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Published ahead of print on March 24, 2004, doi:10.1164/rccm.200304-592OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 70-77, (2004)
© 2004 American Thoracic Society

Burkholderia cenocepacia Lipopolysaccharide, Lipid A, and Proinflammatory Activity

Anthony De Soyza, Charles D. Ellis, C. M. Anjam Khan, Paul A. Corris and Raquel Demarco de Hormaeche

Transplantation and Immunobiology Group, The Freeman Hospital, Newcastle-upon-Tyne, and the Department of Microbiology and Immunology, University of Newcastle, Newcastle, United Kingdom

Correspondence and requests for reprints should be addressed to Anthony De Soyza, M.R.C.P., Transplantation and Immunobiology group, The Freeman Hospital, High Heaton, Newcastle-upon-Tyne NE7 7DN, United Kingdom. E-mail: Anthony.De-Soyza{at}ncl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Organisms from the Burkholderia cepacia complex are important pathogens in cystic fibrosis and are associated with increased rates of sepsis and death. These organisms comprise nine closely related species known as genomovars. B. cenocepacia (genomovar III) is the most prevalent and appears the most virulent. We investigated the biological activity of a reference panel of strains using whole-cell lysates to induce septic-shock related cytokines from differentiated human monocytic cells. We found varying biological activity within and between genomovars, with B. cenocepacia strains possessing the greatest cytokine induction activity. This activity was CD-14 dependent, suggesting that LPS was responsible for the cytokine induction. Cytokine induction was not simply related to the expression of rough or smooth LPS. We purified LPS from two strains, B. cenocepacia LMG 12614 and B. multivorans LMG 14273, each possessing rough LPS. Divergence in biological activity of the two genomovars was preserved when human monocytic cells were stimulated with purified LPS. Lipid A purified from LMG 14273 and LMG 12614 were analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry. Lipid A from the less effective cytokine inducer LMG 14273 was found to be missing a ß-hydroxymyristate (3-OH C14:0) relative to the lipid A of B. cenocepacia LMG 12614.

Key Words: Burkholderia cepacia • LPS • cystic fibrosis • lipid A

Cystic Fibrosis (CF) is the most common lethal single gene disorder in which the majority of patients die of respiratory failure due to pulmonary sepsis. Bilateral lung transplantation has emerged as a viable clinical therapeutic option for selected patients with advanced CF lung disease (1). Burkholderia cepacia, a Gram-negative bacterium, is isolated in sputum of patients with CF with some series reporting infection rates of up to 20% (2). The organism is transmissible between patients, demonstrates multi- and often panresistance to antibiotics, and is associated with a rapid decline in lung function in colonized patients (3). Infection with B. cepacia is distinguished from infection with other major CF pathogens, such as Pseudomonas aeruginosa, by an increase in morbidity among patients with only mild-to-moderate lung disease (4). A fulminant pneumonitis with septicemia that is known as "cepacia syndrome" is the most serious outcome of cepacia infection, although a variable clinical course in those infected with this organism has been noted (5). Importantly, this variable outcome extends to patients infected with the same virulent strain, including the lineage known as ET-12 (6, 7). The ET-12 strain has been responsible for a transatlantic outbreak associated with increased mortality and morbidity.

Taxonomic techniques have revealed that cepacia organisms are a heterogeneous group of at least nine distinct genomovars collectively called the B. cepacia complex (BCC) (8–10). Most of the clinical data available relate to the first five genomovars identified; BCC genomovar II has been renamed B. multivorans, genomovar III was recently renamed B. cenocepacia (3, 12), genomovar IV is known as B. stabilis, and genomovar V as B. vietnamiensis; genomovar I will be known as B. cepacia once genomovar designation is complete. The distribution of putative virulence factors between genomovars is still emerging. Clinically, the most common BCC genomovar isolated is B. cenocepacia (11), which is associated with a more accelerated decline in pulmonary function compared with other genomovars; the ET-12 strain is a B. cenocepacia organism (13, 14). Notably, in some European CF clinics, B. multivorans is the most common species isolated.

We and others have demonstrated a clear association between sepsis and early death after pulmonary transplantation in patients with pretransplant B. cenocepacia infection (15, 16). Pretransplant infection with B. multivorans or B. vietnamiensis organisms was associated with good postoperative survival (15).

There are many bacterial virulence factors that may contribute to clinical outcomes, including bacterial hemolysins (17), proteases (18), and siderophores (19). In addition, BCC organisms are proinflammatory; LPS from this group of organisms has been shown to elicit a ninefold higher release of cytokines from leukocytes compared with that of P. aeruginosa (20). The lipid A component of LPS appears to be the main determinant of proinflammatory activity (21), with the secondary acylation status of lipid A recognized as increasingly important (22, 23). We are unaware of comparative studies of the structure of lipid A between B. cepacia genomovars. Furthermore, little data is available regarding the biological activity of the LPS of BCC organisms categorized by genomovar.

It was hypothesized that LPS from different BCC genomovars may elicit different inflammatory responses in an in vitro system. We further hypothesized that there may be differences in the lipid A component of LPS between species. This variability could be a contributor to the differences noted in vivo for BCC virulence and outcomes. Data presented here have previously been published in abstract form (24).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
BCC Whole Cell Lysates
The reference panel of BCC (genomovars I–V) (14) and additional strains LMG 12614 (B. cenocepacia, ET-12 clone), LMG 14273 (B. multivorans), and LMG16230 (B. vietnamiensis, genomovar V) were obtained (BCCM, Leuven, Belgium) and subcultured onto horse blood agar. Overnight bacterial lawns were harvested into phosphate-buffered saline (PBS) standardized to 0.2 at an optical density of 600 nm. After sonication (amplitude 15 µm for 6 cycles of 30 seconds on/off) on ice, using a Soniprep 150 (MSE, Loughborough, UK), aliquots were subjected to proteinase K digestion (2 mg/ml final concentration) for 2 hours at 60°C, boiled for 20 minutes, and lysates (enzyme-treated or untreated) were frozen prior to use. Additional detail is provided in the online supplement.

Tissue Culture and Cell Stimulation
Myelomonocytic cells (line U937) were prepared as previously described (23). U937 cells were induced to terminal monocytic differentiation with phorbol myristate acetate and stimulated with B. cepacia whole-cell lysates (100 µl of serial dilutions made up in CRPMI), E. coli 055 LPS (Sigma, UK) (10–100 ng/ml) or CRPMI as a negative control. In other experiments, various concentrations of purified BCC LPS were used. Monocytic cell supernatants were separated and collected at time zero (T0) and after 24 hours of incubation (T24); further time points were used in certain experiments.

Experiments to determine the effect of polymixin B treatment on whole-cell lysates prior to use in the tissue culture system were undertaken. Similar experiments were performed with U937 cells (resuspended to a lower density [1 x 105 cells/ml]) preincubated for 30 minutes with anti-CD14 antibody (clone MEM18) at a final concentration of 100 ng/ml (Monosan, Uden, The Netherlands).

Detection of Cytokine Production
ELISA was performed on supernatants of stimulated cells to detect cytokine production. Assays for tumour necrosis factor-{alpha} (TNF-{alpha}; Pharminogen, Oxford, or R&D, Abingdon, UK), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were performed using paired antibodies (R&D, Abingdon, UK) (23).

Analysis of LPS by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
The chemotype of LPS in whole-cell lysates was assessed using 12–16% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining (23, 25).

Lipopolysaccharide Purification
Standard water/phenol extraction was undertaken (23). Extracted LPS was reconstituted and protein contamination assessed (BCA Protein Kit; Perbio Science, Tattenhall, UK). DNA contamination was assessed by absorption ratios at E280/260 as described (23). Repeat DNase and proteinase-K digestion followed by ultracentrifugation were conducted until LPS with less than 5% contamination was obtained.

Isolation of Lipid A
One-liter cultures in nutrient broth containing 0.5% yeast extract (DIFCO, Oxford, UK) grown at 37°C to late log phase (A600 of 1.2) were collected by centrifugation at 1000 x g and resuspended in 40 ml PBS, pH 7.4. Lipid A was extracted (26–28) and multiple species of lipid A were confirmed with thin layer chromatography, as previously described (26).

Mass Spectroscopy of Lipid A Samples
Matrix-assisted laser desorption ionization/time of flight (MALDI/TOF) mass spectrometry analysis was performed using a Kompact MALDI 4 (Kratos Analytical, Manchester, UK) equipped with a nitrogen laser (337 nm) at 20 kV extraction voltage using time-delayed extraction. The samples were prepared for MALDI/TOF, as previously described (26). Spectra were acquired in both the positive and negative ion linear modes with an average of 50 laser hits for each spectrum.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytokine Induction by BCC Whole Cell Lysates
The B. cepacia lysates induced cytokine production in U937 cells, which was consistently demonstrated in repeated experiments using different aliquots of each lysate and/or various lysates prepared on different occasions. The strains displayed various levels of biological activity, as detected in terms of induction of TNF-{alpha}, IL-1ß, and IL-6. Cytokine induction was dose dependent and both proteinase K–treated and untreated lysates caused induction (data not shown). Lysates of most B. cenocepacia strains (8 of 11) induced production of TNF-{alpha} at or above the mean for all strains tested (Figure 1) , with B. cenocepacia strain LMG 12614 inducing the greatest quantity of TNF-{alpha}. The number of above-average TNF-{alpha}–inducing strains was significantly higher in the B. cenocepacia group than in the non–B. cenocepacia group of strains (Fisher's Exact Test, p < 0.01). Three B. cenocepacia strains induced only moderate levels of TNF-{alpha} defined arbitrarily as 50% or less than the average for all strains tested. The B. cenocepacia group average TNF-{alpha} induction was, however, statistically higher than that of the non-B. cenocepacia strains (mean 1.55 ng [range, 0.2–0.3 ng] vs. 0.61 ng [range, 0.4–2.5 ng]; t test, p < 0.05 ). In contrast, lysates of genomovar I, B. multivorans, B. stabiliz, and B. vietnamiensis induced less production of TNF{alpha}. An exception was B. multivorans strain LMG 13010, which was a potent TNF-{alpha} inducer. Lysates across the genomovar designations were generally of similar potency in IL-1ß induction, with no statistical difference between the B. cenocepacia group and the non–B. cenocepacia strains (Figure 2) (mean 1.6 ng [range, 0.38–3.00 ng] vs. 1.4 ng [range, 0.50–2.68 ng]; t test, p > 0.05).



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  		Figure 1. Tumor necrosis factor-{alpha} (TNF-{alpha}) induction elicited by Burkholderia cepacia complex (BCC) whole-cell lysates in U937 monocytic cells at 24 hours post stimulation. Bars represent the mean ± SD of TNF-{alpha} (ng). NS = nonstimulated; EC100 = positive control, purified E. coli O55 LPS; checkerboard bars = genomovar I strains; horizontal-striped bars = B. multivorans strains; solid bars = B. cenocepacia strains; grid-filled bars = B. stabiliz strains, and oblique-filled bars = B. vietnamiensis strains. The dashed horizontal line represents average TNF-{alpha} induction. Significantly more B. cenocepacia strains elicited above average TNF{alpha} induction compared with all other genomovars (p < 0.01).

 


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  		Figure 2. Interleukin (IL)-1ß induction elicited by BCC whole-cell lysates from U937 monocytic cells at 24 hours after stimulation. Bars represent the mean ± SD of IL-1ß (ng). Checkerboard bars = genomovar I strains; horizontal-striped bars = B. multivorans strains; solid bars = B. cenocepacia strains; grid-filled bars = B. stabiliz strains, oblique-filled bars = B. vietnamiensis strains. The dashed horizontal line represents average IL-1ß induction. There was no significant difference between genomovar groupings (p > 0.05).

 
The main biological activity of the lysates (both untreated and proteinase K–treated) was inhibited by preincubation of the lysates with polymixin B (not shown) or pretreatment of the monocytic cells with anti-CD14 antibodies (Figure 3) , strongly suggesting LPS as the main determinant of cytokine induction. The TNF-{alpha} induction values in Figure 3 are less than those in Figure 1, reflecting the lower U937 cell densities used in the inhibition studies.



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  		Figure 3. Inhibition of biological activity of whole-cell lysates by anti-CD 14 antibodies. U937 cells (1 x 105/ml) were stimulated with 1:200 dilutions of prepared whole-cell lysates of B. cepacia genomovar I strain LMG 17997, B. multivorans strain LMG 13010 and B. cenocepacia ET-12 clone strain LMG 16656 (solid bars), or with lysates preincubated with anti-CD 14 antibodies (open bars). *p < 0.05. The TNF-{alpha} induction values depicted here are less than those seen in Figure 1, reflecting the lower U937 cell densities used in the inhibition studies.

 
Analysis by SDS-PAGE of BCC LPS
SDS-PAGE analysis performed on proteinase K–treated lysates showed that B. cepacia genomovar I strains were predominantly smooth LPS chemotype strains; a notable exception was LMG 17997, a non-CF strain with a partial rough chemotype (Table 1) . B. multivorans strains were mostly smooth chemotype (the exceptions being rough LPS strains LMG 13010, a potent cytokine inducer, and LMG 14273, a less effective cytokine inducer). B. cenocepacia strains were rough, partial rough, or smooth chemotype. There were differences in the LPS chemotype of the various ET-12 clones tested; LMG 12614 and LMG 16656 (J2315) were of rough LPS, whereas LMG 18863 (K562) and LMG 18826 (BC7) were of smooth chemotype. B. stabilis and B. vietnamiensis strains had a smooth chemotype.


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  		TABLE 1. Lps chemotypes of selected strains from the burkholderia cepacia complex reference panel

 
Analysis by SDS-PAGE and Biological Activity of Purified LPS
Two rough LPS strains, LMG 12614 (B. cenocepacia–ET12) and LMG 14273 (B. multivorans), with clear biological diversity in TNF-{alpha} induction were chosen for LPS purification. These strains were chosen, in part, because of favorable antibiotic susceptibilities that may allow future genetic manipulation. A yield of 60–90 mg of purified LPS was obtained from 35 g of freeze-dried bacteria. Analysis by SDS-PAGE confirmed that the LPS of both strains were of a rough chemotype but showed different migration characteristics, suggesting compositional and/or structural differences (Figure 4) .



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  		Figure 4. Structural differences in purified LPS in a 16% silver–stained sodium dodecyl sulfate polyacrylamide gel. Lane 1, purified Neisseria gonorrhoea LPS, molecular weight 4.2 kD (23); lanes 2–5, purified B. cenocepacia strain LMG 12614 ET-12 clone LPS, 10, 5, 2.5 and 1.25 µg/lane, respectively; and lanes 6–9, purified B. multivorans strain LMG 14273 LPS, at 10, 5, 2.5 and 1.25 µg/lane, respectively.

 
Using the purified LPS in the U937 culture system, we found that the B. cenocepacia LMG 12614, ET-12 LPS was twice as potent at inducing TNF-{alpha} as was LMG 14273 LPS at almost every time point studied (Figure 5) , confirming the results with whole-cell lysates that showed different cytokine inducing capacities for these strains. An additional difference in cytokine induction patterns was an increase in IL-6 induction beyond 48 hours for B. cenocepacia strain LMG 12614 LPS, whereas the IL-6 induction elicited by B. multivorans strain LMG 14273 LPS diminished after the 48 hour time point (mean of two experiments) (Figure 5).



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  		Figure 5. Inflammatory cytokine induction of TNF-{alpha} (upper panel) and interleukin-6 (IL-6) (lower panel) in U937 cells elicited by purified LPS (100 ng/ml) from selected strains as detected by ELISA compared with standards of appropriate recombinant cytokine. B. multivorans strain LMG 14,273 (open bars); B. cenocepacia strain LMG 12614 (solid bars); and E. coli O55 (cross-filled bars). Results shown are mean values of two separate experiments, ± SD.*Significantly lower TNF-{alpha} or IL-6 induction in B. multivorans when compared with B. cenocepacia (p < 0.05, t test).

 
Lipid A Mass Spectrometry
BCC lipid A derived from B. cenocepacia strain LMG 12614 and B. multivorans strain LMG 14273 were a mixture of several species, as revealed by thin layer chromatography of their crude lipid A obtained after hydrolysis of phospholipid-depleted cell pellets (not shown). B. multivorans and B. cenocepacia had lipid A species that migrated similarly on thin layer chromatographs. However, B. multivorans strain LMG 14273, the less effective inducer, contained additional lipid A species with a slightly retarded migration on the plate (not shown). The migration of these new species in the B. multivorans strain suggested that they may be slightly more hydrophobic species of the lipid A species shared by both strains.

Fractionation of the crude lipid A by diethylaminoethyl-cellulose column chromatography suggested a mixture of species that may be structurally related. Based on their order of elution from the column, these lipids were called Bc-0, Bc-60, and Bc-120. Bc-0 eluted first from the column with the solvent mixture CHCl3, MeOH, dH2O (2:3:1, vol/vol), followed closely by Bc-60, which eluted with the solvent mixture CHCl3, MeOH, 60 mM NH4Ac (2:3:1, vol/vol). Bc-120 required a higher salt concentration and eluted with the solvent mixture CHCl3, MeOH, 120 mM NH4Ac (2:3:1, vol/vol).

MALDI-TOF mass spectrometry provided consistent proof of the structural relationship between these lipid A species. The mass spectra also suggested that BCC lipid A had similarities to that of P. aeruginosa lipid A (29). The lipid A had a disaccharide backbone and was acylated at the 2, 3, 2', and 3' positions by 3-OH fatty acids (Figure 6) . Two acyloxyacyl moieties were associated with the lipid A, and one of these secondary fatty acids may be hydroxylated at C-2. The mass spectra showed that B. cepacia lipid A was a mixture of several species, which was consistent with results of thin layer chromatography. The lipid A species included species containing two aminoarabinose moieties, some of which were deacylated.



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  		Figure 6. Negative ion mode matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis of extracted lipid A from B. cenocepacia strain LMG 12614 (lower panel) and B. multivorans strain LMG 14273 (upper panel). The species at mass/charge ratio (m/z) 1708.4 marked with an asterisk is deacylated, whereas the lipid A species with a m/z of 1803.4 is not. %int = percentage intensity; GII = genomovar II strain/B. multivorans; GIII = genomovar III strain/B. cenocepacia.

 
The spectra of B. cenocepacia strain LMG 12614 and B. multivorans strain LMG 14273 lipid A displayed two identical peaks (Figure 6). These peaks (mass/charge ratio [m/z], 1,935.1 and 1,803.8, respectively) differed by 131.3 amu, which suggested that the peak with an m/z of 1,803.8 had lost an aminoarabinose moiety. In the B. cenocepacia strain, another peak with an m/z of 1,672.9, which was 131 amu less than the peak with the m/z of 1,803.8, was noted. This was consistent with the lipid A species with the m/z of 1,672.9 that was devoid of aminoarabinose. The lipid A species with the m/z of 1,803.8 had one aminoarabinose (at either the 1 or 4' position), whereas that with the m/z of 1,935.1 had two aminoarabinose moieties, possibly appended to the phosphates at the two ends of the disaccharide. The lipid A mass spectrum for B. multivorans strain LMG 13010, a potent cytokine inducer, was the same as that obtained for B. cenocepacia strain LMG 12614 (not shown).

B. multivorans strain LMG 14273, the less effective cytokine inducer, contained, in addition to a lipid A species containing one aminoarabinose (m/z 1,803.4) and two aminoarabinose moieties (m/z 1,935.1), a lipid A species with a mass/charge of 1,708.4. This species corresponded to the loss of a hydroxylated myristic acid chain (3-OH C14:O). Similarly, a deacylated species that had lost an aminoarabinose moiety was suggested by the presence of a peak with an m/z of 1,578.4.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have compared the proinflammatory capacity of a number of BCC strains using whole-cell lysates to stimulate differentiated U937 cells. We used lysates instead of purified LPS because only minor differences in cytokine induction have been noted when comparing purified BCC LPS and whole organisms (30). Work in our laboratory has shown that proteinase K–treated whole-cell lysates from Gram-negative bacteria can elicit a comparable cytokine response from U937 monocytic cells to that elicited by purified LPS (10). We therefore, obtained crude whole-cell lysates from a panel of BCC isolates, including the strains of the international BCC reference panel (14). We also included additional strains such as ET-12 clone LMG 12614, in view of the association between ET-12 and cepacia syndrome (6, 14).

We found that there was a dose-dependent TNF-{alpha} induction elicited by whole-cell lysates from human monocytic cells. Most B. cenocepacia strains consistently elicited high levels of proinflammatory cytokine production, particularly for TNF-{alpha}, although certain genomovar III strains, including some ET-12 clones, such as K56-2 (also designated as LMG 18863), were less effective cytokine inducers using this system, eliciting 50% less TNF-{alpha} than the average. Whereas there is a temptation to assume all ET-12 clones behave similarly in experimental models, our data suggest that great care must be taken when choosing strains for such models. There was variable cytokine induction capacity by the ET-12 clones tested; LMG 12614 and LMG 16656 (J2315) were among the most potent cytokine inducers of all strains tested, whereas LMG 18863 (K562) and LMG 18826 (BC7) elicited at least twofold less TNF-{alpha}. These differences in biological activity are important, as many research groups use one or two of the strains as representative of the whole ET-12 group. Notably, one B. cenocepacia strain LMG 16654, which was associated with the first UK report of cepacia syndrome but not with transmissibility, was a potent TNF-{alpha} inducer (31), lending biological plausibility to the role of LPS in clinical outcomes. In contrast, strains from other genomovars, including B. multivorans strains, caused the induction of less TNF-{alpha} than average with the exception of the B. multivorans strain LMG 13010.

There was no clear correlation between the pattern of cytokine induction and the original source of the strain (e.g., environmental vs. clinical origin). Of the genomovar I strains tested, LMG 17997, a non-CF origin strain (isolated from a urinary tract infection), was the only cytokine inducer of note. Strong cytokine induction was not seen for any other non-CF strains, including B. cenocepacia strain LMG 18832 (urinary tract), B. multivorans strains LMG 17588 (soil), LMG 18823 (laboratory strain), and B. vietnamiensis LMG 18835 (soil) and LMG 10929 (rice). However, the reference panel does not include large numbers of environmental strains, and the role of using large quantities of Burkholderia cepacia complex organisms in bioremediation and its possible effect on human health remains controversial (32).

Observations in the pathogenesis of shock have suggested that TNF-{alpha} is a pivotal cytokine in septic shock (33). In our experimental system, organisms from the genomovar classes that were associated with a benign posttransplant course, namely B. multivorans and B. vietnamiensis, elicited, in general, only a modest TNF-{alpha} response. We therefore investigated LPS chemotypes, and our findings, showing wide distribution of rough and smooth chemotypes within genomovars, are similar to those published (34). There was a general trend toward higher cytokine induction in rough strains that could be explained by the higher proportion of lipid A in the lysates of these strains compared with those of smooth LPS strains. Importantly, however, our data demonstrates that intensity of the proinflammatory activity could not be attributed simply to LPS chemotypes, as B. multivorans strains that had similar cytokine induction profiles had either rough or smooth LPS.

The differences in LPS chemotype and cytokine induction amongst ET-12 clones may offer insights into the variability in clinical outcomes associated with this strain. However, the observed differences in cytokine induction activity could simply be a reflection of ex vivo factors, such as different numbers of passages between ET-12 clones before each strain was entered into the reference repository, which could account for the loss or variation of other factors not yet defined. An alternative explanation may be differing host characteristics, including variability in CF genotype and potential CF modifier genes' defensin levels, which may be important in the specific host–pathogen interactions between Pseudomonas LPS in patients with CF (29, 35). Furthermore, variability in outcomes in patients infected with the same BCC strain may reflect the presence or absence of copathogens. It has, for example, been demonstrated that B. cepacia LPS can prime neutrophil respiratory burst activity to P. aeruginosa and other neutrophil activating moieties during coinfection (36, 37).

Using purified LPS from two rough strains of CF origin, B. cenocepacia strain LMG 12614 (ET-12) and B. multivorans strain LMG 14273, we were able to demonstrate differences in both potency and pattern of cytokine induction for TNF-{alpha} and IL-6. Notably, the LPSs were of very similar molecular mass, suggesting that the lipid A content would be approximately similar. Greater cytokine induction by ET-12 LPS could reflect signaling via additional or atypical LPS receptors, such as Toll-like receptor 2 (TLR2) (38). The biological activity of both LPS moieties was, however, inhibited by anti-CD14 antibodies, suggesting that these differences could be explained by differences in ligand–receptor affinity rather than signaling through additional LPS receptors.

The most biologically active component of LPS is lipid A, and we present the first report of the composition of lipid A from BCC genomovars. All BCC strains studied included lipid A species that contained two aminoarabinose moieties. This observation correlates with the findings of others, in which P. aeruginosa from patients with CF synthesized LPS containing aminoarabinose and a variety of penta- and hexaacylated lipid A structures, which were associated with resistance to cationic antimicrobial peptides (29). These changes may account for the observation that BCC organisms are resistant to the bactericidal effects of human airway defensins and antibiotic peptides, such as polymyxin and colistin (39–41).

Our findings demonstrating that LPS of the weak TNF inducer, B. multivorans strain LMG 14273, has a deacylated lipid A species lacking an acyl chain at the primary acyl chain position is significant as lipid A deacylation is associated with loss of proinflammatory activity. The presence of deacylated species in B. multivorans shows similarities with CF-associated P. aeruginosa in which deacylation of lipid A has been reported (29). This may be due to the presence of a bacterial lipid A deacylase. Enzyme activity of this type has been described in Rhizobium (42), and a lipid A deacylase capable of removing the acyl chain at position 3 of lipid A encoded by the pagL gene has been described in Salmonella (43). It has been suggested that such deacylase activity may protect the bacteria from immunologic responses (43). No pagL homologs have, as yet, been demonstrated in other bacteria, raising speculation that there may be species–specific lipid A deacylases (43). A basic locus alignment search tool (BLAST) search of the B. cenocepacia sequencing project (www.sanger.ac.uk) failed to reveal any homologous deacylase enzyme in B. cenocepacia. The possible biological advantage or disadvantage in a clinical situation to strains, such as the ET-12 clone, that do not possess a deacylated lipid A remains to be elucidated. Whereas prior data demonstrate that secondary acylation of lipid A is important in determining cytokine induction capacity (21–23), our data suggest that the primary acylation status of lipid A of CF pathogens' LPS on the proinflammatory activity may also be important (29).

The presence of a similar lipid A in the high inducer B. multivorans strain LMG 13010 and in strain LMG 12614 demonstrates that the lipid A motifs may not be genomovar-specific. The shared lipid A species across genomovar groups confirms the lack of genomovar specificity in terms of B. cepacia LPS that has previously been shown for other LPS-associated properties, including serology and phage susceptibility (44–46).

Collectively, the data presented on the variable potential to elicit septic shock, promoting cytokines between and within the B. cepacia genomovars, and on the correlation between intensity of cytokine response and lipid A acylation in specific strains, may provide insight into the differences in transplant outcomes dependent on the genomovar status and lipid A species of the colonizing strains.


   Acknowledgments
 
The authors thank Dr. A. McDowell, Queens University, Belfast, and Dr. J. Perry, Freeman Hospital, for technical assistance; Dr. N. Que-Gewirth and Prof. C. R. Raetz, Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, and Dr. S. Ramirez-Kalb and Prof. R. J. Cotter, Middle Atlantic Mass Spectrometry Laboratory, School of Pharmacology and Molecular Science, John Hopkins University School of Medicine, Baltimore, Maryland, for assistance in lipid A analysis.


   FOOTNOTES
 
Supported by the Wellcome Trust, UK, and Breathe North, UK (A.D.S.).

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: A. D-S. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; C.D.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; C.M.A.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; P.A.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.D. D-H. does not have a financial relationship with a commercial entity that has an interest in the subject of this article.

Received in original form April 30, 2003; accepted in final form March 19, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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