Burkholderia cenocepacia ET12 strain activates TNFR1 signalling in cystic fibrosis airway epithelial cells.

Burkholderia cenocepacia is an important pulmonary pathogen in individuals with cystic fibrosis (CF). Infection is often associated with severe pulmonary inflammation, and some patients develop a fatal necrotizing pneumonia and sepsis ('cepacia syndrome'). The mechanisms by which this species causes severe pulmonary inflammation are poorly understood. Here, we demonstrate that B. cenocepacia BC7, a potentially virulent representative of the epidemic ET12 lineage, binds to tumour necrosis factor receptor 1 (TNFR1) and activates TNFR1-related signalling pathway similar to TNF-alpha, a natural ligand for TNFR1. This interaction participates in stimulating a robust IL-8 production from CF airway epithelial cells. In contrast, BC45, a less virulent ET12 representative, and ATCC 25416, an environmental B. cepacia strain, do not bind to TNFR1 and stimulate only minimal IL-8 production from CF cells. Further, TNFR1 expression is increased in CF airway epithelial cells compared with non-CF cells. We also show that B. cenocepacia ET12 strain colocaizes with TNFR1 in vitro and in the lungs of CF patients who died due to infection with B. cenocepacia, ET12 strain. Together, these results suggest that interaction of B. cenocepacia, ET12 strain with TNFR1 may contribute to robust inflammatory responses elicited by this organism.

Cable pili and the 22-kilodalton adhesin are required for Burkholderia cenocepacia binding to and transmigration across the squamous epithelium.

Burkholderia cenocepacia strains expressing both cable (Cbl) pili and the 22-kDa adhesin bind to cytokeratin 13 (CK13) strongly and invade squamous epithelium efficiently. It has not been established, however, whether the gene encoding the adhesin is located in the cbl operon or what specific contribution the adhesin and Cbl pili lend to binding and transmigration or invasion capacity of B. cenocepacia. By immunoscreening an expression library of B. cenocepacia isolate BC7, we identified a large gene (adhA) that encodes the 22-kDa adhesin. Isogenic mutants lacking expression of either Cbl pili (cblA or cblS mutants) or the adhesin (adhA mutant) were constructed to assess the individual role of Cbl pili and the adhesin in mediating B. cenocepacia binding to and transmigration across squamous epithelium. Relative to the parent strain, mutants of Cbl pili showed reduced binding (50%) to isolated CK13, while the adhesin mutant showed almost no binding (0 to 8%). Mutants lacking either cable pili or the adhesin were compromised in their ability to bind to and transmigrate across the squamous epithelium compared to the wild-type strain, although this deficiency was most pronounced in the adhA mutant. These results indicate that both Cbl pili and the 22-kDa adhesin are necessary for the optimal binding to CK13 and transmigration properties of B. cenocepacia.

A novel model to study bacterial adherence to the transplanted airway: inhibition of Burkholderia cepacia adherence to human airway by dextran and xylitol.

BACKGROUND: Lung infection with Burkholderia cepacia complex before lung transplantation in patients with cystic fibrosis is a major risk factor for decreased post-operative survival rates compared with those of patients colonized with the more common opportunistic pathogen Pseudomonas aeruginosa. Because adherence to mucosal surfaces is an important initial step in infection, we investigated the use of non-toxic neutral polysaccharides and a sugar alcohol to prevent adherence of B cepacia complex to allograft airway epithelium. METHODS: We used human airway explants prepared from donor tracheobronchial tissue to test the effect of dextrans and xylitol in inhibiting the binding of Burkholderia cepacia complex. We used immunofluorescence and electron microscopy to determine the distribution of bacteria in the explants. RESULTS: Burkholderia cepacia complex bound to the explants and was found only in the surface mucus layer. Dextran 40 kd applied before adding the bacteria decreased the number of bound organisms by 80% to 99%. Smaller molecular mass dextrans (4 and 20 kd) were ineffective. Xylitol inhibited bacterial binding by 67% to 85%. Both agents seemed to decrease the thickness of the surface mucus, suggesting that they may indirectly inhibit bacterial binding by removing adherent surface mucus. CONCLUSIONS: Treating donor lungs with dextran 40 kd or xylitol before (and possibly after) surgery may inhibit the adherence of Burkholderia cepacia complex to airways and may prevent or decrease subsequent infection of the allografts.

Responses of well-differentiated airway epithelial cell cultures from healthy donors and patients with cystic fibrosis to Burkholderia cenocepacia infection.

Well-differentiated cultures established from airway epithelia of patients with cystic fibrosis (CF cultures) exhibited goblet cell hyperplasia, increased secretion of mucus, and higher basal levels of interleukin-8 than similarly cultured cells from healthy donors. Upon apical infection with low doses (10(4) to 10(5) CFU) of Burkholderia cenocepacia isolate BC7, the two cultures gave different responses. While normal cultures trapped the added bacteria in the mucus layer, killed and/or inhibited bacterial replication, and prevented bacterial invasion of the cells, CF cultures failed to kill and/or supported the growth of bacteria, leading to invasion of underlying epithelial cells, compromised transepithelial permeability, and cell damage. Depletion of the surface mucus layer prior to bacterial infection rendered the normal cultures susceptible to bacterial invasion, but the invading bacteria were mainly confined to vacuoles within the cells and appeared to be nonviable. In contrast, bacteria that invaded cells in CF cultures were found free in the cytoplasm surrounded by intermediate filaments and also between cells. Cultured CF airway epithelium was therefore more susceptible to infection than normal epithelium. This mimics CF tissue in vivo and illustrates differences in the way epithelia in CF patients and normal subjects handle bacterial infection. In addition, we found that the CF and normal cell cultures responded differently not only to isolate BC7 but also to isolates of other B. cepacia complex species. We therefore conclude that this cell culture model is suitable for investigation of B. cepacia complex pathogenesis in CF patients.

Evidence for a second peptide cleavage in the C-terminal domain of rodent intestinal mucin Muc3.

Rat intestinal mucin Muc3 (rMuc3), like its human homologue (MUC3) and several other membrane mucins, contains a C-terminally located SEA (sea urchin sperm protein, enterokinase and agrin) module, with an intrinsic proteolytic site sequence G downward arrow SIVV (where G downward arrow S is the glycine serine cleavage site). As shown previously [Wang, Khatri and Forstner (2002) Biochem. J. 366, 623-631], expression of the C-terminal domain of rMuc3 in COS-1 cells yields a V5 epitope-tagged N-terminal glycopeptide of 30 kDa and a Myc- and His epitope-tagged C-terminal glycopeptide of 49 kDa. The present study shows that the 49 kDa membrane-anchored fragment undergoes a further cleavage reaction which decreases its size to 30 kDa. Western blotting, pulse-chase metabolic incubations, immunoprecipitation and deglycosylation with N-glycosidase F were used to detect and identify the proteolytic products. Both the first and second cleavages are presumed to facilitate solubilization of Muc3 at the apical surface of enterocytes and/or enhance the potential for Muc3 to participate in ligand-receptor and signal transduction events for enterocyte function in vivo.

Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia.

We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.

N-linked oligosaccharides play a role in disulphide-dependent dimerization of intestinal mucin Muc2.

Within the C-terminal domain of many secretory mucins is a 'cystine knot' (CK), which is needed for dimer formation in the endoplasmic reticulum. Previous studies indicate that in addition to an unpaired cysteine, the three intramolecular cystine bonds of the knot are important for stability of the dimers formed by rat intestinal mucin Muc2. The present study was undertaken to determine whether the two N-glycans N9 and N10, located near the first and second cysteines of the knot, also play a role in dimer formation. The C-terminal domain of rat Muc2 (RMC), a truncated RMC mutant containing the CK, and mutants lacking N9 and N10 sites, were expressed in COS-1 cells and the products monitored by radioactive [(35)S]Met/Cys metabolic pulse-chase and immunoprecipitation. Mutation of N9, but not N10, caused increased synthesis of dimers over a 2-h chase period. The N9 mutant remained associated with calreticulin for a prolonged period. About 34-38% of the total labelled products of RMC and its mutants was secreted into the media by 2 h, but the proportion in dimer form was dramatically reduced for the N9 mutant, suggesting lower dimer stability relative to RMC or its N10 mutant. We conclude that under normal conditions the presence of the N9 glycan functions to maintain a folding rate for mucin monomers that is sufficiently slow to allow structural maturation and stability of Muc2 dimers. To our knowledge this report is the first demonstration that a specific N-glycan plays a definitive role in mucin dimer formation.

Identification and molecular analysis of cable pilus biosynthesis genes in Burkholderia cepacia.

Burkholderia cepacia is an opportunistic respiratory pathogen in cystic fibrosis patients. One highly transmissible and virulent clone belonging to genomovar IIIa expresses pili with unique cable morphology, which enable the bacterium to bind cytokeratin 13 in epithelial cells. The cblA gene, encoding the major pilin subunit, is often used as a DNA marker to identify potentially virulent isolates. The authors have now cloned and sequenced four additional genes, cblB, cblC, cblD and cblS, in the pilus gene cluster. This work shows that the products of the first four genes of the cbl operon, cblA, cblB, cblC and cblD, are sufficient for pilus assembly on the bacterial surface. Deletion of cblB abrogated pilus assembly and compromised the stability of the CblA protein in the periplasm. In contrast, deletion of cblD resulted in no pili, but there was no effect on expression and stability of the CblA protein subunit. These results, together with protein sequence homologies, predicted structural analyses, and the presence of typical amino acid motifs, are consistent with the assignment of functional roles for CblB as a chaperone that stabilizes the major pilin subunit in the periplasm, and CblD as the initiator of pilus biogenesis. It is also shown that expression of Cbl pili in Escherichia coli is not sufficient to mediate the binding of bacteria to the epithelial cell receptor cytokeratin 13, and that B. cepacia still binds to cytokeratin 13 in the absence of Cbl pili, suggesting that additional bacterial components are required for effective binding.

Effect of chondroitinase ABC on purulent sputum from cystic fibrosis and other patients.

Cystic fibrosis (CF) patients develop chronic lung infections associated with airway obstruction by viscous and insoluble mucus secretions. Although mucus glycoproteins (mucins) are thought to be responsible for mucus plugs, other glycoconjugate components of airway secretions have not been systematically evaluated. The aim of the present study was to determine whether chondroitin sulfate proteoglycans (CSPG) contribute to the insolubility of CF sputum. Sputa obtained from 18 CF patients were incubated with chondroitinase ABC (ChABC) or buffer (control) for 18 h at 37 degrees C, and after centrifugation at 12,000 g, the volume of the insoluble pellet and turbidity of the supernatant were determined as measures of solubility. ChABC caused a 70-90% reduction in supernatant turbidity and a 60-70% decrease in pellet volume of the 13 purulent CF sputa, but had much less effect on the five nonpurulent CF sputa tested. Similar results were obtained with two non-CF purulent and two non-CF, nonpurulent sputa. Gel electrophoresis, Western blot, and slot blot immunoassays with antichondroitin sulfate and antimucin antibodies revealed that purulent sputa (CF and non-CF) contained more CSPG and less mucin than nonpurulent sputa. In vitro mixing experiments showed that mucin in nonpurulent sputa was reduced upon incubation with purulent sputa, presumably because of degradation or a loss of immunoreactive mucin epitopes from leukocyte and/or bacterial enzymes present in purulent sputa. Our results suggest that CSPG contribute more significantly than mucins to the insolubility of purulent tracheobronchial secretions from CF patients. Because purulent sputa from non-CF patients showed a similar pattern, our observations with CF sputa may have wider applicability.

SEA (sea-urchin sperm protein, enterokinase and agrin)-module cleavage, association of fragments and membrane targeting of rat intestinal mucin Muc3.

In a previous study we showed, by transient expression studies in COS-1 cells, that the C-terminal domain of rat intestinal membrane mucin Muc3 was cleaved between glycine and serine within a GSIVV (one-letter) amino acid sequence during its residence in the endoplasmic reticulum. The extracellular domain fragment remained linked to the membrane-associated fragment by non-covalent interactions. The present study demonstrates that cleavage depends not only on the presence of the G/SIVV site (where G/S is the glycine downward arrow serine cleavage site), but also on more distant C-terminal sequences in the SEA (sea-urchin sperm protein, enterokinase and agrin) module. Inhibition of N-glycosylation by tunicamycin treatment of transfected cells did not prevent re-association of fragments, although cleavage was partially impaired, as some of the non-glycosylated, non-cleaved products were seen to accumulate in cells. Membrane targeting of the Muc3 domain and its cleavage products occurred in transfected cells and was not impaired in mutants in which the cleavage site was mutated. Targeting was also not impaired for products devoid of N-linked oligosaccharides. Our studies thus indicate that (a) cleavage within the SEA module of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the cleavage site, (b) re-association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides, and (c) membrane targeting of the mucin is independent of the SEA-module-cleavage reaction.

Lack of cable pili expression by cblA-containing Burkholderia cepacia complex.

The Burkholderia cepacia complex consists of several closely related bacterial species (or genomovars) which although generally not pathogenic for healthy individuals, contribute significantly to morbidity and mortality among persons with cystic fibrosis (CF). Certain B. cepacia complex strains are more frequently recovered from CF sputum cultures than are others, and these typically reside in genomovar III. The ET12 clone is a genomovar III strain that predominates among CF patients in Canada and the United Kingdom and is characterized by distinctive cblA-encoded pili that have a cable-like morphology. In a previous survey of B. cepacia complex isolates recovered from 606 CF patients in the US, a single genomovar III ET12 isolate (isolate AU0007) was identified; several cblA-containing genomovar I isolates, however, were also detected. In the study reported here, analysis by PFGE revealed several distinct strain types among these genomovar I isolates, and sequence analysis of their cblA genes demonstrated 87.8-88.4% identity to the ET12 cblA sequence. Southern analysis indicated that the cblA variant from each genomovar I isolate resides on a 4 kbp EcoRI fragment, in contrast to ET12 isolates, in which cblA localizes to a 5 kbp EcoRI fragment. Western blot assay indicated expression of the 16 kDa major pilin subunit by ET12 isolates, including AU0007, but neither whole-cell nor surface-protein extracts of the genomovar I reacted. Electron microscopy revealed the complete absence of pili expression by the genomovar I isolates. In contrast to typical ET12 isolates, AU0007 appeared to be hyperpiliated with rigid pili that lacked the cable morphology and did not bind cytokeratin 13, which has been previously identified as the epithelial cell receptor for the ET12 cable-pili-associated adhesin.

C-terminal domain of rodent intestinal mucin Muc3 is proteolytically cleaved in the endoplasmic reticulum to generate extracellular and membrane components.

Although human MUC3 and rodent Muc3 are both membrane-associated intestinal mucins, the present study has explored the possibility that rodent Muc3 might exist in soluble as well as membrane forms. No evidence was obtained for the existence of soluble splice variants; however, experiments with heterologous cells transfected with cDNA encoding the 381-residue C-terminal domain of rodent Muc3 showed that a definitive proteolytic cleavage occurs during processing in the endoplasmic reticulum. The products consisted of a V5-tagged 30 kDa extracellular glycopeptide and a Myc-tagged 49 kDa membrane-associated glycopeptide. Throughout their cellular transport to the plasma membrane, the two fragments remained associated by non-covalent SDS-sensitive interactions. Site-specific mutagenesis pinpointed the need for glycine and serine residues in the cleavage sequence Leu-Ser-Lys-Gly-Ser-Ile-Val-Val, which is localized between the two epidermal-growth-factor-like motifs of the mucin. A similar cleavage sequence (Phe-Arg-Pro-Gly downward arrow Ser-Val-Val-Val, where downward arrow signifies the cleavage site) has been reported in human MUC1 and analogous sites are present in human MUC3, MUC12 and MUC17. Thus early proteolytic cleavage may be a conserved characteristic of many membrane-associated mucins, possibly as a prelude to later release of their large extracellular domains at cell surfaces.

Interaction of cblA/adhesin-positive Burkholderia cepacia with squamous epithelium.

A highly transmissible strain of Burkholderia cepacia from genomovar III carries the cable pilin gene, expresses the 22 kDa adhesin (cblA +ve/Adh +ve), binds to cytokeratin 13 (CK13) and is invasive. CK13 is expressed abundantly in the airway epithelia of cystic fibrosis (CF) patients. We have now investigated whether binding of cblA +ve/Adh +ve B. cepacia to CK13 potentiates bacterial invasion and epithelial damage using bronchial epithelial cell cultures differentiated into either squamous (CK13-enriched) or mucociliary (CK13-deficient) epithelia. Three different B. cepacia isolates (cblA +ve/Adh +ve, cblA +ve/Adh -ve and cblA -ve/Adh -ve) showed minimal binding to mucociliary cultures, and did not invade or cause cell damage. In contrast, the cblA +ve/Adh +ve isolate, but not others, bound to CK13-expressing cells in squamous cultures, caused cytotoxicity and stimulated IL-8 release within 2 h. By 24 h, this isolate invaded and migrated across the squamous culture, causing moderate to severe epithelial damage. A specific antiadhesin antibody, which blocked the initial binding of the cblA +ve/Adh +ve isolate to CK13, significantly inhibited all the pathologic effects. Transmission electron microscopy of squamous cultures incubated with the cblA +ve/Adh +ve isolate, revealed bacteria on the surface surrounded by filopodia by 2 h, and within the cells in membrane-bound vesicles by 24 h. Bacteria were also observed free in the cytoplasm, surrounded by intermediate filaments containing CK13. These findings suggest that binding of B. cepacia to CK13 is an important initial event and that it promotes bacterial invasion and epithelial damage.

Immunolocalisation of Burkholderia cepacia in the lungs of cystic fibrosis patients.

Infection by Burkholderia cepacia is sometimes fatal in patients with cystic fibrosis (CF), as the organism can cause necrotising pneumonia and septicaemia (the cepacia syndrome), and is resistant to antibiotics. To increase knowledge of the pathogenesis of lung infection, the present study investigated the distribution of B. cepacia in lung explants from nine CF recipients of double lung transplants, of which six were colonised with both B. cepacia and Pseudomonas aeruginosa and the other three with P. aeruginosa only. In one case, explants of the donor lung (allograft) became available after the patient succumbed post-operatively to the cepacia syndrome. Further autopsy sections were examined from two patients who had chronic and then acute infection with B. cepacia. A specific antibody reactive with all five genomovars of the B. cepacia complex and another antibody specific for the 22-kDa adhesin of cable pili, were used to localise bacteria in situ. In chronic infection, the organisms were diffusely distributed, but most concentrated in hyperplastic bronchiolar epithelium, inflamed peribronchial and perivascular areas, between adjacent airway epithelial cells and in pathologically thickened alveolar septae and luminal macrophages. In acute infections the distribution was more focal, with B. cepacia on injured airway surfaces and in sites of pneumonia and abscess formation. In autopsy sections from one of the patients with chronic, then acute infection, B. cepacia was also observed in the lumen of blood capillaries. These results suggest that B. cepacia has the capacity to be highly invasive, migrating from the airways across the epithelial barrier to invade the lung parenchyma and capillaries, thereby initiating septicaemia.

Preferential adherence of cable-piliated burkholderia cepacia to respiratory epithelia of CF knockout mice and human cystic fibrosis lung explants.

The Burkholderia cepacia complex consists of at least five well-documented bacterial genomovars, each of which has been isolated from the sputum of different patients with cystic fibrosis (CF). Although the world-wide prevalence of this opportunist pathogen in CF patients is low (1-3%), 'epidemic' clusters occur in geographically isolated regions. Prevalence in some of these clusters is as high as 30-40%. The majority of CF B. cepacia isolates belong to genomovar III, but the relationship between genomovar and virulence has not yet been defined. Because the initial stage of infection involves bacterial binding to host tissues, the present study investigated differences in the binding of representative isolates of all five genomovars to fixed nasal sections of UNC cftr (-/-) and (+/+) mice and to human lung explants, biopsy and autopsy tissue of CF and non-CF patients. Binding was highest for isolates of genomovar III, subgroup RAPD type 2, but only if the isolates expressed the cable pili phenotype. Antibodies to the 22-kDa adhesin of cable pili virtually abolished binding. Binding occurred only to cftr (-/-) nasal sections or to CF lung sections and was negligible in cftr (+/+) or human non-CF, histologically normal lung sections. Unlike normal epithelia, the hyperplastic epithelia of CF bronchioles were enriched in cytokeratin 13, a 55-kDa protein that has previously been shown to act as a receptor in vitro for cable-piliated B. cepacia. These findings may help to explain the high transmissibility ofCbl-positive, genomovar III strains of B. cepacia among CF patients.