 4) and fractions with purified fimbriae (lanes 5 and 6) and after preparative electrophoresis (lanes 7 and 8). The gels (15%) were stained with a silver reagent (lanes 1, 3, 5 and 7) and with Coomassie Brilliant Blue R250 (lanes 2, 4, 6, 8 and 9). Lane 9 represents protein molecular mass standards (LMW, Pharmacia).
major peak corresponding to the single charged, (M + H)+, pseudomolecular ion with m/z = 15,847 and m/z = 18,574, respectively (Fig. 2(a) and (b)). Some spectra (not shown) contained signals of lower intensity ascribed to the dimers and trimers of pilin, with doubled or tripled m/z values, respectively. Interestingly, several preparations of type 3 fimbriae contained also peaks related to the molecular mass of type 1 fimbriae (not shown). In all spectra, a major peak was associated with two less intense peaks, which corresponded to the doubly charged (M + 2H)2+ and triply charged (M + 3H)3+ ions of pilin monomers (Fig. 2(a) and (b)). All spectra contained also a low intensity peak with an m/z value higher by one or two sugar units. This finding was corroborated by sugar analysis of the representative preparation of type 3 fimbriae, thus revealing the presence of mannose, galactose and glucose in a molar ratio of 0.42:1.00:1.09. Further analyses were carried out for the induction of IFN, TNF and IL-6 cytokines in human peripheral blood cells by purified fimbriae. A certain level of these cytokines was measured in the supernatants over the cells which were treated with 5, 10 and 50 lg doses of protein for 20 h. Both types of the Klebsiella fimbriae were moderate, dose-dependent inductors of interferon and IL-6 (Fig. 3(a) and (c)). Interestingly, these fimbriae were inactive with respect to TNF stimulation (Fig. 3(b)). Lipopolysaccharides isolated from these Klebsiella strains were less active inductors of IFN, TNF and IL-6 than the reference LPS E. coli O111. 3.2. Preparation of conjugates, immunochemical analysis and testing of their immunogenicity Fimbrial proteins were then conjugated with a core oligosaccharide of E. coli C600K12 lipopolysaccharide. The glycine-containing oligosaccharide fraction [12] was used for conjugation with fimbrial proteins.
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Fig. 2. MALDI-TOF mass spectra of fimbrial preparations of Klebsiella pneumoniae 304 (type 1) (a), K. oxytoca 666 (type 3) (b) and of conjugate of E. coli C600K12 LPS core oligosaccharide with fimbriae of type 3 (c).
Analysis of the conjugates by MALDI-TOF mass spectrometry revealed that approximately 10% of the pilin monomers possessed an attached oligosaccharide, due to the appearance of an ion (m/z = 20,518, Fig. 2(c)) corresponding to species with a molecular mass by 1953 Da higher than that of pilin monomer type 3. This mass increment similarly to that of type 1 pilin conjugate
(not shown) is consistent with the molecular mass of oligosaccharide hapten. The rabbit anti-conjugate sera obtained for type 1 and type 3 fimbriae conjugates were then analyzed in immunological tests. Control sera from healthy non-immunized animals were negative. In immunoblotting experiments, exemplified in Fig. 4, the rabbit anti-type 3 fimbrial conjugate serum reacted with
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Fig. 4. SDS–PAGE analysis (a) and immunoblotting (b) of enterobacterial lipopolysaccharides with rabbit serum against E. coli C600K12 LPS core oligosaccharide conjugated to fimbriae of type 3.
Fig. 3. Induction of IFN (a), TNF (b) and IL-6 (c) by Klebsiella fimbriae of type 1 (strain 304) and type 3 (strain 666) and by fimbrial conjugates with E. coli C600K12 LPS core oligosaccharide (B-304 and B-666). Stimulation was achieved with 5, 10 or 50 lg doses of fimbriae or conjugates and with 5 lg of LPS of E. coli O111, K. pneumoniae 304 and K. oxytoca 666. Blood samples treated with PBS served as control. Measurements were made in triplicate in each experiment, and the experiments were repeated on 4–8 individual blood samples.
the carbohydrate epitope which is present in several enterobacterial lipopolysaccharides. The most intensive reactivity of this anti-conjugate serum was observed with LPS of H. alvei 537, 1188 and E. coli O56, O24 and homologous LPS E. coli C600K12. The reactivity
with these enterobacterial LPS antigens was proved quantitatively by the ELISA test (not shown). This serum reacted specifically with fimbriae of type 3, and recognized also the oligosaccharide epitope situated in its conjugate with BSA and in a native lipopolysaccharide of E. coli C600K12 (Fig. 5). The ELISA and immunoblotting data showed that the oligosaccharide-fimbriae conjugates were immunogenic when injected to rabbits and were able to induce the antibodies directed against the sugar moiety of glycoconjugates. The reactivity of fimbrial conjugate antisera with native protein carriers was also analyzed, as exemplified on Fig. 5 (lane 1). Anti-type 3 serum reacted only with the homologous protein whereas anti-type 1 fimbrial conjugate serum reacted with both types of fimbriae (not shown). When the conjugates were tested for the induction of cytokines, this activity was noticeably increased with respect only to TNF stimulation (as compared to nonconjugated fimbriae) (Fig. 3(b)). The results of the bactericidal test shown in Table 1 indicate that the antibodies against glycoconjugates induced in rabbits are bactericidal for the wild, S-type bacteria of different species. The H. alvei, Salmonella, E. coli and Sh. sonnei strains tested are uncapsulated. This
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Fig. 5. Immunoblotting of rabbit serum against E. coli C600K12 LPS core oligosaccharide conjugated to type 3 Klebsiella fimbriae – with LPS of E. coli C600K12 (1), fimbriae of type 3 (2) and E. coli C600K12 LPS core oligosaccharide conjugated to BSA (3). SDS–PAGE was carried out in 15% acrylamide gel. Lane 4 stained with Coomassie Brilliant Blue R250 represents protein molecular mass standards (LMW, Pharmacia).
bactericidal activity does not depend on LPS type, on the serotype of the protein or on the type of the LPS core region. The preimmune sera were not bactericidal for these strains. The bactericidal activity was only considered in relation to the complement resistant strains and highly increased in the presence of antibodies. The other strains studied (E. coli C600K12, Sh. sonnei PhII, K. oxytoca 666 and H. alvei 537) were complement sensitive, and they were not considered in the bactericidal experiments with antibodies.
4. Discussion Two pathogenic strains of uncapsulated, fimbriae producing Klebsiella were chosen for the preparation
of type 1 and type 3 fimbrial major structural proteins. The strains have distinct characteristics of adherence, namely type 1 has mannose-specific tissue receptors and type 3 has receptors at the intercellular matrix [6]. There are several methods for the purification of fimbriae; they include a mild homogenization of the cells, salt precipitation, ion-exchange chromatography, the use of detergents, and ultracentrifugation in the gradient of sucrose or cesium chloride [3,28–31]. Trace amounts of LPS are difficult to remove due to the hydrophobic interaction with fimbriae. Chromatography on phenylSepharose was used previously by Honda et al. [14] for the purification of CFAII pili from enterotoxigenic E. coli. We adopted the same optimized procedure for the purification of structural protein from both type 1 and type 3 fimbriae, yielding both proteins of high purity. The same result was obtained from preparative electrophoresis. Hence, these conditions may as well apply to the purification of different types of fimbriae. Sensitive silver staining is well suited for monitoring the trace amounts of lipopolysaccharide. With MALDI-TOF mass spectrometry it was possible to determine the molecular mass of fimbrial monomers, pilin. The actual molecular masses obtained with this technique differ by about 2 kDa from higher values estimated by SDS– PAGE. Mass spectrometry revealed also a microheterogeneity caused by glycosylation, which not only proved the purity of the preparations but also made it possible to analyze the products of chemical modification of pilin. Klebsiella fimbriae were found to be moderate inducers of IL-6 and interferon and inactive toward stimulation of TNF-a production in our experiments. After conjugation, it was only the stimulating activity of TNF that showed noticeably elevated levels. In that regard, it is postulated that fimbriae should be the candidate for practical use as a carrier in conjugate vaccine, due to their low activity in inducing proinflammatory cytokines. It is interesting to note that LPS from our both Klebsiella strains (of serotype O3) posses poor activity of inducing of proinflammatory cytokines in relation to standard E. coli O111 endotoxin. It might be in accord with data on the role of Klebsiella O3
Table 1 Bactericidal activity of rabbit sera against conjugates of core oligosaccharide (fraction B) of LPS of E. coli C600K12 with Klebsiella fimbriae 666 and 304, respectively Bacterial strain
Serum dilution causing bacterial killing (%) Anti C600B-666 serum
H. alvei 1196 S. Toucra O48 E. coli O104 Sh. sonnei PhI NB, not bactericidal.
Anti C600B-304 serum
P20%
P60%
P90%
P20%
P60%
P90%
10,240 10 1280 10,240
10,240 NB 1280 10,240
1280 NB 1 NB
10,240 NB 10,240 10,240
10,240 NB 5120 5120
NB NB 10 NB
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mannan polysaccharide enhancing the adjuvant activity of LPS, whereas lipid A from this LPS was completely devoid of an adjuvant effect typical for enterobacterial lipid A [32]. One may also speculate that Klebsiella fimbriae and lipopolysaccharide surface structures are less immunogenic possibly due to the presence of these strains as saprophytes in the intestinal tract [33], thus enhancing a virulence potential of Klebsiella. Increased immunogenicity of fimbrial conjugates may beneficially improve the anti Klebsiella immunity. It was estimated by mass spectrometry that 10% of the pilin monomers possessed an attached oligosaccharide, although at the same conditions of conjugation, lactose was bound to bovine serum albumin in high yields [25]. This may be due to low content of lysine residues in fimbrial protein molecule, namely 8 in type 1 [3] and 14 in type 3 [31] pilin and the fact that only a part of the monomer molecule is exposed on the surface of the fimbrial superstructure normally present in solution. Most likely only limited number of total free amino groups is available for conjugation. However, this amount of the conjugated hapten was sufficient for induction of anti oligosaccharide antibody. When rabbits were injected with fimbrial proteins conjugated with a glycine containing core oligosaccharide isolated from E. coli C600K12 lipopolysaccharide, the antibodies were induced against oligosaccharide and fimbriae. This means that the coupling method did not destroy the immunogenicity either of the protein carrier or of the sugar moiety containing an important oligosaccharide epitope. The glycine substituent of the core oligosaccharide is alkali and acid labile [12]. This newly elaborated method of conjugation without an activating or coupling reagent [25] is therefore suitable for binding carbohydrates to the fimbrial carriers. The antigenic epitopes present on the fimbriae after conjugation were available to immune cells. The glycine containing core oligosaccharide used for coupling was expected to have a common epitope [12]. The broad reactivity of the sera with enterobacterial lipopolysaccharides seems to substantiate this supposition. The sera were bactericidal for several enterobacterial organisms and it seems reasonable to assume that the bactericidal activity is due to the cross-reactive antibodies. Preliminary work (A. Gamian, T. Lipin´ski, unpublished) showed that the epitope is not destroyed in conjugate. Analyzing pure fimbriae of different species by serological tests and MALDI mass spectrometry might elucidate the problem of heterogeneity of fimbriae. In our experiments, the MALDI-TOF-MS spectra of some type 3 fimbriae preparations contained also a peak related to type 1 pilin [34]. Antibodies to fimbriae can facilitate phagocytosis of bacteria [6,11]. Fimbrial conjugates have been used for the preparation of vaccines against experimental and natural diseases caused by E. coli, Neisseria gonorrhoeae, N.
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meningitidis, Bordetella pertussis, Pseudomonas aeruginosa, Haemophilus influenzae and Porphyromonas gingivalis [35–37]. The protective efficacy of fimbriae might be increased and extended by coupling with an epitope of broad specificity. Significant value of these Klebsiella fimbrial conjugates would be for monitoring of the status of immunoglobulin deficiency, especially in children.
Acknowledgements This work was supported under Grant Nos. 4P05A04018 and 6P05B13420 from the Committee for Scientific Research (KBN), Warsaw, Poland and Grant 249 from the Ministe´re des Affaires Etrange´res, Paris, France.
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[26] Ługowski, C., Kułakowska, M. and Romanowska, E. (1986) Characterization and diagnostic application of a lipopolysaccharide core oligosaccharide-protein conjugate. J. Immunol. Methods 95, 187–194. [27] Jennings, H.J., Gamian, A. and Ashton, F.E. (1987) N-Propionylated group B meningococcal polysaccharide mimics a unique epitope on group B Neisseria meningitides. J. Exp. Med. 165, 1207–1211. [28] Karkhanis, Y.D. and Bhogal, B.S. (1986) A single-step isolation of K99 pili from B-44 strain of Escherichia coli. Anal. Biochem. 155, 51–55. [29] Salit, I.E., Vavougios, J. and Hofmann, T. (1983) Isolation and characterization of Escherichia coli pili from diverse clinical sources. Infect. Immun. 42, 755–762. [30] Hanson, M.S., Hempel, J. and Brinton Jr., C.C. (1988) Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein. J. Bacteriol. 170, 3350–3358. [31] Korhonen, T.K., Nurmiaho, E.L., Ranta, H. and Svanborg-Ede´n, C. (1980) New method for isolation of immunologically pure pili from Escherichia coli. Infect. Immun. 27, 569–575. [32] Rietschel, E.T., Brade, L., Schade, U., Seydel, U., Za¨hringer, U., Kusumoto, S. and Brade, H. (1988) Bacterial endotoxins: properties and structure of biologically active domains In: Surface Structures of Microorganisms and Their Interactions with the Mammalian Host (Schrinner, E., Richmond, M., Seibert, G. and Schwarz, U., Eds.), pp. 1–41. VCH, Weinheim. [33] Berg, R.D. (1995) Bacterial translocation from the gastrointestinal tract. Trends Microbiol. 3, 149–154. [34] Gamian, A., Witkowska, D., Mieszała, M., Staniszewska, M. and Przondo-Mordarska, A. (1999) Fimbriae as a carrier for antibacterial vaccines. Nova Acta Leopoldina 80, 265–272. [35] Choi, J.I., Schifferle, R.E., Yoshimura, F. and Kim, B.W. (1998) Capsular polysaccharide-fimbrial protein conjugate vaccine protects against Porphyromonas gingivalis infection in SCID mice reconstituted with human peripheral blood lymphocytes. Infect. Immun. 66, 391–393. [36] Dick Jr., E.W. and Beurret, M. (1989) Glycoconjugates of bacterial carbohydrate antigens In: Conjugate Vaccines Contribution in Microbiology and Immunology (Cruse, J.M. and Lewis, R.E.J., Eds.), vol. 10, pp. 48–114. Karger, Basel. [37] Reddin, K.M., Crowley-Luke, A., Clark, S.O., Vincent, P.J., Gorringe, A.R., Hudson, M.J. and Robinson, A. (2001) Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitides serogroup C conjugate vaccines. FEMS Immunol. Med. Microbiol. 31, 153–162.
Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization Danuta Witkowska a, Małgorzata Mieszała a, Andrzej Gamian a,*, Magdalena Staniszewska a, Anna Czarny a, Anna Przondo-Mordarska b, Michel Jaquinod c, Eric Forest d a
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wrocław, Weigla 12, Poland b Department of Microbiology, Medical University, 50-368 Wrocław, Chałubin´skiego 4, Poland c Laboratoire de Chimie des Prote´ines, CEA, 17, avenue des Martyrs, 38054 Grenoble, France d Institut de Biologie Structurale, CEA-CNRS, LSMP, F-38027 Grenoble, France Received 14 January 2005; received in revised form 22 March 2005; accepted 5 April 2005 First published online 26 May 2005
Abstract Fimbriae are filamentous structures present on the cell surface of many bacteria, including genus Klebsiella. The use of fimbriae as protein carriers in conjugates may allow to formulate effective multivalent vaccines and suitable diagnostics. However, the evidences have been reported that fimbriae may enhance the inflammatory response. This prompted us to examine the degree of cytokine induction by the type 1 and type 3 Klebsiella fimbriae and their conjugates. Fimbriae were assessed as carrier proteins for Escherichia coli K12 endotoxin core oligosaccharide. MALDI-MS revealed the molecular mass of fimbrial monomer major protein, which was 15,847 Da for type 1 and 18,574 Da for type 3 fimbriae of Klebsiella. These two types of fimbriae were moderate inductors of IL-6 and interferon and almost inactive with regard to the stimulation of TNF when tested in human whole blood assay. Coupling of fimbriae with E. coli K12 core oligosaccharide gave immunogenic conjugates with respect to a saccharide ligand and protein carrier, although only 10% of the pilin monomers possessed the attached oligosaccharide. Rabbit antiserum reacted with a broad spectrum of lipopolysaccharides, as measured by ELISA and immunoblotting assays. The antibodies against glycoconjugates were bactericidal for the wild, S-type bacteria of some species. Regarding the induction of cytokines by conjugates only the TNF level was noticeably elevated. These results prompt for the practical use of fimbriae, as effective protein carriers for conjugates to obtain broad-spectrum antisera for diagnostic applications. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Klebsiella; Fimbriae; Conjugate; Carrier protein; Fimbrillin; Endotoxin; Diagnostics; MALDI mass spectrometry; Cytokine; Pilin
1. Introduction Fimbriae are bacterial surface structures which mediate the attachment of many pathogenic bacteria to the *
Corresponding author. Tel.: +48 71 337 11 72; fax: +48 71 337 13
82. E-mail address: [email protected] (A. Gamian).
host cells. They are thin appendages exposed outside the lipopolysaccharide and capsular polysaccharide layers of the cell envelope. Fimbriae are built mainly of the monomers of major structural protein of average molecular mass of 20 kDa, and adhesin subunits usually of lectin properties [1]. The type 1 mannose-specific fimbriae are expressed in many enterobacterial species, whereas the type 3 fimbriae specific for collagen IV
0928-8244/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2005.04.005
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and V, are present in the majority of Klebsiella strains. Type 1 fimbriae, which are present in Klebsiella are closely related to the type 1 fimbriae of Escherichia coli [2,3]. Fimbrial proteins, with major structural monomer FimA or MrkA in types 1 and 3, respectively, and adhesin subunits FimH or MrkD in these fimbriae of Klebsiella, may be encoded by chromosomal and plasmid genes [4]. Klebsiella pneumoniae and K. oxytoca are opportunistic bacteria, which may cause urinary tract and respiratory tract infections. They are often involved in hospital outbreaks of nosocomial infections, which may be the cause of septicemia [5]. Klebsiella are also considered to participate in an intestinal translocation to the blood stream. In these bacteria, fimbriae are important pathogenic factors by the mechanism of adhesion to host tissue [1,4,6]. Prompt and specific diagnostic identification of Klebsiella antigens is important to control the disease. For this purpose, the search for common epitopes suitable for the construction of immunogen to obtain diagnostic antisera with a broad specificity for a genus, seems to be the proper approach. Fimbriae induce production of cytokines in a differential manner in various host cells, though the proinflammatory molecules are induced most efficiently, in that case they may contribute to the pathogenic effects [7–10]. Fimbriae mediate also phagocytosis of bacteria by phagocytic cells and this process is complemented by opsonophagocytosis in the presence of serum opsonins. It was shown that the interaction between Klebsiella fimbriae and human polymorphonuclear leukocytes depends on the type of fimbriation [11]. Type 3 fimbriae coated on latex particles stimulated leukocytes to a higher extent than did type 1 fimbriae, while in the presence of human serum opsonins the response of leukocytes was significantly higher with type 1 than with type 3 fimbriae [11]. The induction of antibodies may be important when opsonic activity is impaired at the initial stages of inflammation or in the neonatal period. In spite to the numerous capsular K- and lipopolysaccharide O-specific antigens in Klebsiella, these bacteria have only a few types of fimbrial structures, therefore, fimbriae are more conservative as compare to K and O antigens and as protein carriers might be as well the common antigens of a broad specificity. The aim of the present study was to prepare and characterize the major structural fimbrial proteins in view of their use as carriers and immunogens of broad specificity. Another aim was to answer the question whether Klebsiella fimbriae are involved in cytokine induction and therefore contribute to the inflammation. Studies into the stimulation of immune mediators require immunologically pure fimbrial preparation, which criterion might be relevant for possible vaccine construction. Finally, our present study aimed to prepare immunogenic conju-
gates of fimbriae with an oligosaccharide hapten from E. coli K12 endotoxin. This core oligosaccharide contains glycine which has been found in several bacterial lipopolysaccharides [12].
2. Materials and methods 2.1. Bacterial strains Two non-capsular strains were used for the preparation of fimbriae: K. pneumoniae 304 (K3 , fimbriae of type 1) and K. oxytoca 666 (K58 , fimbriae of type 3) [11]. Bacteria were cultivated on nutrient agar plates, washed and centrifuged for the preparation of fimbriae. In order to cultivate fimbriae-enriched bacteria, use was made of serial passages [11]. The degree of fimbriation was checked in the hemagglutination test and by electron microscopy. For the preparation of lipopolysaccharides, E. coli C600 K12 and some strains of Shigella, Hafnia, Salmonella and E. coli were derived from the Polish Collection of Microorganisms (PCM), Institute of Immunology and Experimental Therapy, Wrocław. Bacterial growth in liquid medium, as well as isolation and purification of the lipopolysaccharide (LPS), was carried out as detailed previously [12]. 2.2. Preparation and purification of fimbriae The procedure is based mainly on the methods developed by Fader et al. [3], Gerlach and Clegg [13] and Honda et al. [14]. Bacterial wet mass (20 g) was homogenized with 5 mM Tris–HCl buffer pH 7.5, containing 1 M NaCl (100 ml) in a blender homogenizer, in an ice-cold bath for 15 min. The cells were centrifuged and homogenized for the second time. Combined supernatants were salted out with 5% ammonium sulfate at room temperature for 30 min. The pellet containing fragments of bacteria was discarded and the supernatant was adjusted to 30% of concentration with ammonium sulfate and kept at 4 C for 20 h. Pelleted fimbriae were dialyzed against water, concentrated by ultrafiltration and purified on a Sepharose 4B column (1 cm · 27 cm), with 50 mM Tris–HCl buffer pH 7.0 containing 6 M urea as eluant. Fimbriae were eluted in a void volume, dialyzed against water, concentrated by ultrafiltration and subjected to preparative electrophoresis or hydrophobic chromatography on a Phenyl-Sepharose CL-4B column (0.5 cm · 20 cm) in 5 mM Tris–HCl buffer pH 7.0 with an NaCl gradient from 4 to 0 M. Protein was determined by the method described by Lowry et al. [15]. Fractions were monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with Coomassie Brilliant Blue staining for protein and with a silver reagent specific for both protein and LPS [16].
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2.3. Preparative SDS–PAGE Preparative separation of the proteins was performed at the conditions of Laemmli [17], using 80 ml of 10% or 12.5% resolving gel and 20 ml of 5% stacking gel prepared in 37 mm ID tube. The Model 491 Prep Cell (Bio-Rad) was set up for electrophoresis using an electrode buffer containing 25 mM Tris, 0.192 M glycine, 1% SDS pH 8.3 in cathode and anode reservoir and the same buffer as an elution buffer. After application of the 30–40 mg sample of fimbriae to the top of the stacking gel, electrophoresis was run at maximum setting 260 V and 109 mA current. Elution was started when the bromophenol blue indicator band reached the base of the separating gel. Fractions of 1.4 ml were collected according to the continuously monitored protein with UV detector set at 280 nm and checked in SDS–PAGE. Fractions with fimbrial proteins were dialysed against water, pooled and concentrated using vacuum centrifugation. 2.4. Analytical methods Sugar analysis was carried out as previously described [12,16,18]. Sugar derivatives in the form of alditol acetates were analyzed by gas–liquid chromatography–mass spectrometry (GLC–MS), using a Hewlett– Packard MSD 5971A with an HP-1 column (0.2 mm · 12 m) and programmed temperature (150–270 C, 8 C min 1).
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were dispensed to 24-well cell culture polystyrene plates (Linbro). Fimbrial, conjugate or LPS preparations were added (0.1 ml) in doses of 5, 10 and 50 lg. The samples were cultured in a cell culture incubator (at 37 C in the presence of 5% CO2). Supernatants were collected after 20 h and stored at 70 C. Samples of human peripheral blood treated with PBS served as controls. The titers of IFN, TNF and IL-6 were determined in the supernatants by biological microtests, using cell lines A549, L929 and 7TD1 as previously [22–24], where the specificity and sensitivity of these bioassays were also determined. 2.7. Determination of TNF activity Monolayer cultures of murine fibroblasts L929 were prepared on 96-well plastic plates (Linbro) by adding 4 · 104 cells/well in 100 ll Eagle medium. The plates were incubated in a cell culture incubator for 24 h. On separate plates, sequential twofold dilutions of the tested supernatants were prepared in the culture medium containing 5 lg ml 1 of actinomycin D (Sigma) and then 100 ll of each dilution were transferred to the wells with L929 cell monolayers. The cells in the medium with and without actinomycin D served as controls. After 24 h of incubation in a cell culture incubator, cell death was estimated using the colorimetric method described by Hansen et al. [23]. TNF-a activity in the supernatants was calculated by comparing the OD of the samples under test with those of the different concentrations of the standard TNF-a (Genentech).
2.5. MALDI mass spectrometry 2.8. Determination of IFN level Matrix-assisted laser-desorption ionization (MALDI) mass spectra were recorded on a time-of-flight (TOF) instrument from Perseptive Biosystems, equipped with a delayed extraction ion source [19,20]. Spectra were recorded from 256 laser shots (nitrogen laser, 337 nm) with an accelerating voltage of 20 kV in a linear mode. As a matrix, 2,5-dihydroxybenzoic acid was dissolved in aqueous acetonitrile solution (70%) containing 0.1% TFA. Then 1 ll of a 2,5-dihydroxybenzoic acid matrix was added to 1 ll of the sample and mixed together. The sample was then simply placed on top of the matrix surface and allowed to dry either by itself or in a gentle stream of nitrogen. The spectra were calibrated using 1 pmol ll 1 solution of myoglobin (m/z = 16,952), under the same conditions. 2.6. Induction of cytokines A whole-blood model was used in principle as described by Wang et al. [21]. Samples of whole peripheral blood from healthy donors were collected to a 0.015% solution of EDTA and diluted fivefold with an antibiotic-enriched RPMI 1640 medium. Volumes of 1 ml
Interferon was determined with a microtest, using the human lung carcinoma cell line A549 and the strain Col MM of the EMC virus. Dilutions of the tested supernatants (100 ll/well) were prepared on 96-well plates (Nunc), and then transferred to 100 ll cultures of A549 cells (2 · 104 cells/well) in the Dulbeco culture medium supplemented with 10% calf serum and antibiotics. The control culture was with or without supernatant over human blood cells not treated with fimbrial preparations. The cultures were incubated in a cell culture incubator (37 C, 5% CO2) for 24 h and then 50 ll of virus ColMM was added at a concentration of 100 TCI D50/ml. The results were read after 48 h of incubation, using the colorimetric method [23]. The preparation B69/19 of HuIFN-Med.Res.Council was used as a standard. 2.9. Determination of IL-6 activity The activity of IL-6 in the tested supernatants was estimated by measuring the growth of the IL-6 dependent cell line 7TD1 [24]. Cell suspensions (5 · 103 cells
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in 100 ll of Iscove culture medium supplemented with 10% fetal calf serum, antibiotics, hypoxantine and thymidine) were distributed into 96-well cell culture plates (Nunc) and then 100 ll of the tested supernatants were added to each well. The control culture was with or without supernatant over human blood cells not treated with fimbrial preparations. After incubation for 72 h (37 C, 5% CO2), the proliferation of the cells was determined with the colorimetric method [23]. IL-6 activity in the tested supernatants was expressed by comparing the OD of the samples and those of the different standard IL-6 concentrations. 2.10. Isolation of LPS core oligosaccharide and preparation of conjugates Lipopolysaccharide of E. coli K12 C600 (strain PCM 2184) was subjected to mild acid hydrolysis (1% CH3COOH, 100 C, 1 h). The carbohydrate material present in the supernatant was separated by gel filtration on a Bio-Gel P-4 column (1.6 cm · 100 cm) in order to isolate the core oligosaccharide fraction containing glycine, as detailed previously [12]. Core oligosaccharide was coupled to fimbrial proteins by a high-temperature method of conjugation as described elsewhere [25]. The oligosaccharide (0.5 mg) and fimbriae (0.5 mg) were dissolved in distilled water (0.5 ml). The solution was frozen in an acetone-dry ice bath in a glass test tube and lyophilized. Dry lyophilizate was then heated in an air oven at 105 C for 15 min. The tube was allowed to cool down to room temperature and the powder was dissolved in PBS and dialyzed against PBS. The conjugates were analyzed for sugars [16] and protein [15] and then subjected to SDS–PAGE and serological assays [18] as well as to MALDI-MS analysis. 2.11. Preparation of rabbit antisera and immunoblotting analysis Immune sera were obtained after multipoint intradermal threefold immunization of rabbits with the conjugates suspended in Freund complete adjuvant (1:1, v/v) [26]. Prior to the experiments, samples of rabbit sera were checked by immunoblotting assay for the presence of antibodies against fimbriae and lipopolysaccharides. SDS–PAGE and immunoblotting experiments were performed as previously described [18]. SDS–PAGE was carried out in 15% acrylamide gel. LPS suspension (1 mg ml 1) in sample buffer was boiled for 5 min and 2 ll samples were applied to the gel. After electrophoresis, the gel was stained with a silver reagent. For immunoblotting, after SDS–PAGE, the gel was electrophoretically transferred to a nitrocellulose membrane (Schleicher-Schuell, 0.45 lm). The blot was blocked with 20 mM Tris–HCl buffer with 50 mM NaCl (TBS) containing 3% (w/v) of gelatin at 36 C for 1 h, followed
by incubation at 36 C overnight with rabbit antiserum diluted 1:200 with TBS-1% (w/v) gelatin. The nitrocellulose membrane was then incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (diluted 1:5000 in TBS-1% (w/v) gelatin), at 36 C for 1 h and stained with a 4-chloro-1-naphthol substrate solution in the presence of H2O2. 2.12. ELISA The experiments were performed with lipopolysaccharides as described previously [18]. The wells of the microtiter plates (Nunc) were coated at 37 C for 3 h with solutions of various lipopolysaccharides (2 lg/100 ll) in 0.05 M carbonate buffer pH 9.6. The plates were then blocked at room temperature for 10 min. with 0.2% casein in TBS buffer containing 0.05% Tween 20, pH 7.5 (T-TBS). Thereafter, the wells were filled with 100 ll of serial dilutions of antiserum in PBS and the plates were incubated at room temperature for 2.5 h. After washing, 50 ll of a peroxidase-labeled goat antirabbit IgG (ICN, USA) diluted 1:10000 with PBS were added to each well. Following incubation at room temperature for 1 h, the plates were washed three times with T-TBS and the o-phenylenediamine substrate (50 ll well 1) was added. The reaction was stopped with 50 ll of H2SO4 and the optical densities were read at 492 nm, using a Behring EL311 Microplate Reader. 2.13. Bactericidal assay The experiment was essentially performed as detailed previously [27]. The bactericidal assay was carried out on tissue culture 96-well polystyrene plates (Costar, No. 3595). The E. coli C600 K12 (PCM 2184), E. coli O104 (PCM 270), Shigella sonnei PhI (PCM 2336), Sh. sonnei PhII (PCM 1984), K. oxytoca 666, Hafnia alvei PCM 1196, H. alvei PCM 537 and Salmonella enterica ser. Toucra O48 (PCM 2515) strains used in this assay were from the PCM, Institute of Immunology and Experimental Therapy, Wrocław. Bacteria were grown on nutrient agar plates at 37 C for 19 h. Suspensions of bacterial strains in HanksÕ balanced salt solution (HBSS) containing 1% of casein hydrolysate were prepared to give an OD600 = 0.26–0.29. A final working dilution of bacteria was obtained by further 1:400,000 or 1:800,000 dilutions. Twofold dilutions of rabbit polyclonal sera were performed directly on the plate to a final volume of 50 ll well 1, using HBSS-1% casein hydrolysate. Freshly thawed guinea pig complement was added to each well (20 ll) followed by 30 ll of the working dilution of bacteria (100 CFUs/well). Controls included 70 ll HBSS-1% casein hydrolysate and 30 ll of bacteria or 50 ll HBSS-1% casein hydrolysate and 20 ll of complement and 30 ll of bacteria. The tissue culture plates were incubated at 37 C for 30 min,
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then the contents of each well were mixed and 30 ll was plated onto the nutrient agar (2 plates/well). The plates were incubated at 37 C overnight and the number of colonies (CFU) was counted. The bactericidal activity of the serum expressed as the percent of the bacteria killed by the serum was calculated with respect to the mean value of the HBSS/bacteria/complement control well in the following manner: percentage of killing = [(CFUcontrol CFUserum)/CFUcontrol] · 100%. 2.14. Statistical evaluation The results were analyzed as medians and absolute ranges (Min–Max). The significance of measurements was compared using the WilcoxonÕs matched-pair signed-ranks test (Statistica software).
3. Results 3.1. Preparation of fimbriae and their immunochemical characterization To isolate pure fimbriae, use was made of a procedure which involved homogenization, ammonium sulfate precipitation, gel filtration in the presence of 6 M urea and hydrophobic chromatography. The latter step might be replaced by preparative electrophoresis. In every step, purity was checked by SDS–PAGE, with Coomassie Brilliant Blue for protein and silver staining specific for proteins and lipopolysaccharides. The yield of fimbrial protein varied between 0.5 and 2.5 mg of the obtained 20 g of wet bacterial mass. Fragments of bacterial membranes were removed via precipitation with 5% ammonium sulfate. After gel filtration on Sepharose 4B in the presence of urea, the void volume peak contained fimbriae with some proteins and LPS. The purification steps are exemplified for type 1 fimbriae (Fig. 1, lanes 1 and 2). Fimbriae could be separated from lipopolysaccharide and nonfimbrial proteins on a hydrophobic column (Fig. 1, lanes 3–6), where – with a linear gradient of NaCl – LPS and fimbriae were eluted with 3.8 and 0.2 M of sodium chloride, respectively. Nonfimbrial proteins were then eluted with ethanol. The staining of the gels with a silver reagent allowed a sensitive detection of the lipopolysaccharide. Fractions containing fimbriae were rechromatographed when necessary. Alternative to hydrophobic chromatography was preparative electrophoresis, the efficient way of one step separation of major structural fimbrial protein as a single component (Fig. 1, lines 7 and 8). Purified fimbriae were analyzed with MALDI-TOF mass spectrometry to determine the molecular mass of pilin monomers. The positive ion MALDI-TOF spectra of type 1 pilin monomers of K. pneumoniae and of type 3 pilin monomers of K. oxytoca generally revealed one
Fig. 1. SDS–PAGE analysis of fimbrial preparations of Klebsiella pneumoniae 304 (type 1) after gel filtration on Sepharose 4B column in the presence of 6 M urea (lanes 1 and 2), after hydrophobic chromatography on phenyl-Sepharose CL-4B of LPS-contaminated fractions (lanes 3 and 4) and fractions with purified fimbriae (lanes 5 and 6) and after preparative electrophoresis (lanes 7 and 8). The gels (15%) were stained with a silver reagent (lanes 1, 3, 5 and 7) and with Coomassie Brilliant Blue R250 (lanes 2, 4, 6, 8 and 9). Lane 9 represents protein molecular mass standards (LMW, Pharmacia).
major peak corresponding to the single charged, (M + H)+, pseudomolecular ion with m/z = 15,847 and m/z = 18,574, respectively (Fig. 2(a) and (b)). Some spectra (not shown) contained signals of lower intensity ascribed to the dimers and trimers of pilin, with doubled or tripled m/z values, respectively. Interestingly, several preparations of type 3 fimbriae contained also peaks related to the molecular mass of type 1 fimbriae (not shown). In all spectra, a major peak was associated with two less intense peaks, which corresponded to the doubly charged (M + 2H)2+ and triply charged (M + 3H)3+ ions of pilin monomers (Fig. 2(a) and (b)). All spectra contained also a low intensity peak with an m/z value higher by one or two sugar units. This finding was corroborated by sugar analysis of the representative preparation of type 3 fimbriae, thus revealing the presence of mannose, galactose and glucose in a molar ratio of 0.42:1.00:1.09. Further analyses were carried out for the induction of IFN, TNF and IL-6 cytokines in human peripheral blood cells by purified fimbriae. A certain level of these cytokines was measured in the supernatants over the cells which were treated with 5, 10 and 50 lg doses of protein for 20 h. Both types of the Klebsiella fimbriae were moderate, dose-dependent inductors of interferon and IL-6 (Fig. 3(a) and (c)). Interestingly, these fimbriae were inactive with respect to TNF stimulation (Fig. 3(b)). Lipopolysaccharides isolated from these Klebsiella strains were less active inductors of IFN, TNF and IL-6 than the reference LPS E. coli O111. 3.2. Preparation of conjugates, immunochemical analysis and testing of their immunogenicity Fimbrial proteins were then conjugated with a core oligosaccharide of E. coli C600K12 lipopolysaccharide. The glycine-containing oligosaccharide fraction [12] was used for conjugation with fimbrial proteins.
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Fig. 2. MALDI-TOF mass spectra of fimbrial preparations of Klebsiella pneumoniae 304 (type 1) (a), K. oxytoca 666 (type 3) (b) and of conjugate of E. coli C600K12 LPS core oligosaccharide with fimbriae of type 3 (c).
Analysis of the conjugates by MALDI-TOF mass spectrometry revealed that approximately 10% of the pilin monomers possessed an attached oligosaccharide, due to the appearance of an ion (m/z = 20,518, Fig. 2(c)) corresponding to species with a molecular mass by 1953 Da higher than that of pilin monomer type 3. This mass increment similarly to that of type 1 pilin conjugate
(not shown) is consistent with the molecular mass of oligosaccharide hapten. The rabbit anti-conjugate sera obtained for type 1 and type 3 fimbriae conjugates were then analyzed in immunological tests. Control sera from healthy non-immunized animals were negative. In immunoblotting experiments, exemplified in Fig. 4, the rabbit anti-type 3 fimbrial conjugate serum reacted with
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Fig. 4. SDS–PAGE analysis (a) and immunoblotting (b) of enterobacterial lipopolysaccharides with rabbit serum against E. coli C600K12 LPS core oligosaccharide conjugated to fimbriae of type 3.
Fig. 3. Induction of IFN (a), TNF (b) and IL-6 (c) by Klebsiella fimbriae of type 1 (strain 304) and type 3 (strain 666) and by fimbrial conjugates with E. coli C600K12 LPS core oligosaccharide (B-304 and B-666). Stimulation was achieved with 5, 10 or 50 lg doses of fimbriae or conjugates and with 5 lg of LPS of E. coli O111, K. pneumoniae 304 and K. oxytoca 666. Blood samples treated with PBS served as control. Measurements were made in triplicate in each experiment, and the experiments were repeated on 4–8 individual blood samples.
the carbohydrate epitope which is present in several enterobacterial lipopolysaccharides. The most intensive reactivity of this anti-conjugate serum was observed with LPS of H. alvei 537, 1188 and E. coli O56, O24 and homologous LPS E. coli C600K12. The reactivity
with these enterobacterial LPS antigens was proved quantitatively by the ELISA test (not shown). This serum reacted specifically with fimbriae of type 3, and recognized also the oligosaccharide epitope situated in its conjugate with BSA and in a native lipopolysaccharide of E. coli C600K12 (Fig. 5). The ELISA and immunoblotting data showed that the oligosaccharide-fimbriae conjugates were immunogenic when injected to rabbits and were able to induce the antibodies directed against the sugar moiety of glycoconjugates. The reactivity of fimbrial conjugate antisera with native protein carriers was also analyzed, as exemplified on Fig. 5 (lane 1). Anti-type 3 serum reacted only with the homologous protein whereas anti-type 1 fimbrial conjugate serum reacted with both types of fimbriae (not shown). When the conjugates were tested for the induction of cytokines, this activity was noticeably increased with respect only to TNF stimulation (as compared to nonconjugated fimbriae) (Fig. 3(b)). The results of the bactericidal test shown in Table 1 indicate that the antibodies against glycoconjugates induced in rabbits are bactericidal for the wild, S-type bacteria of different species. The H. alvei, Salmonella, E. coli and Sh. sonnei strains tested are uncapsulated. This
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Fig. 5. Immunoblotting of rabbit serum against E. coli C600K12 LPS core oligosaccharide conjugated to type 3 Klebsiella fimbriae – with LPS of E. coli C600K12 (1), fimbriae of type 3 (2) and E. coli C600K12 LPS core oligosaccharide conjugated to BSA (3). SDS–PAGE was carried out in 15% acrylamide gel. Lane 4 stained with Coomassie Brilliant Blue R250 represents protein molecular mass standards (LMW, Pharmacia).
bactericidal activity does not depend on LPS type, on the serotype of the protein or on the type of the LPS core region. The preimmune sera were not bactericidal for these strains. The bactericidal activity was only considered in relation to the complement resistant strains and highly increased in the presence of antibodies. The other strains studied (E. coli C600K12, Sh. sonnei PhII, K. oxytoca 666 and H. alvei 537) were complement sensitive, and they were not considered in the bactericidal experiments with antibodies.
4. Discussion Two pathogenic strains of uncapsulated, fimbriae producing Klebsiella were chosen for the preparation
of type 1 and type 3 fimbrial major structural proteins. The strains have distinct characteristics of adherence, namely type 1 has mannose-specific tissue receptors and type 3 has receptors at the intercellular matrix [6]. There are several methods for the purification of fimbriae; they include a mild homogenization of the cells, salt precipitation, ion-exchange chromatography, the use of detergents, and ultracentrifugation in the gradient of sucrose or cesium chloride [3,28–31]. Trace amounts of LPS are difficult to remove due to the hydrophobic interaction with fimbriae. Chromatography on phenylSepharose was used previously by Honda et al. [14] for the purification of CFAII pili from enterotoxigenic E. coli. We adopted the same optimized procedure for the purification of structural protein from both type 1 and type 3 fimbriae, yielding both proteins of high purity. The same result was obtained from preparative electrophoresis. Hence, these conditions may as well apply to the purification of different types of fimbriae. Sensitive silver staining is well suited for monitoring the trace amounts of lipopolysaccharide. With MALDI-TOF mass spectrometry it was possible to determine the molecular mass of fimbrial monomers, pilin. The actual molecular masses obtained with this technique differ by about 2 kDa from higher values estimated by SDS– PAGE. Mass spectrometry revealed also a microheterogeneity caused by glycosylation, which not only proved the purity of the preparations but also made it possible to analyze the products of chemical modification of pilin. Klebsiella fimbriae were found to be moderate inducers of IL-6 and interferon and inactive toward stimulation of TNF-a production in our experiments. After conjugation, it was only the stimulating activity of TNF that showed noticeably elevated levels. In that regard, it is postulated that fimbriae should be the candidate for practical use as a carrier in conjugate vaccine, due to their low activity in inducing proinflammatory cytokines. It is interesting to note that LPS from our both Klebsiella strains (of serotype O3) posses poor activity of inducing of proinflammatory cytokines in relation to standard E. coli O111 endotoxin. It might be in accord with data on the role of Klebsiella O3
Table 1 Bactericidal activity of rabbit sera against conjugates of core oligosaccharide (fraction B) of LPS of E. coli C600K12 with Klebsiella fimbriae 666 and 304, respectively Bacterial strain
Serum dilution causing bacterial killing (%) Anti C600B-666 serum
H. alvei 1196 S. Toucra O48 E. coli O104 Sh. sonnei PhI NB, not bactericidal.
Anti C600B-304 serum
P20%
P60%
P90%
P20%
P60%
P90%
10,240 10 1280 10,240
10,240 NB 1280 10,240
1280 NB 1 NB
10,240 NB 10,240 10,240
10,240 NB 5120 5120
NB NB 10 NB
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mannan polysaccharide enhancing the adjuvant activity of LPS, whereas lipid A from this LPS was completely devoid of an adjuvant effect typical for enterobacterial lipid A [32]. One may also speculate that Klebsiella fimbriae and lipopolysaccharide surface structures are less immunogenic possibly due to the presence of these strains as saprophytes in the intestinal tract [33], thus enhancing a virulence potential of Klebsiella. Increased immunogenicity of fimbrial conjugates may beneficially improve the anti Klebsiella immunity. It was estimated by mass spectrometry that 10% of the pilin monomers possessed an attached oligosaccharide, although at the same conditions of conjugation, lactose was bound to bovine serum albumin in high yields [25]. This may be due to low content of lysine residues in fimbrial protein molecule, namely 8 in type 1 [3] and 14 in type 3 [31] pilin and the fact that only a part of the monomer molecule is exposed on the surface of the fimbrial superstructure normally present in solution. Most likely only limited number of total free amino groups is available for conjugation. However, this amount of the conjugated hapten was sufficient for induction of anti oligosaccharide antibody. When rabbits were injected with fimbrial proteins conjugated with a glycine containing core oligosaccharide isolated from E. coli C600K12 lipopolysaccharide, the antibodies were induced against oligosaccharide and fimbriae. This means that the coupling method did not destroy the immunogenicity either of the protein carrier or of the sugar moiety containing an important oligosaccharide epitope. The glycine substituent of the core oligosaccharide is alkali and acid labile [12]. This newly elaborated method of conjugation without an activating or coupling reagent [25] is therefore suitable for binding carbohydrates to the fimbrial carriers. The antigenic epitopes present on the fimbriae after conjugation were available to immune cells. The glycine containing core oligosaccharide used for coupling was expected to have a common epitope [12]. The broad reactivity of the sera with enterobacterial lipopolysaccharides seems to substantiate this supposition. The sera were bactericidal for several enterobacterial organisms and it seems reasonable to assume that the bactericidal activity is due to the cross-reactive antibodies. Preliminary work (A. Gamian, T. Lipin´ski, unpublished) showed that the epitope is not destroyed in conjugate. Analyzing pure fimbriae of different species by serological tests and MALDI mass spectrometry might elucidate the problem of heterogeneity of fimbriae. In our experiments, the MALDI-TOF-MS spectra of some type 3 fimbriae preparations contained also a peak related to type 1 pilin [34]. Antibodies to fimbriae can facilitate phagocytosis of bacteria [6,11]. Fimbrial conjugates have been used for the preparation of vaccines against experimental and natural diseases caused by E. coli, Neisseria gonorrhoeae, N.
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meningitidis, Bordetella pertussis, Pseudomonas aeruginosa, Haemophilus influenzae and Porphyromonas gingivalis [35–37]. The protective efficacy of fimbriae might be increased and extended by coupling with an epitope of broad specificity. Significant value of these Klebsiella fimbrial conjugates would be for monitoring of the status of immunoglobulin deficiency, especially in children.
Acknowledgements This work was supported under Grant Nos. 4P05A04018 and 6P05B13420 from the Committee for Scientific Research (KBN), Warsaw, Poland and Grant 249 from the Ministe´re des Affaires Etrange´res, Paris, France.
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