Abstract

The Burkholderia cepacia complex is a group of Gram-negative bacteria that are opportunistic pathogens for humans especially in cystic fibrosis patients. Lipopolysaccharide (LPS) molecules are potent virulence factors of Gram-negative bacteria organisms essential for bacterial survival. A complete analysis of the bacterial lipopolysaccharide structure to function relationship is required to understand the chemical basis of the inflammatory process. We have therefore investigated the structures of lipopolysaccharides from clonally identical Burkholderia multivorans strains (genomovar II) isolated pre- and post-lung transplantation through compositional analysis, mass spectrometry, and 2D NMR spectroscopy. We tested the LPS proinflammatory activity as a stimulant of human myelomonocytic U937 cell cytokine induction and assessed TLR4/MD2 signaling. Marked changes between the paired strains were found in the lipid A-inner core region. Such structural variations can contribute to the bacterial survival and persistence of infections despite the loss of a CF milieu following lung transplantation.

Introduction

We report the complete structure of the Lipopolysaccharide (LPS) endotoxin from the Gram-negative Burkholderia multivorans clinical isolates for the first time. We have also investigated the structural modifications that B. multivorans LPS undergoes after lung transplantation and the proinflammatory activity of such clinical isolates. B. multivorans is one of the most common and dangerous opportunistic pathogens of the Burkholderia cepacia complex species isolated so far in CF centers.

Burkholderia cepacia” has long been identified as a plant pathogen where it causes onion rot (Speert 2002). More recently it has been recognized as an opportunistic human pathogen for individuals with cystic fibrosis (CF) and chronic granulomatous disease. Previously, infections with “Burkholderia cepacia” were attributed to a single species. Indeed, such infections are caused by a variety of bacterial species, now grouped into the “Burkholderia cepacia complex (BCC)” (Mahenthiralingam et al. 2000). Currently this complex comprises 10 related species named genomovars which are phenotypically similar but genotypically different. The most prevalent clinical isolates from CF patients are B. cenocepacia strains (genomovar III) and B. multivorans strains (genomovar II) (De Soyza, Morris, et al. 2004; Wellinghausen and Köthe 2006). B. cenocepacia appears the most pathogenic with accelerated lung disease reported in patients with previously well-preserved lung function. Approximately 20% of patients develop “cepacia syndrome” a fulminating necrotizing pneumonitis with associated septicaemia (De Soyza and Corris 2003). B. cenocepacia is the species most commonly associated with the “cepacia syndrome” although some cases are due to B. multivorans (Blackburn et al. 2004). BCC genomovars also possess a potentially transmissible nature: B. cenocepacia is the most notorious genomovar causing epidemics although B. multivorans epidemics have also been reported.

Lung transplantation, whilst intensive and aggressive, is the only treatment that improves both the quantity and quality of life of CF patients with advanced lung disease. It has been shown that BCC infections are the second most prevalent pretransplantation infection. Transplanting such patients remains controversial as poor outcomes are common in those patients with preoperative B. cenocepacia infection. In our experience recipients infected with B. cenocepacia died early posttransplant in a fashion similar to “cepacia syndrome” (De Soyza et al. 2001; Jones et al. 2004). In contrast, excellent survival rates in patients with pretransplantation B. multivorans (genomovar II) and B. vietnamiensis (genomovar V) infection were noted. Lung transplantation outcomes appear to be dependent on a number of factors including preoperative B. cepacia complex genomovar status (De Soyza et al. 2001).

The pathogenic mechanisms involved in BCC infections are still unclear. An investigation of the structure and biological activity of lipopolysaccharides (LPSs) is required as they are critically important bacterial virulence factors (Holst 1999; Alexander and Rietschel 2001; Raetz and Whitfield 2002). LPSs are amphiphilic macromolecules indispensable for the growth and the survival of Gram-negative bacteria. LPS has been identified as a pathogen associated molecular pattern (PAMP) and is recognized by host pathogen-recognition receptors to induce a marked inflammatory response via TLR4/MD2-mediated signaling pathways (Akira et al. 2006).

We have previously demonstrated that lipid A changes in B. cenocepacia (De Soyza, Ellis, et al. 2004) similar to those reported in CF-associated P. aeruginosa strains (Ernst et al. 1999), suggesting an important interaction between the CF microenvironment and infecting pathogen. Deacylation of P. aeruginosa LPS in bacterial strains from CF patients results in reduced activation of TLR-4/MD2 (Hajjar et al. 2002). Despite the prevalence of B. multivorans as a CF-related pathogen, limited data exist regarding LPS in B. multivorans.

In this work we define the complete primary structure and proinflammatory activity of the LPS extracts from B. multivorans. Lung transplantation offers interesting insights into assessing possible host–pathogen interactions as following transplantation any bacterial interaction will be with a lung allograft that does not bear cystic fibrosis epithelium or CFTR defects. We investigated the structure of the LPS from clinical isolates prior to and shortly after lung transplantation. Such data are essential to elucidate the molecular modifications involved in the inflammatory process that occur pre- and postsurgery. Understanding how bacteria modify and adapt their LPS under new physiological conditions may allow greater understanding of LPS-associated signaling. These data may also allow new therapeutic targets to be identified including the design of antimicrobial compounds against chemically different targets.

Results

Strain selection and SDS electrophoresis analysis of B. multivorans LOSs

We identified three patients who had paired pre- and posttransplantation strains of BCC that demonstrated differences in rough LPS (lipo-oligosaccharide; LOS) migration patterns upon silver staining of 16% SDS gels (LOSs from B. multivorans strains are shown in Figure 1A). This change was not seen in the remainder of the transplant cohort analyzed. Of these three patients, one patient had B. multivorans infection, one had B. vietnamensis infection, and one had B. cenocepacia infection. The data reported will be limited to the patient with B. multivorans infection. The pretransplantation strain was isolated immediately pretransplant and the posttransplantation strain was isolated at 1 week after transplantation (Figure 1B). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) revealed that the extracted B. multivorans lipopolysaccharide was a rough-type LPS or lipooligosaccharide (LOS) (Figure 1A).

Fig. 1

Evaluation LPS from B. multivorans pre- and posttransplantation. (A) SDS gel of extracted Lipo-oligosaccharide from clonal strains suggested migration differences when comparing pre-transplant (lane 1) to posttransplant (lane 2). (B) Typical pulsed field gel electophoretogram of paired pre- and posttransplant strain after SpeI enzyme digestion. The index case was known to have B. multivorans infection pretransplant. Lane 1 represents molecular weight markers. PFGE analysis demonstrated that the pretransplant (lane 2) and posttransplant strain (lane 3) were clonally identical (adapted from De Soyza, Morris, et al. 2004).

Fig. 1

Evaluation LPS from B. multivorans pre- and posttransplantation. (A) SDS gel of extracted Lipo-oligosaccharide from clonal strains suggested migration differences when comparing pre-transplant (lane 1) to posttransplant (lane 2). (B) Typical pulsed field gel electophoretogram of paired pre- and posttransplant strain after SpeI enzyme digestion. The index case was known to have B. multivorans infection pretransplant. Lane 1 represents molecular weight markers. PFGE analysis demonstrated that the pretransplant (lane 2) and posttransplant strain (lane 3) were clonally identical (adapted from De Soyza, Morris, et al. 2004).

Isolation and compositional analysis of LOS, Fr1, and Fr2 from B. multivorans pre- and posttransplantation

LPS fractions from both clinical isolates were extracted and analyzed by SDS–PAGE. Both fractions consisted of rough LPS chemotype (lipo-oligosaccharide, LOS) as suggested by the run at the bottom of the gel (Figure 1A).

Monosaccharide and fatty acid analyses of LOS isolated from B. multivorans pre- and posttransplantation showed the same content in sugar and fatty acids but the presence of an additional 8-substituted d-glycero-d-talo-oct-2-ulosonic acid (Ko) residue was detected only in the posttransplantation strain. The full compositional analysis of all fractions is reported in the text and Table I of Supplementary Data.

Structural characterization of Fr1 product from the B. multivorans pretransplantation strain

The usual mild acid treatment on pretransplantation strain LPS yielded two fractions, Fr1 and Fr2. The monosaccharide analysis of both fractions is shown in the text and Table I available in Supplementary Data.

In order to assign all the spin systems of Fr1 and to define the monosaccharide sequence, a combination of homo- and heteronuclear 2D NMR experiment (DQF-COSY, TOCSY, ROESY, NOESY, 1H-13C HSQC, 1H-13C HSQC-TOCSY and 1H-13C HMBC, see supporting Figure 3A–F) was undertaken. In the anomeric region of the 1H-NMR spectrum (Figure 2A), 12 anomeric signals were identified (A–N, Table II available in Supplementary Data). Furthermore, the signals at 1.94/2.05 ppm were identified as the H-3 methylene protons of the Kdo residue (K) whereas residue J of α-Ko was assigned starting from its oxymethine H-3 signal. The relative intensities of anomeric signals suggested the existence of a mixture of oligosaccharides with different length of the carbohydrate chain.

Fig. 2

NMR structural investigation of Fr1 product. (A) 1H NMR spectrum of Fr1 product with a zoom of the anomeric region. (B) Zoom of the ROESY (red) and TOCSY (black) spectra of oligosaccharide Fr1. Monosaccharide labels are indicated in Supplementary Data Table II whereas complete NMR experimental conditions are given in Supplementary Data.

Fig. 2

NMR structural investigation of Fr1 product. (A) 1H NMR spectrum of Fr1 product with a zoom of the anomeric region. (B) Zoom of the ROESY (red) and TOCSY (black) spectra of oligosaccharide Fr1. Monosaccharide labels are indicated in Supplementary Data Table II whereas complete NMR experimental conditions are given in Supplementary Data.

Fig. 3

Negative-ion MALDI mass spectrum of Fr1 and Fr2 oligosaccharides. (A) Species X, X-QuiNAc, and Y, matching with NMR analysis, are the main peaks. Ions identified as lipid A species (indicates with asterisks) are also visible. (B) High-resolution negative MALDI mass spectrum of Fr2 oligosaccharide acquired in reflectron mode. Peaks R and S are related to oligosaccharides X and Y lacking the Ko moiety, respectively. Ions due to species missing QuiNAc and Hep residues are also present.

Fig. 3

Negative-ion MALDI mass spectrum of Fr1 and Fr2 oligosaccharides. (A) Species X, X-QuiNAc, and Y, matching with NMR analysis, are the main peaks. Ions identified as lipid A species (indicates with asterisks) are also visible. (B) High-resolution negative MALDI mass spectrum of Fr2 oligosaccharide acquired in reflectron mode. Peaks R and S are related to oligosaccharides X and Y lacking the Ko moiety, respectively. Ions due to species missing QuiNAc and Hep residues are also present.

In accordance with the chemical analysis, spin systems A, B, E, G were all identified as l-glycero-d-manno-heptose whereas spin systems C and I were identified as 2-deoxy-2-amino-galactose, both acetylated at C-2 position. Spin systems M and N were identified as glucose residues while D, H, and H′ were recognized as α-rhamnose residues. Residue L (H-1 at 4.68 ppm) was recognized as a β-quinovosamine acetylated at C-2 position.

The oligosaccharide sequence reported below (species X) was established identifying the interresidual NOE contacts (Figure 2B) and the long-range correlations present in the HMBC spectrum:

 
formula

An alternative glycoform of the α-rhamnose D was found, namely residue F, identified as a 3-α-Rha residue. This was glycosylated by residue I of β-GalNAc that was in turn substituted at O-3 by the α-GalNAc C. Additionally, residue C was glycosylated at position 3 by the terminal β-Glc N. The linkage between these residues was attested by NOE contacts (Figure 2B) and confirmed by scalar correlations in the HMBC spectrum. Thus, the oligosaccharide sequence (species Y) reported below differed from the previous one for the presence of the additional terminal trisaccharide:

 
formula

MALDI-MS characterization of Fr1 and Fr2 products from B. multivorans pretransplantation

Matrix-assisted laser desorption ionisation-mass spectrometry (MALDI-MS) confirmed the oligosaccharide structures hypothesized above. The negative mass spectrum (Figure 3A) showed two peaks, at m/z 1883.2 and at m/z 2264.1, matching with the oligosaccharide X and Ym/z ≅ 568 Th from 1696.3) assigned by NMR analysis and a further peak at m/z 1696.3 that differed from X by a β-QuiNAc residue. Likewise, Fr2 fraction underwent mass spectrometric investigation. The negative-ion MALDI mass spectrum of the oligosaccharide mixture Fr2 (Figure 3B) showed ion peaks related to Fr1 oligosaccharides but lacked the Ko residue (Δm/z ≅ 236 Th).

Collectively, these results yielded the full oligosaccharide sequence of the B. multivorans pretransplantation LPS as depicted below:

 
formula

MALDI-MS characterization of oligosaccharide product from the B. multivorans posttransplantation strain

As compared to the pretransplantation strain, the LOS extracted from the B. multivorans posttransplantation strain underwent the same treatment and one oligosaccharide fraction, namely Fr, was isolated by gel-permeation chromatography. The compositional analysis of the fractions is reported in Table I available in Supplementary Data. Both the NMR and the MS data gave evidence of a saccharide sequence identical to the Fr2 fraction found in the pretransplantation strain (data not shown). The matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectra of Fr fraction are reported in Supplementary Data Figure 2.

Structural characterization by MALDI mass spectrometry of the intact LOS from B. multivorans pretransplantation

Further investigation of lipid A and core oligosaccharide structures was conducted on intact LOS using MALDI-MS with our sample preparation procedure specifically set up for such amphiphilic molecules (Sturiale et al. 2005). The negative-ion MALDI mass spectrum obtained before transplantation is shown in Figure 4. At molecular masses from 1000 to 2500 Th (Figure 4A), two peaks related to the core oligosaccharide were found. Peak OS1 at m/z 1677.6 matched with a nonasaccharide constituted of four Hep, one Hex, two dHex, one Kdo, and one Ko residue whereas peak OS2 at m/z 2245.3 corresponded to the dodecasaccharide carrying the adjunctive Hex-HexNAc-HexNAc trisaccharide (Δm/z = 568 Th from OS1), confirming above structural assignments. The same mass region also comprised ion peaks deriving from a mixture of tetra- and penta-acylated lipid A (see also Figure 3A); species L1 at m/z 1444.0 matched with a tetra-acylated bis-phosphorylated disaccharide backbone carrying in ester linkage one 14:0 (3-OH) and in amide linkage two 16:0 (3-OH) acyl chains, one of which, on the GlcN II was further substituted by a secondary 14:0 fatty acid (Silipo et al. 2002). Species L2 and L3 were tetra-acylated Lipids A carrying one and two Ara4N (4-deoxy-4-amino-arabinose) residues (see Supplementary Data Table I and Figure 4). Species L4 (m/z 1670.2), L5 (m/z 1801.3), and L6 (m/z 1932.5) were the penta-acylated Lipids A carrying two ester-linked 14:0 (3-OH) and zero, one, and two Ara4N residues, respectively.

Fig. 4

Negative-ion MALDI mass spectrum of intact LOS from B. multivorans pretransplantation. (A) Low mass range (1000–2500 Th): in this region, ion fragments due to core oligosaccharides OS1 and OS2 and to lipid A species are present. (B) High mass range (2500–4500 Th): in this region peaks due to intact LOS molecular ions are present.

Fig. 4

Negative-ion MALDI mass spectrum of intact LOS from B. multivorans pretransplantation. (A) Low mass range (1000–2500 Th): in this region, ion fragments due to core oligosaccharides OS1 and OS2 and to lipid A species are present. (B) High mass range (2500–4500 Th): in this region peaks due to intact LOS molecular ions are present.

The location of the secondary fatty acid 14:0 on the GlcN II was defined by mass spectrometry of the lipid A moiety after acetate buffer hydrolysis (see the text in Supplementary Data).

Assignment of the main LOS molecular ions (Figure 4B) emerged from the combination of the lipid A and the core oligosaccharides. Species composed by tetra-acylated and penta-acylated lipid A and OS1 were found. A minor peak was further identified at m/z 3439.3 and assigned to the species L2+OS1 bearing in addition the QuiNAc unit on the core region. Higher masses species were also present reflecting a combination of OS2 and tetra- and penta-acylated lipid A.

Structural characterization by MALDI-MS of the intact LOS from B. multivorans posttransplantation

MALDI-MS analysis of LOS isolated from the bacterial strain after lung transplantation was performed as well, as shown in Figure 5. Even in this case either peaks attributable to intact LOS or ions related to fragments core and lipid A were visible. In the mass range between 1000 and 2500 Th (Figure 5A), species OS1 at m/z 1677.7 and OS2 at m/z 2245.1 were identical to those observed in the LOS from B. multivorans pretransplantation (see also MS and NMR analyses on Fr species) derived from both the lipid A and the core oligosaccharide. In accordance to compositional analysis, a peak at m/z 1808.5 (Δm/z ≅ 131 Th) corresponding to OS1 oligosaccharide carrying an Ara4N unit linked to Ko was identified. The predominant lipid A moieties found were the tetra-acylated L1 and L2 species differing by one Ara4N residue, with L3, L4, and L5 lipid A present only in minor quantities. We were unable to detect evidence of a penta-acylated species with two Ara4N residues L6. The MS profile related to LOS molecular ions (Figure 5B) showed peaks reflecting quantitatively the relative amount of lipid A species. The main ion peaks were given by the combination of OS1 with tetra-acylated lipid A (L1 + OS1 at m/z 3122.2, L2 + OS1 at m/z 3253.1, and L3 + OS1 at m/z 3384.1). The possibility of having an Ara4N residue nonstoichiometrically linked to the lipid A or to the core region led to multiple assignments, as indicated in Figure 5B. An ion peak at m/z 3439.9 was present, reflecting the nonstoichiometric amount of terminal QuiNAc residue.

Fig. 5

Negative-ion MALDI mass spectrum of intact LOS from B. multivorans posttransplantation. (A) In the low mass range, the presence of oligosaccharide specie OS1 carrying an additional Ara4N residue is noteworthy. (B) High mass region of the same spectrum: the majority of peaks originate from multiply assignments due to nonstoichiometric linkage of a Ara4N residue with the core portion or the lipid A species.

Fig. 5

Negative-ion MALDI mass spectrum of intact LOS from B. multivorans posttransplantation. (A) In the low mass range, the presence of oligosaccharide specie OS1 carrying an additional Ara4N residue is noteworthy. (B) High mass region of the same spectrum: the majority of peaks originate from multiply assignments due to nonstoichiometric linkage of a Ara4N residue with the core portion or the lipid A species.

The above MS results on intact LOSs are consistent with the previous structural hypotheses allowing a precise molecular characterization. It is also noteworthy that such careful mass spectrometry approaches disclose new avenues on the analysis on the intact LPS/glycolipid-like biomolecules excluding any structural alteration owing to the chemical manipulation of samples. Thus, the lipo-oligosaccharide from B. multivorans strains isolated pre- and post-lung transplantation are reported in Figure 6.

Fig. 6

The full structure of R-type LPS from B. multivorans pre- and posttransplantation strains. Dotted lines indicate the nonstoichiometric substitutions which are present in very different amounts between LOSs from B. multivorans pre- and posttransplantation (see text, figures, and Supplementary Data). The Ara4N moiety in the circle was found exclusively in LOS isolated from the posttransplantation strain while the one in the rectangle is prevalently present in pretransplantation lipid A moiety.

Fig. 6

The full structure of R-type LPS from B. multivorans pre- and posttransplantation strains. Dotted lines indicate the nonstoichiometric substitutions which are present in very different amounts between LOSs from B. multivorans pre- and posttransplantation (see text, figures, and Supplementary Data). The Ara4N moiety in the circle was found exclusively in LOS isolated from the posttransplantation strain while the one in the rectangle is prevalently present in pretransplantation lipid A moiety.

Biological activity of B. multivorans LOSs

The LOS molecules isolated pre- and post-lung transplantation were tested for their proinflammatory activity of eliciting TNF-alpha induction from human myelomonocytic U937 cells (Figure 7A).

Fig. 7

Biological assays of pre- and post- transplantation isolated LOS and lipid A moieties. (A) TNF induction from U937 cells elicited by standard LPS and paired clonal strains. Key: histograms represent TNF induction (ng) at 24 h. U937 cells were stimulated with 100 ng of purified LPS including E. coli O55 as positive control. US represents unstimulated cells (negative control). Purified LPS from strains LMG 14273 (B. multivorans) and LMG 12614 (B. cenocepacia ET-12 strain) as previously reported (De Soyza et al. 2004) was also used as comparators. “Pre” represents the TNF induction elicited by the pretransplantation clonal B. multivorans strain. “Post” represents the biological activity of 100 ng of extracted LOS from the posttransplantation strain which was significantly lower than the pretransplant strain (p < 0.001). (B, C) In these figures, the NF-KB induction elicited by extracted lipid A and LPS/LOS in transfected TLR 4/MD2/CD 14 HEK cell lines is reported. In figures, pre- and post- represent a B. multivorans strain pre- and posttransplantation, respectively, at doses of 10 ng/mL (vertical striped bar) or 100 ng/mL (checkerboard bar). Negative control (culture media only) is represented by C (open bar) and E. coli (closed bar) represents the biological activity of 10 ng/mL of the positive control E. coli 055 LPS. The reporter assay is measured in relative light units (RLU).

Fig. 7

Biological assays of pre- and post- transplantation isolated LOS and lipid A moieties. (A) TNF induction from U937 cells elicited by standard LPS and paired clonal strains. Key: histograms represent TNF induction (ng) at 24 h. U937 cells were stimulated with 100 ng of purified LPS including E. coli O55 as positive control. US represents unstimulated cells (negative control). Purified LPS from strains LMG 14273 (B. multivorans) and LMG 12614 (B. cenocepacia ET-12 strain) as previously reported (De Soyza et al. 2004) was also used as comparators. “Pre” represents the TNF induction elicited by the pretransplantation clonal B. multivorans strain. “Post” represents the biological activity of 100 ng of extracted LOS from the posttransplantation strain which was significantly lower than the pretransplant strain (p < 0.001). (B, C) In these figures, the NF-KB induction elicited by extracted lipid A and LPS/LOS in transfected TLR 4/MD2/CD 14 HEK cell lines is reported. In figures, pre- and post- represent a B. multivorans strain pre- and posttransplantation, respectively, at doses of 10 ng/mL (vertical striped bar) or 100 ng/mL (checkerboard bar). Negative control (culture media only) is represented by C (open bar) and E. coli (closed bar) represents the biological activity of 10 ng/mL of the positive control E. coli 055 LPS. The reporter assay is measured in relative light units (RLU).

These findings are in agreement with our prior data and showed that B. cenocepacia ET-12 LMG 12614 epidemic strain and E. coli 055 were more potent TNF-alpha inducers than any of the B. multivorans strains tested (LMG 14273 and the paired pre- and posttransplant strains).

Assessment of lipid A and LOS activity using transient transfection of TLR 4/MD2/CD 14 into HEK cells

There was, in general, a greater biological activity at induction of NF-KB observed for the LOS as compared to the extracted lipid A (Figure 7B and C). This implies a biological role for the core region. All stimulants had relatively potent proinflammatory activity attaining similar levels of NF-KB at ng concentrations in similar to that of the positive control E. coli O55 LPS (10 ng/mL). The NF-KB induction activity of the B. multivorans strains was however less potent than that achieved by the B. cenocepacia ET-12 strain (not shown).

The B. multivorans lipid A isolated prior to transplantation induced greater activation in the NF-KB reporter assay than after transplantation. Using the NF-KB luciferase assay the proinflammatory activity of extracted lipid A from the B. multivorans pretransplantation strain (A pre 10) was significantly higher than that of lipid A from the posttransplant strain (A post 10), pairwise t-test comparison of data with pooled standard deviations, p < 0.05. This was also significantly tested at both concentrations from the B. multivorans strain. This confirms the significant differences observed in the cytokine output in the U937 assay that were likely to reflect signaling through the TLR4/MD2 complex.

Discussion

Infection with the B. cepacia complex (BCC) is problematic in patients with cystic fibrosis reflecting a remarkable antibiotic resistance and the potential for epidemic spread. B. multivorans, a member of the BCC, is prevalent in European CF centers. Interestingly, this organism appears to be associated with less severe clinical outcomes than those seen in patients infected with B. cenocepacia. Prior in vitro data have suggested that B. multivorans genomovar strains have a reduced capacity to elicit proinflammatory cytokines as compared to B. cenocepacia (De Soyza, Ellis, et al. 2004). Recent studies have demonstrated divergent proinflammatory signaling pathways for these endotoxins. In an in vitro model with macrophages as target cells, B. multivorans LPS promoted a My-D88 independent inflammatory pathway whilst that elicited by B. cenocepacia LPS was My-D88 dependent (Bamford et al. 2007). This qualitative divergence in signaling pathways could be a possible explanation of our prior data. Previously, we noted qualitative differences in cytokine induction patterns: B. multivorans LOS induced IL-6 production from U937 macrophages peaking at 24–48 h whilst B. cenocepacia LOS induced production continued to increase up to 60 h poststimulation (De Soyza, Ellis, et al. 2004). These combined observations in conjunction offer possible explanations for the different clinical outcomes observed in patients infected with B. cenocepacia and B. multivorans (Bamford et al. 2007) (De Soyza, Ellis, et al. 2004).

The full primary structure of lipopolysaccharides from BCC genomovars and LPS structure/function relationships are therefore clinically relevant. Such studies represent a contributive step in the understanding of molecular mechanisms involved in the inflammatory process. We report for the first time the complete structure of the LOS from B. multivorans clinical isolates and also report the structural modifications that B. multivorans LOS undergoes after a change in host microenvironment as seen following lung transplantation.

The CF airway is known to be associated with inducing specific changes in the lipid A of both Pseudomonas and Burkholderia species (Ernst et al. 1999; De Soyza, Ellis, et al. 2004). It is not, however, known if these changes are reversible nor over what timescale any reversibility occurs. Studying strains that have coexisted in vivo with CF airway epithelia and that are subsequently required to interact with non-CF (normal) airway epithelial following transplantation may offer powerful insights into host–pathogen relationships.

The analysis of core oligosaccharide from B. multivorans pre- and post-lung transplantation clinical isolates revealed a novel oligosaccharide structure. The Burkholderia genus has a highly conserved LPS inner core structure with several characteristics replicated in the current study. The main feature is the presence of the [3,4-α-l,d-Hep-(1→5)-α-d-Kdo-(4→2)-α-d-Ko] trisaccharide in the inner core. Notably, within the genus the Ko residue is frequently not terminal but further links an Ara4N residue that, in the present case, was only present in the posttransplantation strain. Usually the heptose residue bears another heptose linked at C-3 and glycosylated at C-4 by a β-d-Glc; this latter can be optionally glycosylated either by terminal α-Glc (Molinaro et al. 2002; Silipo et al. 2007) or as in the present case by a terminal α-Hep, composing a Hep-(1→6)-Glc disaccharide. Moreover, in the outer core region, the presence of a nonstoichiometric [β-d-Glc-(1→3)-β-d-GalNAc-(1→3)-α-d-GalNAc] trisaccharide sitting on a rhamnose residue is noteworthy as it is likely preassembled and then attached.

The main structural changes between the clonal strains were found in the different distribution of the lipid A species. The lipid A moiety from both species possessed a carbohydrate backbone characterized by a [P→4-β-d-GlcpN-(1→6)-α-d-GlcpN-1→P→Ara4N] sequence, already found in other BCC genomovars such as B. cenocepacia and B. pyrrocinia (Silipo et al. 2006, 2007). The lipid A from B. multivorans pretransplantation isolate comprised a mixture of penta- and tetra-acylated species in similar proportions carrying up to two Ara4N residues. The posttransplantation B. multivorans strains’ LPS demonstrated marked changes in the distribution and composition of lipid A species with an overall lower level of acylation and substitution by Ara4N. In particular, there was a negligible amount of penta-acylated species with a predominance of tetra-acylated lipid A. Furthermore, each species carried no more than one Ara4N residue.

The core oligosaccharide sequence of the B. multivorans species studied was distinct from that of B. cenocepacia (Silipo et al. 2007). It has been postulated that B. cenocepacia biological activity and survival in vivo is in part dependent on the presence of a complete LOS as a B. cenocepacia mutant strain lacking a complete LPS core oligosaccharide is sensitive to antimicrobial peptides and polymyxin B (Loutet et al. 2006). Further evidence for an important biological activity of the core oligosaccharide was suggested by the higher levels of NF- induction KB by the complete LOS as compared to lipid A (Figure 7B and C). The differences in the core oligosaccharide between the two genomovars could contribute to clinical phenotypes (inflammatory syndromes).

The core portion in B. multivorans is likely to contribute to bacterial resistance to antimicrobial compounds. The net charge surface on the external membrane of Gram-negative bacteria is felt to play a key role in pathogenesis. LPSs from the Burkholderia genus are frequently positively charged or in an isoelectric state. This is closely related to the abundance of aminoarabinose (Ara4N) residues present in the lipid A-inner core and confers resistance to antibiotic compounds and host cationic antimicrobial peptides. Under physiological conditions, Ara4N moieties are positively charged and reduce the net charge surface on the external membrane weakening the ionic attraction for respiratory tract antimicrobial defensins. The inherent resistance of these microorganisms to polymyxin B, a cyclic polycationic antibiotic peptide that has high affinity for negatively charged bacterial LPS, is likely to be Ara4N dependent. These residues prevent the antimicrobial action of polymyxin B, increasing permeability in a bacterial outer membrane (Shimomura et al. 2003).

Peritransplantation processes that promote increased bacterial defensin resistance are suggested by our observation of a further Ara4N residue on the inner core portion of LOS from B. multivorans posttransplantation. There are many possible explanations for this observation including the effect of antibiotics administered during the transplant operation or an as yet, undescribed effect of immunosuppressant agents. However, it is interesting to speculate that, posttransplantation, the replacement of CF epithelium with a normal airway epithelium would increase defensin activity by normalizing high airway sodium content (a known inhibitor of defensin activity).

Differences in SDS gel migration patterns of LOSs were not observed in every clonal-paired strain from our transplant cohort (data not shown). It was observed in one of the four recipients with pretransplant B. multivorans infection. This suggests that the phenomenon may be strain specific and was not a uniform effect as may be expected if peritransplant immunosuppressive drug therapy or antimicrobials were a cause.

Lipid A contributes to the majority of endotoxic activity of LPS. A number of factors influence the lipid A biological activity including the number and the distribution of acyl chains, the phosphorylation pattern, and the presence of charged groups on the polar heads. The correlation between increasing acylation of lipid A and a greater cytokine induction capacity has previously been reported in Pseudomonas (Ernst et al. 1999) and Burkholderia (De Soyza, Ellis, et al. 2004). The lipid A from B. multivorans pretransplantation had a comparable amount of penta- and tetra-acylated lipid A species and a notable abundance of Ara4N moieties. These data in part confirm our previous report comparing B. multivorans and B. cenocepacia with higher levels of lipid A acylation noted in B. cenocepacia (De Soyza, Ellis, et al. 2004). Interestingly, the lipid A from B. multivorans posttransplantation was less toxic in our U937 cell and HEK TLR4/MD2 bioreporter assays consistent with the observed relative reduction in penta-acylated lipid A species. These data confirm that the lipid A changes are recognized at the human TLR-4 receptor. A reduction in cytokine induction may allow bacteria to survive in a more hostile environment (Munford and Varley 2006; Reife et al. 2006; Raetz et al. 2007).

Other novel findings presented suggest that expressed lipid A species are relatively rapidly altered to reflect changes in host–pathogen interfaces (Munford and Varley 2006; Raetz et al. 2007). The mechanisms responsible for controlling lipid A biosynthesis remain unclear though recent reports have elucidated the pathways for aminoarabinose biosynthesis (Ortega et al. 2007). A critical role of airway adaptation in cystic fibrosis-related Pseudomonas aeruginosa strains has been seen (Ernst et al. 1999; Hajjar et al. 2002). Interestingly, these studies demonstrated that a specific penta-acylated LPS was found in mild/moderate CF lung disease and hepta-acylated species were found in severe CF disease. Such data in conjunction with our observations suggest that increasing acylation of lipid A in cystic fibrosis may be a slow adaptation to the CF environment (reflecting disease progression) that can be more rapidly downregulated once selective pressures are removed (e.g., removal of the CF microenvironment). Notably, the finding of species-specific recognition of the CF Pseudomonas adaptation to the CF airway suggests that bacterial adaptation to the airway reflects the host innate immune system (Hajjar et al. 2002). In the new microenvironment post transplantation, B. multivorans decreases the immunostimulant activity of lipid A by decreasing its acylation pattern and reducing the consequent host immune response. A further adaptation that increases the antimicrobial resistance is the addition of an Ara4N residue to the outer core Ko residue. Further elucidation of controllers of acylation status may offer the development of therapeutic strategies and new treatments for the management of Gram-negative infections in cystic fibrosis.

In conclusion, in this work we have chemically and biologically defined for the first time the LPS endotoxin from B. multivorans. We have also investigated the molecular modifications that B. multivorans LOS undergoes after lung transplantation. This study improves the understanding of the endotoxin structure–activity relationship, which is of pivotal importance in the comprehension of the overall process of pathogenesis of such important microorganisms.

Material and methods

Bacterial growth and LPS extraction

Bacterial strains isolated from recipients were stored on microbeads. These were streaked on to LB-agar. Standard hot phenol/water LPS extraction was undertaken as previously described (Westphal and Jahn 1965). Large volume extractions were carried out as described and the complete experimental details are given in Supplementary Data. LPS fractions were analyzed by SDS–PAGE on 16% gels, which were stained with silver nitrate.

Patient selection

Cystic fibrosis patients were listed for transplantation according to international guidelines (Orens et al. 2006). Patients who had paired B. cepacia complex isolates pre- and posttransplantation that were recoverable from storage repositories were considered eligible for study selection.

Strain preparation and selection

Sputum was collected from patients immediately before surgery. Posttransplant lower airway (bronchoalveolar) lavages were collected from recipients at day 7. Presumed B. cepacia complex bacteria were isolated by culture. For this study we restricted investigations to B. multivorans strains.

Pulsed-field gel electrophoresis

Confirmation of the clonal nature of paired pre- and posttransplant strains has previously been reported (De Soyza, Morris, et al. 2004). B. cepacia complex strains were blinded to the investigators and genotyped by macrorestriction of whole genomic DNA with the restriction enzyme SpeI (New England Biolabs, UK), followed by separation of the fragments by pulse field gel electrophoresis (PFGE) (CHEF DRII system; Bio-Rad, CA).

SDS electrophoresis analysis and biological activity of B. multivorans LOSs

Extracted LPS or LOS were subjected to SDS–PAGE on 16% SDS gels and silver stained as previously (De Soyza et al. 2004). B. multivorans LOS was tested for its proinflammatory activity for TNF-alpha induction in human myelomonocytic U937 cells as described (De Soyza, Morris, et al. 2004). Detailed methodology is available in Supplementary Data.

Assessment of lipid A and LOS activity using transient transfection of TLR 4/MD2/CD 14 into HEK cells

HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were seeded at 3 × 104/well of a 96-well plate and transiently transfected 2 days later. Expression vectors containing cDNA encoding TLR4, MD-2, and CD14 (1 ng/well of each), a NFκB transcription reporter vector encoding firefly luciferase (5 ng/well pNFκB-luc, Clontech), and a constitutively active reporter vector encoding Renilla luciferase (5 ng/well phRG-TK, Promega), together with empty vector to ensure an optimal amount of DNA were mixed with JetPEI (Polyplus transfection) according to the manufacturer's instructions. After 48 h cells were stimulated with LOS and lipid A at two concentrations 10 ng and 100 ng of stimulant for 6 h and compared to control (media only) and compared to a positive control of transfected cells stimulated with 10 ng of E. coli 055 strain LPS (Sigma, UK). The effect of stimulation was assessed using a NF-KB luciferase reporter assay (Dual Luciferase assay, Promega, Southampton, UK). The luciferase results were normalized to the constitutively active renilla transfection control activity and results were expressed as a percentage of the positive control. Experiments were repeated in quadruplicate.

Structural characterization of oligosaccharides Isolation of Oligosaccharide Fr1, Fr2, and Fr

The lipid A fractions and core oligosaccharides (Fr1, Fr2, and Fr) from B. multivorans pre- and posttransplantation LPS were obtained by acetate buffer hydrolysis at 100°C for 3 h as described (Silipo et al. 2007).

General and analytical methods

Determination of sugars residues, their absolute configuration, methylation analysis, fatty acid, and their absolute configurations were all determined as described previously and completely described in Supplementary Data (Hakomori 1964; Rietschel 1976; Leontein and Lönngren 1978; Silipo et al. 2007).

NMR spectroscopy

In all structural assignments, 1D and 2D 1H-NMR spectra were recorded with a solution of 0.5–1 mg in 0.5 mL of D2O, at 300 K, at pD 7 (uncorrected value) on Bruker 600 DRX equipped with a cryo probe. Spectra were calibrated with internal acetone (δH 2.225, δC 31.45). 31P NMR experiments were carried out using a Bruker DRX-400 spectrometer, aqueous 85% phosphoric acid was used as external reference (0.00 ppm). The full structural determination and NMR experiments used are reported in Supplementary Data.

MALDI-TOF mass spectrometry

MALDI-TOF mass spectra were recorded in negative or in positive polarity on a Perseptive (Framingham, MA) Voyager STR instrument equipped with delayed extraction technology. High-resolution mass spectra were acquired in reflector mode on a 4800 Proteomics analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA). Sample preparations and details of mass spectra executed are fully described in Supplementary Data.

Supplementary Data

Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.

Funding

Italian Cystic Fibrosis Research Foundation (grant FFC 11/2006 to A.M.)

A.D.S was supported by the Special Trustees of The Newcastle upon Tyne Teaching Hospitals and a Higher Education Funding Council for England (HEFCE) Senior lecturership. We are grateful to Professor John Govan, University of Edinburgh, UK, for the pulsed field gel electrophoresis experiments as previously published, and Christine Aldridge for technical support. We are also grateful to Dr T. J. McKinely, Department of Veterinary Medicine, University of Cambridge, for statistical advice.

Conflict of interest statement

None declared.

Abbreviations

    Abbreviations
  • Ara4N

    4-deoxy-4-amino-L-arabinose

  • BCC

    Burkholderia cepacia complex

  • CF

    cystic fibrosis

  • DQF-COSY

    double quantum filtered correlation spectroscopy

  • HMBC

    heteronuclear multiple bond correlation

  • HSQC

    heteronuclear single quantum coherence

  • Kdo

    3-deoxy-d-manno-oct-2-ulosonic acid

  • Ko

    d-glycero-d-talo-oct-2-ulosonic acid

  • LOS

    lipo-oligosaccharide LPS, lipopolysaccharides

  • MALDI-MS

    matrix-assisted laser desorption ionisation-mass spectrometry

  • NOE

    nuclear Overhauser effect

  • NOESY

    nuclear Overhauser enhancement spectroscopy

  • PAMP

    pathogen-associated molecular pattern

  • ROESY

    rotating frame Overhauser enhancement spectroscopy

  • SDS–PAGE

    sodium dodecyl sulphate–polyacrylamide gel electrophoresis

  • TOCSY

    total correlation spectroscopy

  • TOF

    time-of-flight

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Supplementary data