The CepIR quorum-sensing system contributes to the virulence of Burkholderia cenocepacia respiratory infections.

The cepIR genes encode an N-acyl homoserine lactone (AHL)-dependent quorum-sensing system consisting of an AHL synthase that directs the synthesis of N-octanoyl-L-homoserine lactone (ohl) and n-hexanoyl-L-homoserine lactone and a transcriptional regulator. The virulence of cepIR mutants was examined in two animal models. Rats were infected with agar beads containing Burkholderia cenocepacia K56-2, K56-I2 (cepI : : Tp(r)) or K56-R2 (cepR : : Tn5-OT182). At 10 days post-infection, the extent of lung histopathological changes was significantly lower in lungs infected with K56-I2 or K56-R2 compared to the parent strain. Intranasal infections were performed in Cftr((-/-)) mice and their wild-type siblings. K56-2 was more virulent in both groups of mice. K56-I2 was the least virulent strain and was not invasive in the Cftr((-/-)) mice. OHL was readily detected in lung homogenates from Cftr((-/-)) mice infected with K56-2 but was only detected at levels slightly above background in a few mice infected with K56-I2. Lung homogenates from mice infected with K56-2 had significantly higher levels of the inflammatory mediators murine macrophage inflammatory protein-2, KC/N51, interleukin-1beta and interleukin-6 than those from K56-I2-infected animals. These studies indicate that a functional CepIR quorum-sensing system contributes to the severity of B. cenocepacia infections. A zinc metalloprotease gene (zmpA) was shown to be regulated by CepR and may be one of the factors that accounts for the difference in virulence between the cepI mutant and the parent strain.

P-113D, an antimicrobial peptide active against Pseudomonas aeruginosa, retains activity in the presence of sputum from cystic fibrosis patients.

Antimicrobial peptides are a source of novel agents that could be useful for treatment of the chronic lung infections that afflict cystic fibrosis (CF) patients. Efficacy depends on antimicrobial activity against the major pathogens of CF patients, Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in the environment of the CF patient's airway. We describe the in vitro efficacies of derivatives of histatins, which are histidine-rich peptides produced by the salivary glands of humans and higher primates. P-113, a peptide containing 12 of the 24 amino acid residues of the parent molecule, histatin 5, retained full antibacterial activity and had a good spectrum of activity in vitro against the prominent pathogens of CF patients. However, P-113 was not active in the presence of purulent sputum from CF patients. In contrast, P-113D, the mirror-image peptide with the amino acid residues in the D configuration, was stable in sputum, was as active as P-113 against pathogens of CF patients in the absence of sputum and retained significant activity in the presence of sputum from CF patients. Recombinant human DNase, which effectively liquefies sputum, enhanced the activity of P-113D in undiluted sputum against both exogenous (added) bacteria and endogenous bacteria. Because of its properties, P-113D shows potential as an inhalant in chronic suppressive therapy for CF patients.

Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(-/-) mice.

Progressive pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the molecular basis for this susceptibility remains incompletely understood. To study this problem, we developed a model of chronic pneumonia by repeated instillation of a clinical isolate of Burkholderia cepacia (genomovar III, ET12 strain), an opportunistic gram-negative bacterium, from a case of CF into the lungs of Cftr (m1unc-/-) (Cftr(-/-)) and congenic Cftr(+/+) controls. Nine days after the last instillation, the CF transmembrane regulator knockout mice showed persistence of viable bacteria with chronic severe bronchopneumonia while wild-type mice remained healthy. The histopathological changes in the lungs of the susceptible Cftr(-/-) mice were characterized by infiltration of a mixed inflammatory-cell population into the peribronchiolar and perivascular spaces, Clara cell hyperplasia, mucus hypersecretion in airways, and exudation into alveolar airspaces by a mixed population of macrophages and neutrophils. An increased proportion of neutrophils was observed in bronchoalveolar lavage fluid from the Cftr(-/-) mice, which, despite an increased bacterial load, demonstrated minimal evidence of activation. Alveolar macrophages from Cftr(-/-) mice also demonstrated suboptimal activation. These observations suggest that the pulmonary host defenses are compromised in lungs from animals with CF, as manifested by increased susceptibility to bacterial infection and lung injury. This murine model of chronic pneumonia thus reflects, in part, the situation in human patients and may help elucidate the mechanisms leading to defective host defense in CF.

Cable-piliated Burkholderia cepacia binds to cytokeratin 13 of epithelial cells.

Although the Burkholderia cepacia complex consists of several genomovars, one highly transmissible strain of B. cepacia has been isolated from the sputa of cystic fibrosis (CF) patients throughout the United Kingdom and Canada. This strain expresses surface cable (Cbl) pili and is thought to be the major strain associated with the fatal "cepacia syndrome." In the present report we characterize the specific 55-kDa buccal epithelial cell (BEC) protein that binds cable pilus-positive B. cepacia. N-terminal sequences of CNBr-generated internal peptides identified the protein as cytokeratin 13 (CK13). Western blots of BEC extracts probed with a specific monoclonal antibody to CK13 confirmed the identification. Mixed epidermal cytokeratins (which contain CK13), cytokeratin extract from BEC (which consists essentially of CK13 and CK4), and a polyclonal antibody to mixed cytokeratins inhibited B. cepacia binding to CK13 blots and to normal human bronchial epithelial (NHBE) cells. Preabsorption of the antikeratin antibody with the BEC cytokeratin fraction reversed the inhibitory effect of the antibody. A cytokeratin mixture lacking CK13 was ineffective as an inhibitor of binding. Colocalization of CK13 and B. cepacia by confocal microscopy demonstrated that intact nonpermeabilized NHBE cells express small amounts of surface CK13 and bind Cbl-positive B. cepacia in the same location. Binding to intact NHBE cells was dependent on bacterial concentration and was saturable, whereas a Cbl-negative isolate exhibited negligible binding. These findings raise the possibility that surface-accessible CK13 in respiratory epithelia may be a biologically relevant target for the binding of cable piliated B. cepacia.

Burkholderia (basonym Pseudomonas) cepacia binding to lipid receptors.

Piliated Burkholderia (formerly Pseudomonas) cepacia from sputa of cys tic fibrosis patients in Toronto, Canada, were shown earlier to bind to purified mucins and to a protein receptor on epithelial cells via a 22-kDa adhesin located on unique cable pili. However, a second receptor, thought to be lipid in nature, was also identified on cells and appeared to serve as the major cell receptor for poorly piliated or nonpiliated isolates. In the present study in vitro approaches were used to identify putative lipid receptors for B. cepacia and to explore the nature of the binding interaction. As judged by thin-layer chromatography overlay assays, the best receptors were digalactosylceramide and globotriosylceramide (Gb(3)). Both contain and unsubstituted terminal Gal alpha 1-4Gal sequence. B cepacia also bound moderately to galactosylceramide, gangliotriosylceramide, and gangliotetraosylceramide. Binding to glycolipids was not affected by tetramethylurea, a hydrophobic-bond-breaking adhesin for GB(3). Binding to glycolipids was not affected by tetramethylurea, a hydrophobic-bond-breaking agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the carbohydrate epitope to the bacteria. Gb(3) was also the major receptor in lipid extracts of human erythrocytes, human buccal epithelial cells and HEp-2 laryngeal epithelial cells. In a receptor-based enzyme-linked immunosorbent assay, binding to Gb(3) within a phospholipid-cholesterol mixture (a membrane-like environment) increased and then approached saturation as a direct function of increasing bacterial concentration. The calculated value of K(a) (3.06 X 10(-8) ml/CFU), the affinity constant, was almost identical to the K(a) calculated earlier for B. cepacia binding to a set of lipid receptors in buccal epithelial cells (1.5 X 10(-8) to 2.0 X 10(-8) ml/CFU). Our findings suggest that within cell membranes, galactose-containing glycolipids, particularly Gb(3) are good candidates for receptors for B. cepacia, particularly for isolates in which cable pili are poorly expressed.

The emergence of a highly transmissible lineage of cbl+ Pseudomonas (Burkholderia) cepacia causing CF centre epidemics in North America and Britain.

The rapid increase in Pseudomonas (Burkholderia) cepacia infection in cystic fibrosis (CF) patients suggests epidemic transmission, but the degree of transmissibility remains controversial as conflicting conclusions have been drawn from studies at different CF centres. This report provides the first DNA sequence-based documentation of a divergent evolutionary lineage of P. cepacia associated with CF centre epidemics in North America (Toronto) and Europe (Edinburgh). The involved epidemic clone encoded and expressed novel cable (Cbl) pili that bind to CF mucin. The sequence of the cblA pilin subunit gene carried by the epidemic isolates proved to be invariant. Although it remains to be determined how many distinct, highly transmissible lineages exist, our results provide both a DNA sequence and chromosomal fingerprint that can be used to screen for one such particularly infectious, transatlantic clone.

Cable (cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers.

Previous studies have shown that appendage pili of Burkholderia cepacia strains isolated from patients with cystic fibrosis (CF) at The Hospital for Sick Children, Toronto, Canada, mediate adherence to mucus glycoproteins and also enhance adherence to epithelial cells. The specific pilin-associated adhesin molecule is a 22-kDa protein. In the present study we purified the major subunit pilin (17 kDa) and immunolocalized it to peritrichously arranged pili. On the basis of their novel morphological appearance as giant intertwined fibers, we refer to them as cable (Cbl) pili. Using an oligonucleotide probe corresponding to regions of the N-terminal amino acid sequence of the pilin subunit, we detected the encoding cblA gene in a chromosomal DNA library. Sequencing revealed this structural gene to be 555 bp in length, encoding a leader sequence of 19 amino acids, a cleavage site between the alanine at position 19 and the valine at position 20, and a mature pilin sequence of 165 amino acids. The calculated molecular mass is 17.3 kDa. Hydrophobic plus apolar amino acids account for 60% of the total residues. The pilin exhibits some similarities in its amino acid sequence to colonization factor antigen I and CS1 fimbriae of Escherichia coli. With the cblA gene used as a probe, hybridization assays of 59 independent isolates, including those from several geographically separated CF centers, plus environmental and clinical (non-CF) strains, gave positive results with all of the 15 CF-associated B. cepacia isolates from Toronto, plus a single strain from one other CF center (Jackson, Mississippi). The cblA gene is the first pilin subunit gene of B. cepacia to be identified.

Structurally variant classes of pilus appendage fibers coexpressed from Burkholderia (Pseudomonas) cepacia.

One or more of five morphologically distinct classes of appendage pili were determined to be peritrichously expressed by Burkholderia (formerly Pseudomonas) cepacia isolated from disparate sources. B. cepacia-encoded cblA pilin gene hybridization-based analysis revealed that one associated class, cable (Cbl) adhesin type IIB. cepacia pili, correlates with epidemically transmitted strains from a single cystic fibrosis (CF) center. When only phenotypic assays were available, correlations between the source and the pilus type were nonetheless observed: filamentous (Fil) type IIIB. cepacia pili correlated with CF-associated nonepidemic isolates, spine (Spn) type IVB. cepacia pili correlated with clinical (non-CF) isolates, and spike (Spk) type VB. cepacia pili correlated with environmental isolates. Further, Cbl, Fil, or Spk pili typically appear as an internal framework for constitutively coexpressed, peritrichously arranged dense mats of fine, curly mesh (Msh) type IB. cepacia pili. Constitutive coexpression of dense mats of Msh type IB. cepacia pili in association with a labyrinth of either Cbl, Fil, or Spk pili suggests possible cooperative pilus interactions mediating adhesion-based colonization in the differing environments from which the strains were isolated. Despite such correlations, phylogenetic analyses indicate that with the exception of the epidemically transmitted clusters of isolates, the remaining B. cepacia strains from the other three sources exhibited an equal degree of genetic relatedness independent of origin. As previously found for Escherichia coli, this discrepancy could be accounted for by selection-driven, in vivo horizontal transfer events between distantly related members of the species B. cepacia, leading to the genetic acquisition of environmentally appropriate adhesion-based colonization pilus operons.

Intestinal mucins inhibit rotavirus replication in an oligosaccharide-dependent manner.

Rotaviruses are important causes of infant morbidity and mortality worldwide. It has been previously shown that mucinous glycoproteins can inhibit rotavirus replication. However, the structure-function relationships of this inhibition have not been completely elucidated. Mucins were purified from epithelial scrapings of rat and human intestine by CsCl density-gradient ultracentrifugation and tested for the inhibition of rotavirus replication in MA-104 cells. Native human and rat intestinal mucins inhibited the replication of human and animal rotaviruses at low concentrations. Antiviral activity was most prominent in the densely glycosylated part of the rat and human mucins. Activity was retained after thiol reduction and alkylation, chloroform methanol partition, and partial removal of oligosaccharides. However, total deglycosylation of the mucins destroyed antiviral activity. Intestinal mucins from humans and other animals are potent inhibitors of rotavirus replication, and this inhibition is dependent on specific mucin-viral interactions.

Role of a 22-kilodalton pilin protein in binding of Pseudomonas cepacia to buccal epithelial cells.

Previously we have shown that many isolates of Pseudomonas cepacia obtained from the sputum of patients with cystic fibrosis exhibit specific binding to purified mucins. The binding was mediated by a 22-kDa protein located on peritrichous pili of the bacteria. Nonpiliated bacteria did not bind to mucin. In the present study we found that both piliated and nonpiliated P. cepacia bind to buccal epithelial cells (BECs) obtained from health human volunteers. Scatchard plot analyses of binding data with the LIGAND computer program suggest the presence of at least two classes of cell receptors (A and B) for piliated P. cepacia (isolates PC 5 and PC 7) and a single class of receptors (A) for nonpiliated P. cepacia (isolates PC 45 and PC 61). The affinity constants for receptor A varied from 1.7 x 10(-9) to 4.7 x 10(-8) ml/CFU. Receptor B had a lower affinity constant (2.5 x 10(-10) to 1.2 x 10(-9) ml/CFU) but a greater saturation capacity. Receptor B was similar in affinity to the mucin receptor for piliated P. cepacia (3.3 x 10(-10) to 1.3 x 10(-9) ml/CFU). Purified mucin partially inhibited the binding of piliated bacteria to BECs by competing with BEC receptor site B. The purified 22-kDa pilin adhesin and an antiadhesin antibody also caused partial inhibition. One BEC receptor for piliated isolates of P. cepacia was identified as a 55-kDa protein as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of BEC homogenate supernatants, Western blotting (immunoblotting), and bacterial overlay assays. Preincubation of piliated bacteria with either mucin or the antiadhesin antibody abolished binding to the 55-kDa BEC receptor. In summary, our results indicate that piliated P. cepacia interacts with BECs by using at least two different adhesin-receptor systems. One adhesin (not examined) is common to piliated and nonpiliated P. cepacia, but the other system is the pilus-localized 22-kDa mucin-binding adhesin and its 55-kDa BEC receptor protein. Because it mediates adherence to both mucin and epithelial cells, the 22-kDa adhesin may be an important virulence determinant in cystic fibrosis lung infections in which mucins are abnormally adhesive on mucosal surfaces.

Human intestinal mucin-like protein (MLP) is homologous with rat MLP in the C-terminal region, and is encoded by a gene on chromosome 11 p 15.5.

A cDNA specific for a human intestinal mucin (MLP) was amplified by PCR from cDNA of cultured human colonic adenocarcinoma cells, LS174T. The human cDNA shared high sequence homology with a corresponding rat intestinal mucin (MLP) cDNA in the 3' terminal region, and hybridized to the same mRNA (approximately 9.0 Kb) that was recognized by a probe for the MUC-2 human intestinal mucin gene. The gene encoding our human mucin peptide also mapped to chromosome 11 p 15.5, the known locus of MUC-2. Our findings suggest that human MLP and MUC-2 are encoded by the same gene and that rat and human intestinal mucin share a common C-terminal amino acid structure.

Binding of nonmucoid Pseudomonas aeruginosa to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis.

Lung infections due to Pseudomonas aeruginosa and Pseudomonas cepacia are common in patients with cystic fibrosis. Initial colonization is due to nonmucoid P. aeruginosa, while later mucoid variants emerge and are associated with chronic infection. P. cepacia colonization tends to be more prevalent in older patients. The present study was conducted to discover whether highly purified mucins (from cystic fibrosis sputum and control intestinal secretions) exhibited specific binding of nonmucoid P. aeruginosa. In vitro solid phase microtiter binding assays (with or without a blocking agent) as well as solution phase assays were conducted. Bacteria bound to both mucins via bacterial pili, but no differences in binding capacity were noted between the mucins. Unlike P. cepacia (described in the accompanying manuscript) there was also no preferential binding of P. aeruginosa to mucins versus bovine serum albumin, casein, gelatin, or a host of structurally unrelated proteins and glycoproteins. Carbohydrate hapten inhibition studies did not suggest the existence of specific mucin carbohydrate receptors for P. aeruginosa. In solid phase assays a low concentration (0.05 M) of tetramethylurea abolished P. aeruginosa bacterial binding to both mucins as well as to BSA, whereas in solution phase assays mucin binding to bacteria was not completely disrupted by tetramethylurea. Specific monoclonal antipilus antibodies did not inhibit binding to a greater extent than did Fab fragments of normal mouse IgG. Binding of strains PAO1 and PAK (and isolated PAK pili) to buccal epithelial cells was not influenced by the presence of mucin in binding assay mixtures. Our findings do not support the widely held notion that specific mucin receptors are responsible for the attachment of P. aeruginosa pili, nor do they support the idea that there is a competitive interference by mucins of bacterial binding to respiratory cells. In patients with cystic fibrosis, it would seem unlikely therefore that initial colonization of the lungs by P. aeruginosa is due to a 'selective tropism' of these bacteria for respiratory mucin.

Binding of Pseudomonas cepacia to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis.

Although not as prevalent as Pseudomonas aeruginosa, Pseudomonas cepacia is another opportunistic pathogen which colonizes the lungs of at least some patients with cystic fibrosis. A subgroup of these patients exhibits the "cepacia syndrome", i.e., a rapid clinical deterioration and death within one year. To investigate potential early sites of bacterial attachment, we have measured the specific binding of P. cepacia isolates from cystic fibrosis (CF) sputa to both CF and non-CF mucins purified from respiratory and intestinal secretions, respectively. As shown in microtiter binding assays, clinical isolates from 19/22 patients were found to bind to both mucins, with the highest specific binding exhibited by isolates from eight patients, seven of whom later died with the cepacia syndrome. No differences were observed in the binding capacity of the two (CF versus non-CF) mucins. Binding was specific, saturable, and not influenced by tetramethylurea, a disruptor of hydrophobic associations. Individual sugars were ineffective as hapten inhibitors, as were several lectins. Mucins treated by reduction/alkylation or chloroform/methanol extraction showed enhanced bacterial binding, findings which were attributed to exposure of underlying binding sites. Deglycosylation procedures indicated that mucin receptors for P. cepacia include N-acetylglucosamine and N-acetylgalactosamine, probably linked together as part of core oligosaccharide structures. P. cepacia isolates also bound to buccal epithelial cells, and mucin partially inhibited the binding of those isolates of P. cepacia that also had the ability to bind to mucin. We speculate that specific binding of P. cepacia to secreted mucins may be an early step in the pathogenesis of the cepacia syndrome.