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1 Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain
2 Departamento de Microbiología y Parasitología Sanitarias, Facultad de Farmacia, Universidad de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
3 Dipartimento di Chimica e Biochimica, Università Federico II di Napoli, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy
Correspondence
Juan M. Tomás
jtomas{at}ub.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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wabH waaL and 52145waaL mutants, but not those from from K. pneumoniae 52145wabG waaL mutant, contained both LPS and capsular polysaccharide, even after hydrophobic chromatography. The two polysaccharides were dissociated by gel-filtration chromatography, eluting with detergent and metal-ion chelators. From these results, it is concluded that the K2 capsular polysaccharide is associated by an ionic interaction to the LPS through the negative charge provided by the carboxyl groups of the GalA residues.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). K. pneumoniae infections can occur in almost all body sites, but the highest incidence is in the urinary and the respiratory tracts. The main populations at risk are neonates, immunocompromised hosts, and patients predisposed by surgery, diabetes, malignancy, etc. (Emori & Gaynes, 1993; Hansen et al., 1999; Hervás et al., 1993). The existence of multiple antibiotic-resistant K. pneumoniae strains is well known, and they have complicated therapy. Mortality rates of up to 50 % have been found in respiratory tract infections. As an alternative to antibiotic treatment, prevention and/or treatment of K. pneumoniae infections by immunotherapy have received increased attention in recent years.
K. pneumoniae strains typically express smooth LPS, LPS with O-antigen polysaccharide (O-PS), and antigenic capsular polysaccharide (K-PS) on their surface; both O-PS and K-PS contribute to the pathogenesis of the species. The K. pneumoniae K-PS plays a critical role in the ability of the organism to resist complement-mediated opsonophagocytic killing (Williams & Tomás, 1990). Furthermore, an extracellular complex containing LPS and capsule has been suggested to be involved in lung damage (Straus et al., 1985). More than 90 different capsular types (K-antigens) have been described in Klebsiella, but only the gene clusters involved in K2 (wcaK2) and K57 (wcaK57) antigens have been described (Arakawa et al., 1995). The two known wca gene clusters contain genes involved in K-PS residue biosynthesis, linkage, polymerization and export. In addition, the production of the K2 requires a wcaK2 plasmid-borne capsular gene transcriptional activator (Arakawa et al., 1995; Lai et al., 2003).
In a few Gram-negative K-PS-containing bacteria, the basis of K-antigen attachment to the cell surface involves an interaction with a lipid moiety, either the LPS domain lipid A core (Jann et al., 1992), or a glycerolipid (Gotschlich et al., 1981; Kuo et al., 1985). However, despite its importance as a pathogenic factor, little is known about the linkage of the K. pneumoniae K-PS to the cell surface. Knowledge of the chemical structures of the O1-antigen, core LPS, lipid A (Vinogradov et al., 2002; Süsskind et al.1995; Vinogradov & Perry, 2001, Helander et al., 1996), and K2 (Corsaro et al., 2005) antigens from K. pneumoniae, as well as the genes involved in their biosynthesis (Arakawa et al., 1995; Kelly et al., 1993; Regué et al., 2001, 2005a), have enabled us to study the basis of the K2-capsule–cell-surface interaction.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Bacterial strains were grown in LB broth and on LB agar (Miller, 1972). LB media were supplemented with kanamycin (50 µg ml–1), ampicillin (100 µg ml–1), chloramphenicol (20 µg ml–1) and tetracycline (25 µg ml–1) when needed. Bacteriophages FC3-9, 
1 and 2, specific for capsular serovars K66, K1 and K2, respectively, have been described previously (Camprubí et al., 1992; Regué et al., 2004).
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; Regué et al., 2004).
General DNA methods.
General DNA manipulations were done essentially as described (Sambrook et al., 1989). DNA restriction endonucleases, T4 DNA ligase, Escherichia coli DNA polymerase (Klenow fragment), and alkaline phosphatase were used as recommended by the suppliers.
Mutant construction.
K. pneumoniae individual genes were mutated by creating in vitro in-frame deletions of each gene. Each mutated gene was transferred to the chromosome by homologous recombination using the temperature-sensitive suicide plasmid pKO3, containing the counterselectable marker sacB (Link et al., 1997). Mutations were made in waaL and wabI J G and H. The plasmids containing the engineered in-frame deletions derived from DL1 (pKO3waaLDL1), and C3 (pKO3wabIC3 and pKO3wabJC3) sequences were transformed into K. pneumoniae C3 and DL1, respectively, by electroporation. Mutants were selected based on growth on LB agar containing 10 % sucrose, and loss of the chloramphenicol resistance marker of vector pKO3. The mutations were confirmed by sequencing of the whole constructs in amplified PCR products. The DL1waaL mutant was constructed by asymmetric PCR amplifications using DL1 chromosomal DNA and primers LADL1 (5'-CGCGCGGCCGCAGCGCGCTGAAAAACAGTAT-3'), LBDL1 (5'-CCCATCCACTAAACTTAAACAACCCTGTCTGGCGAAGTTA-3'), LCDL1 (5'-TGTTTAAGTTTAGTGGATGGGGCGGAAAAACGCTAACAAA-3') and LDDL1 (5'-GAGCGGCCGCCTGCTATTGCGTCTGGATGA-3'). The primers include NotI sites (single underlined). DNA fragments of 803 (LADL1–LBDL1) and 1037 (LCDL1–LDDL1) bp were obtained. DNA fragment LADL1–LBDL1 included nt 3430, inside wabI, to nt 4200, corresponding to the third base of the eighth codon of waaL. DNA fragment LCDL1–LDDL1 included the last four codons of waaL (nt 5315) to nt 6320, inside wabJ. DNA fragments LAC3–LBC3 and LCDL1–LDDL1 were annealed at their overlapping region (double-underlined in primers LBDL1 and LCDL1), and amplified by PCR as a single fragment, using primers LADL1 and LDDL1. The fusion product was purified, NotI digested, ligated into NotI-digested and phosphatase-treated pKO3 vector, electroporated into E. coli DH5
, and plated on chloramphenicol LB agar at 30 °C to obtain plasmid pKO3waaLDL1. Similarly, primers sets I [IAC3 (5'-CGCGCGGCCGCAGCCTGTTTTACAACCGCC-3'), IBC3 (5'-CCCATCCACTAAACTTAAACACAGGGATCCCATCAGAGC-3'), ICC3 (5'-TGTTTAAGTTTAGTGGATGGGATCTTCGAAAAGGTTGCGTTT-3') and IDC3 (5'-CGCGCGGCCGCGTATGGTGCCAGATGCTCAG-3')] and J [JAC3 (5'-CGCGCGGCCGCACCATACGCTGGTTCTCACC-3'), JBC3 (5'-TGTTTAAGTTTAGTGGATGGGGCCGGCTTATTTCTGTATCG-3'), JCC3 (5'-CCCATCCACTAAACTTAAACAATGTGGCCCGTGATTAAAGAG-3') and JDC3 (5'-CGCGCGGCCGCCTTATTGAATACAGCGACG-3')] were used to construct pKO3wabIC3 and pKO3wabJC3, respectively. Plasmid pKO3wabIC3 included nt 2675, inside waaC, to the third base of the fourth codon of wabI (nt 3285), an in-frame 21 bp linker, and nt 4138, inside wabI, to nt 4974, inside waaL, including the last wabI 14 codons. Plasmid pKO3wabJC3 included nt 4581, inside waaL, to nt 5426, containing the last wabJ 17 codons, an in-frame 21 bp linker, and nt 6357, corresponding to the third base of the 19th codon of wabJ, to nt 7128, inside wabN. Due to the extensive identity among wabI and wabJ between strains C3 and DL1, the same sets of four primers and DL1 chromosomal DNA were used to construct pKO3wabIDL1 and pKO3wabJDL1. For the same reason, primers used in the construction of pKO3-engineered deletions of wabG and wabH in strains C3 and 52145 were used to construct DL1wabG and DL1wabH mutants, respectively.
Plasmid constructions for mutant complementation studies.
For complementation studies, waaLDL1, wabHDL1 and wabGDL1 were amplified, ligated to pGEM-T Easy (Promega), and transformed into E. coli DH5.
Cell surface extraction, and electrophoresis.
K. pneumoniae cells grown in trypticase soy broth (TSB) at 37 °C were dried (0.6 g) and extracted. The phenol/chloroform/light petroleum ether (PCP) method (Galanos et al., 1969) and a modified phenol/water (PW) procedure (Westphal & Jann, 1965; Rhan et al., 2003) were used. The PCP and PW methods are used for the extraction of O-PS-containing and -deficient LPS, respectively. The PW extract was processed by two different approaches. First, 100 mg PW extract was purified by hydrophobic chromatography on a Butyl Sepharose column (Pharmacia). The column (40x2.5 cm) was equilibrated with 200 mM sodium acetate (NaOAc), pH 4.7, and eluted initially with 150 ml of the same buffer, and then with 450 ml of a linear gradient to NaOAc : n-propanol (50 : 50, v/v) (Muck et al., 1999). Fractions of 2.5 ml were collected, and monitored for carbohydrates (phenol/sulfuric acid test, A490). Eluted fractions were pooled, dialysed and freeze-dried, yielding fraction pools 1, 2 and 3, of 11, 22 and 26 mg, respectively. Only fraction pools 1 and 3 were further analysed, because fraction pool 2 contained nucleic acid. In the second approach, the water phase obtained was ultracentrifuged (105 000 g) at 15 °C for 17 h. The supernatant was treated with DNase, RNase and proteinase K (Sigma) in order to eliminate both nucleic acid and proteins. The sample was dialysed and lyophilized (20 mg), and chromatographed on a Sephacryl S-200 column (Pharmacia).The column was equilibrated with 10 mM Tris buffer, pH 8, containing 1.25 % deoxycholate (DOC), 0.2 M NaCl and 5mM EDTA, and the eluent was monitored with a Knauer differential refractometer. On the basis of the chromatographic profile, the eluted fractions were pooled, dialysed and lyophilized. Fraction pools 1 and 2, of 7 and 3 mg, respectively, were obtained. For screening purposes, LPS was obtained after proteinase-K digestion of whole cells (Hitchcock & Brown, 1983). PCP and PW cell-surface extracts, and LPS preparations obtained by the method of Hitchcock & Brown (1983), were separated by SDS-PAGE or Tricine {N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine} SDS-PAGE, and visualized by silver staining, as described (Pradel & Schnaitman, 1991; Tsai & Frasch, 1982).
Isolation of oligosaccharides (OS).
The LPS-containing PCP extract from K. pneumoniae 52145waaL wabH (15 mg) was hydrolysed with 1 % acetic acid (100 °C for 1 h). The resulting precipitate (6 mg) was removed by centrifugation (10 000 g), and the supernatant (4 mg) was analysed by MS. Another PCP-LPS sample (15 mg) was deacylated and purified, as described (Holst et al., 1991, 1993), obtaining 2 mg alditol OS. Peaks 1 and 3 from Butyl Sepharose chromatography were hydrolysed separately with 1 % acetic acid, containing 0.1 % SDS, at 100 °C for 3 h. After cooling, the samples were centrifuged (10 000 g) for 20 min. The supernatants were lyophilized, washed with ethanol, and analysed by MS. Samples (1 mg) of peaks 1 and 2 of gel-filtration chromatography were separately stirred in anhydrous hydrazine (0.5 ml) at 37 °C for 1.5 h (Holst et al., 1991). The reaction mixtures were cooled in an ice bath, and cold acetone was added in small portions until the precipitation was complete. The samples were then centrifuged (6300 g, 4 °C, 15 min), the pellets were washed twice with cold acetone, dissolved in water for lyophilization, and analysed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS.
Glycosyl and lipid analysis.
Samples (1 mg) of PCP and PW extracts were dried over P2O5 overnight, and treated with 1 M HCl/CH3OH (1 ml) at 80 °C for 20 h. The crude reactions were extracted twice with hexane; the two extracts were pooled, dried under a stream of air, and treated with acetic anhydride (100 µl) at 100 °C for 15 min, and the acetylated fatty acids methyl esters were analysed by GC-MS. The remaining methanol phase after hexane extraction was neutralized with Ag2CO3, dried, acetylated, and the methylglycoside derivatives were analysed by GC-MS. The same analysis was performed on relevant fractions from Butyl Sepharose and Sephacryl gel chromatography.
GC-MS analysis.
Alditol acetates, methyl glycoside acetates, partially methylated alditol acetates and acetylated methyl esters lipids were analysed on a Agilent Technologies 5973N MS instrument equipped with a 6850A Gas Chromatograph and an RTX-5 capillary column (Restek; 30 mx0.25 mm i.d., flow rate 1 ml min–1, helium carrier gas), as previously described (Izquierdo et al., 2002).
MS studies.
Positive- and negative-ion MALDI-TOF spectra were acquired on a Voyager DE-PRO instrument (Applied Biosystems) equipped with a delayed extraction ion source, in both linear and reflector modes. Ion acceleration voltage was 20 kV, grid voltage was 14 kV, mirror voltage ratio was 1.12, and delay time was 100 ns. Samples were irradiated at a frequency of 5 Hz by 337 nm photons from a pulsed nitrogen laser. Mass calibration was obtained with a malto-oligosaccharide mixture from corn syrup (Sigma). A solution of 2,5-dihydroxybenzoic acid in 20 % CH3CN in water at a concentration of 25 mg ml–1 was used as the MALDI matrix. Matrix solution (1 µl) and sample (1 µl) were premixed, and then deposited on the target. The droplet was allowed to dry at an ambient temperature. Spectra were calibrated and processed under computer control using the Applied Biosystems Data Explorer software.
Murine pneumonia model.
The experiments were performed as described (Cortés et al., 2002). Briefly, ICR-CDI mice (Harlan Ibérica) were anaesthetized, and intubated intratracheally with a blunt-ended needle. Approximately 107 c.f.u. of exponentially growing K. pneumoniae cells were suspended in 50 µl PBS, and inoculated through the blunt-ended needle. The mice were observed daily, and bacteraemia was assessed on days 2, 4 and 6 by culturing blood obtained from the tail vein (approx. 20 µl) on LB agar. Lung and spleen tissues from surviving animals and dead animals were aseptically removed, homogenized, and plated for quantitative bacterial cultures. Each experiment was performed with nine animals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Vinogradov & Perry, 2001) (Fig. 1a, b). Knowledge of the role of K. pneumoniae genes involved in core LPS biosynthesis (Frirdich et al., 2004; Izquierdo et al., 2002, 2003; Regué et al., 2001, 2005a, b) enabled the construction of a set of isogenic non-polar waaL mutants in K. pneumoniae strains producing either type 1 [C3 (O8 : K66) and DL1 (O1 : K1)] or type 2 [52145 (O1 : K2)] core LPS. The waaL gene encodes the O-PS : lipid-A-core ligase, and, as expected, these waaL mutants were devoid of O-PS, as determined by SDS-PAGE analysis of the corresponding LPS preparations (see Fig. 2, lane 2, for 52145waaL). Mutant strains 52145waaL, DL1waaL and C3waaL were sensitive to capsule-specific phages 2 (K2) (Table 2), 1 (K1) and FC3-9 (K66), respectively. No difference in capsule-specific phage e.o.p. was found between each waaL mutant and its isogenic parent strain, suggesting that the level of functional capsule production is independent of the O-PS ligase. To demonstrate that the waaL mutants and their isogenic parent strains produced the same capsular type, ELISAs using specific capsular antibodies were performed. Cells from mutant strains 52145waaL (Table 2), DL1waaL and C3waaL reacted with anti-K2, -K1 and -K66 antibodies, respectively. In addition, the presence of cell-bound capsule was observed in the three waaL mutant strains in electron microscope (EM) studies (data not shown). Each mutant was complemented by introduction of the corresponding wild-type waaL. These results strongly suggest that the O-PS ligase is not involved in the association of the K1, K2 and K66 capsule polysaccharides to the cell surface in K. pneumoniae.
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). These mutants were constructed in strain 52145 (type 2 core). Mutants 52145waaC, 52145waaF and 52145wabG were resistant to phage 2, and their cells did not react with anti-K2 capsule serum in ELISAs using capsule K2-specific antibodies. But, K2-PS-like material was detected in the culture supernatants of these mutants by ELISA (Table 2). These results suggest that these three mutants synthesize capsular polysaccharide, but that it cannot be attached to the cell surface. By contrast, mutants 52145wabH, 52145wabK and 52145wabM were phage 2 sensitive, and contained cell-associated K2-PS (Table 2). Comparison of the known core-OS from mutants 52145wabH (Fig. 1c) and 52145wabG (Fig. 1d) suggests an essential role for the core OS GalA residues in K2 interaction with the cell surface.
Capsule-K2–LPS co-elution
Cells from 52145wabH waaL double mutant were extracted by the PCP method (Galanos et al., 1969). Chemical composition of this PCP extract showed the presence of the characteristic core LPS sugars 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), L-glycero-D-manno-heptopyranose (Hep), D-glucopyranose (Glc) and GalA. The same cells were first treated with PCP, and then with a modified PW procedure (Rahn et al., 2003). The PW extract contained glucuronic acid (GlcA) and mannose (Man) from the K2 capsule (Corsaro et al., 2005), in addition to core LPS sugars. Protein was not detected in these preparations.
Hydrophobic chromatography of the PW extract on a Butyl Sepharose column (Pharmacia) separated this material into three major peaks (1, 2 and 3) (Fig. 3a). Peak 2 was discarded because it contained nucleic acid only. Chemical analysis of peaks 1 and 3 revealed the presence of both K2 and core-LPS residues. The integration results of GC-MS analysis showed a GalA : GlcA ratio of 0.25 for peak 1, whereas this ratio was 3.3 for peak 3, indicating that K2-PS (GlcA) was more abundant in peak 1 while LPS (GalA) was more abundant in peak 3. These results suggest that at least K2 and core LPS co-elute in the same peaks (Fig. 3b). Material from both peaks 1 and 3 was characterized by hydrolysis with 1 % acetic acid, followed by positive-ion MALDI-TOF and 1H-NMR analyses of the recovered OS. The comparison of the MALDI-TOF spectrum of the acetic acid hydrolysis product of purified LPS with those of peaks 1 and 3 suggested the co-elution of K2 and LPS for peak 3 (Fig. 4). The assignment of core-LPS signals is summarized in Fig. 4 (insert above figure), and was obtained as previously reported (Regué et al., 2005b). In the mass spectrum of peak 1 (Fig. 4), the difference () between the clusters of signals corresponded to hexose or hexuronic acid (162 or 176 Da), which are constituents of the K2 capsule. In the mass spectrum of peak 3, signals of core OS were present, together with those of K2-PS (Fig. 4). By contrast, the comparison of 1H-NMR spectra of purified K2 structure and acetic-acid-released core LPS with those of peaks 1 and 3 suggest the co-elution of the two polysaccharides in both peaks 1 and 3 (Fig. 5). The absence of core LPS signals in the mass spectrum of peak 1 could be attributable to the low abundance of LPS in this peak, and to a lower ionization ability of the core LPS than that of K2-PS.
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Capsule-K2–LPS ionic interaction
To determine how the K2 polysaccharide was associated to the core LPS, the 52145wabH waaL PW extract was treated with DNase, RNase and proteinase K, and further purified by high-speed centrifugation. This preparation was subjected to gel-filtration chromatography on a Sephacryl S-200 column (Pharmacia) using as the eluent a buffer with an increased ionic strength (0.1 M NaCl and 10 mM Tris), which contained the metal-ion chelator EDTA, and the dissociating detergent DOC (see Methods). This approach allowed the fractionation of the PW extract into two main peaks (1 and 2) (Fig. 6a). SDS-PAGE analysis showed the presence of only LPS molecules in peak 2, while in peak 1, both K2 and LPS molecules were present (Fig. 6b). Chemical analysis confirmed the presence of LPS residues in peak 2, and of both K2 and LPS residues in peak 1. Both peak 1 and 2 samples were characterized by O-delipidation and negative-ion MALDI-TOF MS analysis (Fig. 6). Comparison of mass spectra signals of peak 2 and purified O-deacylated LPS showed the presence of only O-deacylated LPS in this peak (Fig. 7). Signals corresponding to K2-PS and O-deacylated LPS were present in peak 1 (Fig. 7). In addition, ELISA analyses of peaks 1 and 2 with specific K2 antiserum showed the presence of K2 molecules in peak 1 only, thus confirming the above results. The complete separation between LPS and K2 antigen was achieved by three successive gel-filtration chromatographs of the peak 1 material in the presence of EDTA and DOC (Fig. 8). This result indicates that the ionic strength and the amount of EDTA are crucial to completely remove cation bridges between LPS and K2 capsule.
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waaL and 52145wabH mutants. However, some changes in the relative intensities of the minor OMPs were observed, but the major OMPs remained unchanged in their relative intensity [OmpK36, K35, OmpA-like, OmpK17 (Albertí et al., 1995; Climent et al., 1997; Hernández-Allés et al., 1999; Martínez-Martínez et al., 1996)]. Nevertheless, truncation of the core LPS beyond the outer core GalA residue in the wabH mutant might alter the presence or the topology of other outer-membrane (OM) minor molecules, which could block capsule attachment to other(s) target(s) in the OM. To test this possibility, 52145waaL cells, containing full-length type 2 core OS, were extracted successively with PCP and PW. PW extracts from these cells also contained LPS and K2-PS co-purifying through hydrophobic chromatography. Both LPS and K2-PS were fully separated into two peaks after three consecutive gel-filtration chromatography assays in the presence of EDTA and DOC, as determined by SDS-PAGE, ELISA and chemical analysis (data not shown). This result suggests that the postulated ionic interaction between core OS and K2-PS is not due to 5245wabH mutant pleiotropic effects on the OM.
Other Klebsiella capsular types (K1 and K66)–core-LPS interaction
To study if the K2–core-LPS interaction is a feature shared by other K. pneumoniae capsular serotypes, core-LPS-deficient mutants were constructed in K. pneumoniae strains C3 (O8 : K66) and DL1 (O1 : K1). The capsular phenotypes of these mutants were determined using capsule K66- and K1-specific bacteriophages FC3-9 and 1, respectively, ELISAs with specific antiserum against K66 and K1, and EM studies. Strains with mutations in the genes waaC (C3waaC, DL1waaC), waaF (C3waaF, DL1waaF) and wabG (C3wabG, DL1wabG) were devoid of cell-surface-associated capsule, although K-PS-like material was detected in their culture supernatants by ELISA. Strains with mutations in the genes wabH (C3wabH, DL1wabH), wabI (C3wabI, DL1wabI) and wabJ (C3wabJ, DL1wabJ) were capsulated. Double-deletion mutants C3waaL wabG and DL1waaL wabG were non-capsulated, while C3waaL wabH and DL1waaL wabH contained cell-associated K66- and K1-PS, respectively. These results suggest that capsule–core-LPS interaction could be a general phenomenon in K. pneumoniae, because this interaction determines the presence of cell-associated capsule for the studied capsular types K1, K2 and K66. Furthermore, the capsule–core-LPS interaction appears to require the core OS GalA residues.
Virulence studies
Virulence was tested in a murine model of pneumonia by intratracheal injection. When the virulence of the strains was assayed in the murine pneumonia model, we obtained the results shown in Table 3. The 52145wabG mutant was completely avirulent in this model, while 52145waaL and 52145wabH mutants, and the wild-type strain, showed similar values. Introduction of the corresponding genes in the mutants rendered them as virulent as the wild-type strain. However, mutant 52145wabG transformed with the plasmid vector alone (pGEMT) remained avirulent in this animal model.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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K. pneumoniae core-LPS mutants containing the GalA core residues contained functional K2-PS. By contrast, mutants devoid of core GalA residues were devoid of functional K2-PS. PCP extraction of waaL wabH mutant did not enable us to recover K2-PS, while PW extraction recovered both LPS and K2-PS. This is in agreement with the amphipathic and hydrophilic natures of LPS and K2-PS, respectively.
Characterization of PW extracts from K. pneumoniae 52145waaL wabH showed that K2 capsule and LPS co-purify after hydrophobic-interaction chromatography. MS of the chromatographic peaks agreed with a non-covalent association between both molecules. The possibility that phenol used in both extraction methods could hydrolyse the putative ester linkage between K2-PS and core LPS is unlikely since these methods allow the isolation of LPS molecules containing O-acylated residues (Knirel & Kochetkov, 1994).
Gel-filtration chromatography and elution in a buffer with EDTA and DOC enabled the isolation of a chromatographic peak with LPS only, according to chemical composition and MS analysis. The other chromatographic peak containing K2-PS associated to core LPS was fully separated on successive chromatographic assays. The same results were obtained using 52145waaL containing a full-length core OS. These results indicate that the K2-PS is associated to the cell surface by an ionic interaction between K2-PS and core OS. The role of Mg2+ ions in establishing ionic bridges between adjacent LPS molecules through negatively charged core residues has been established (Nikaido & Vaara, 1985). In a similar way, we suggest that the divalent ions Mg2+ and/or Ca2+ could play a role in the ionic interaction between GlcA (K2-PS) and GalA residues (core OS). This ionic interaction could also explain the formation of the extracellular toxic complex, containing both K2-PS and LPS, in K. pneumoniae (Straus et al., 1985). Furthermore, the similar behaviour of waaL wabH and waaL wabG mutants from K. pneumoniae belonging to capsular serotypes K1 and K66 suggests that the capsule–core-LPS ionic interaction is a general K. pneumoniae phenomenon. In the three K. pneumoniae capsular serotypes analysed, the presence of GalA residues in core OS was necessary for capsule association to cell surface.
The effects of the wabG, wabH and waaL mutations in virulence experiments were studied in the K. pneumoniae 52145 background because this strain is highly virulent in the model used. The wabG mutant was completely avirulent in the experimental model of pneumonia (Table 3). A similar result was obtained using a non-capsulated mutant of strain 52145 (Cortés et al., 2002). On the other hand, a K. pneumoniae waaL mutant, with a full inner and outer core, but devoid of O-antigen, showed similar virulence when compared with the wild-type strain. A similar result was obtained for the wabH mutant. All the changes observed in the K. pneumoniae wabG and H, and waaL mutants were rescued by introduction of the corresponding single wild-type gene, while the introduction of the plasmid vector alone was unable to perform the rescue. The capsule is essential in the K. pneumoniae experimental model of pneumonia, as previously indicated (Cortés et al., 2002). Furthermore, the K2 present in wabH and waaL mutants enables them to be as virulent as the wild-type strain in the experimental model of pneumonia. These studies clearly confirmed the presence of virulent functional K2 in waaL and wabH mutants, and its absence in the wabG mutant.
Summarizing, K. pneumoniae lacks phosphoryl residues in its core, but instead contains GalA as a critical source of negative charge. These results indicate that the negative charge provided by the carboxyl groups of GalA play an essential role in capsule attachment by an ionic interaction. Further work will be necessary to obtain a detailed knowledge of this interaction, which could contribute to the design of more efficient methods of prevention and/or treatment of K. pneumoniae infections.
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Received 20 October 2005;
revised 5 February 2006;
accepted 6 February 2006.
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