Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for ∼10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e.g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 Å crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5.     

Structural characterization of the Pseudomonas aeruginosa 1244 pilin glycan

An antigenic similarity between lipopolysaccharide (LPS) and glycosylated pilin of Pseudomonas aeruginosa 1244 was noted. We purified a glycan-containing molecule from proteolytically digested pili and showed it to be composed of three sugars and serine. This glycan competed with pure pili and LPS for reaction with an LPS-specific monoclonal antibody, which also inhibited twitching motility by P. aeruginosa bearing glycosylated pili. One-dimensional NMR analysis of the glycan indicated the sugars to be 5N beta OHC(4)7NfmPse, Xyl, and FucNAc. The complete proton assignments of these sugars as well as the serine residue were determined by COSY and TOCSY. Electrospray ionization mass spectrometry (MS) determined the mass of this molecule to be 771.5. The ROESY NMR spectrum, tandem MS/MS analysis, and methylation analysis provided information on linkage and the sequence of oligosaccharide components. These data indicated that the molecule had the following structure: alpha-5N beta OHC(4)7NFmPse-(2-->4)beta-Xyl-(1-->3)-beta-FucNAc-(1-->3)-beta-Ser.     

Cloning and expression of Pseudomonas aeruginosa flagellin in Escherichia coli.

The flagellin gene was isolated from a Pseudomonas aeruginosa PAO1 genomic bank by conjugation into a PA103 Fla- strain. Flagellin DNA was transferred from motile recipient PA103 Fla+ cells by transformation into Escherichia coli. We show that transformed E. coli expresses flagellin protein. Export of flagellin to the E. coli cell surface was suggested by positive colony blots of unlysed cells and by isolation of flagellin protein from E. coli supernatants.     

Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin

Pseudomonas aeruginosa (PA) is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. CF patients with chronic PA infections have a more rapid deterioration of their lung function and the bacteria become impossible to eradicate from the lungs. Antibiotic resistance among PA strains in CF patients is steadily increasing. Specific chicken (IgY) antibodies against PA have been shown to have potential to prevent PA infections in CF. Anti-Pseudomonas IgY reduces PA adhesion to epithelia, but the mechanism has not been fully elucidated. To gain further insight into the prophylactic effect of these antibodies, the immunoreactivity was investigated by 2D electrophoresis of PA strains, immunoblotting and MALDI-TOF-MS. To confirm the identity of the proteins, the tryptic peptides were analyzed by MALDI-TOF-MS to accurately measure their monoisotopic masses as well as determine their amino acid sequences. In order to facilitate fragmentation of the peptides they were N-terminally or C-terminally labeled. Several strains were investigated and anti-Pseudomonas IgY was immunoreactive against all of these strains, which strengthens its potential as a prophylactic treatment against PA. Flagellin was identified as the major antigen. Flagellin is the main protein of the flagella and is crucial for establishing infections in hosts as well as being involved in PA chemotaxis, motility, adhesion and inflammation. Furthermore, secreted flagellin elicits an inflammatory response. In conclusion, anti-Pseudomonas IgY binds flagellin, which may prevent PA infections in CF patients by hindering host invasion.     

Comparison of flagellin genes from clinical and environmental Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa, an important opportunistic pathogen, was isolated from environmental samples and compared to clinically derived strains. While P. aeruginosa was isolated readily from an experimental mushroom-growing unit, it was found only rarely in other environmental samples. A flagellin gene PCR-restriction fragment length polymorphism analysis of the isolates revealed that environmental and clinical P. aeruginosa strains are not readily distinguishable. The variation in the central regions of the flagellin genes of seven of the isolates was investigated further. The strains used included two strains with type a genes (998 bp), four strains with type b genes (1,258 bp), and one strain, K979, with a novel flagellin gene (2,199 bp). The route by which flagellin gene variation has occurred in P. aeruginosa is discussed.     

Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO.

Two independently derived, exotoxin A-deficient (Tox- phenotype), nitroso-guanidine-induced mutants of Pseudomonas aeruginosa PAO1 were isolated by using sensitive immunological assays. One mutant, designated PAOT10, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT10 and the parental strain. The mutation in PAOT10 (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.     

A new type of flagellin gene in Pseudomonas putida

Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.     

Topical flagellin protects the injured corneas from Pseudomonas aeruginosa infection

Among bacterial pathogens, Pseudomonas (P.) aeruginosa infection is the most sight threatening. The corneal innate immune responses are key mediators of the host’s defense to P. aeruginosa. Using a mouse model of Pseudomonas keratitis, we evaluated the protective effects of topical application of flagellin, a ligand for Toll-Like receptor 5 (TLR5), on the development of Pseudomonas keratitis and elucidated the underlying mechanisms. Topical application of purified flagellin 6 and 24 h prior to P. aeruginosa inoculation on injured mouse corneas significantly attenuated clinical symptoms of P. aeruginosa keratitis, decreased bacterial burden, and suppressed infection induced inflammation in the B6 mouse cornea. Topical application of flagellin on wounded cornea induced PMN infiltration and markedly upregulated cathelicidin-related antimicrobial peptide (CRAMP) expression. In PMN depleted mice, flagellin promoted bacterial clearance in the cornea compared to that of the PBS treated mice, but was unable to prevent corneal perforation and systemic bacterial dissemination and sepses. Deletion of CRAMP increased corneal susceptibility to P. aeruginosa and abolished flagellin-induced protection in B6 mice. Our findings illustrate the profound protective effect of flagellin on the cornea innate defense, a response that can be exploited for prophylactic purposes to prevent contact lens associated Pseudomonas keratitis.     

Sequence polymorphism in the glycosylation island and flagellins of Pseudomonas aeruginosa

A genomic island consisting of 14 open reading frames, orfA to orfN was previously identified in Pseudomonas aeruginosa strain PAK and shown to be essential for glycosylation of flagellin. DNA microarray hybridization analysis of a number of P. aeruginosa strains from diverse origins showed that this island is polymorphic. PCR and sequence analysis confirmed that many P. aeruginosa strains carry an abbreviated version of the island (short island) in which orfD, -E and -H are polymorphic and orfI, -J, -K, -L, and -M are absent. To ascertain whether there was a relationship between the inheritance of the short island and specific flagellin sequence variants, complete or partial nucleotide sequences of flagellin genes from 24 a-type P. aeruginosa strains were determined. Two distinct flagellin subtypes, designated A1 and A2, were apparent. Strains with the complete 14-gene island (long island) were almost exclusively of the A1 type, whereas strains carrying the short island were associated with both A1- and A2-type flagellins. These findings indicate that P. aeruginosa possesses a relatively low number of distinct flagellin types and probably has the capacity to further diversify this antigenic surface protein by glycosylation.     

Regulation of matrilysin expression in airway epithelial cells by Pseudomonas aeruginosa flagellin

Matrilysin (matrix metalloproteinase-7) is expressed by mucosal epithelia throughout the body and functions in host defense by activating murine intestinal alpha-defensins. In normal adult human lung, matrilysin is expressed at low levels in the airway epithelium, but is markedly up-regulated in cystic fibrosis (CF). Because CF lungs support a heavy bacterial load, we assessed if relevant CF pathogens regulate matrilysin expression in human lung epithelial cells. Indeed, acute infection with Pseudomonas aeruginosa (but not Staphylococcus aureus, Haemophilus influenzae, or Klebsiella pneumoniae) induced the expression of matrilysin in Calu-3 lung epithelial cells. Increased matrilysin mRNA levels were detectable at 3 h post-infection and peaked at a 25-fold induction between 6 and 8 h. Both P. aeruginosa CF isolates and laboratory strains induced matrilysin expression to similar levels. Flagellin, the monomeric precursor of bacterial flagella, was identified as the inductive factor released by P. aeruginosa that regulated matrilysin expression. In addition, flagellin-null mutants failed to stimulate matrilysin expression in cultured cells or in lungs infected in vivo. These data show that P. aeruginosa (and specifically flagellin) potently stimulates matrilysin expression in lung epithelial cells and may mediate the overexpression of this proteinase in CF lungs.     

Identification and genetic characterization of metallo-beta-lactamase-producing strains of Pseudomonas aeruginosa in Tehran, Iran

Metallo-beta-lactamases (MBLs) are being reported with increasing frequency worldwide. The aim of this study was to investigate the prevalence of blalMP-1, blaVIM-1,2 and blaSPM-1 genes encoding metallo-beta-lactamases (MBLs) among a collection of Pseudomonas aeruginosa strains isolated from patients at different hospitals in Tehran and to trace the disseminated clones at these hospitals by pulsed field gel electrophoresis (PFGE). Susceptibility of 610 P aeruginosa to 14 different antibiotics was determined using disc diffusion method. Isolates showing resistance to imipenem and ceftazidime were subjected to micro broth dilution assay to determine their MIC values. The blaIMP-1, blaVIM-1, blaVIM-2, and blaSPM-1, genes were amplified by PCR. Isolates containing blaVIM-1 were analyzed by PFGE. Sixty-eight isolates were resistant to imipenem (MIC > or = 4 microg/ml) of which 16 isolates carried blaVIM-1 gene using PCR assay. No other MBL genes were detected in this study. Three different unrelated patterns were found for isolates containing blaVIM-1 gene by PFGE of which pattern A was predominant. All isolates were susceptible to colistin and polymixin B. blaVIM-1 was the main gene encoding MBL among the isolates of P aeruginosa in our study. Clonal spread of isolates containing blaVIM-1 had occurred at Tehran hospitals. However, heterogeneous clones also were involved in the outbreaks.     

Structural and functional characterization of TgpA,a critical protein for the viability of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen associated with severe diseases, such as cystic fibrosis. During an extensive search for novel essential genes, we identified tgpA (locus PA2873) in P. aeruginosa PAO1, as a gene playing a critical role in bacterial viability. TgpA, the translated protein, is an internal membrane protein with a periplasmic soluble domain, predicted to be endowed with a transglutaminase-like fold, hosting the Cys404, His448, and Asp464 triad. We report here that Cys404 mutation hampers the essential role of TgpA in granting P. aeruginosa viability. Moreover, we present the crystal structure of the TgpA periplasmic domain at 1.6?Å resolution as a first step towards structure–activity analysis of a new potential target for the discovery of antibacterial compounds.     

Pseudomonas aeruginosa 1244 pilin glycosylation: glycan substrate recognition

The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics and because the sugars at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (beta-N-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the wbjE gene produced an incomplete O subunit, consisting only of the monosaccharide at the reducing end (beta-d-N-acetyl fucosamine), indicating that this moiety contained substrate recognition elements for WaaL. Expression of pilAO(1244) in PA103 wbjE::aacC1, followed by Western blotting of extracts of these cells, indicated that pilin produced has been modified by the addition of material consistent with a single N-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. These data suggest that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O repeat and that structures of the remaining sugars are irrelevant.     

Purification and characterization of a staphylolytic enzyme from Pseudomonas aeruginosa

A strain of Pseudomonas aeruginosa has been shown to produce an enzyme that lyses viable cells of Staphylococcus aureus. The maximal yield of the enzyme was obtained from shake flask cultures of P. aeruginosa which were grown for 18 to 22 hr at 37 C in Trypticase Soy Broth. A 333-fold purification of the enzyme was obtained by acetone precipitation of the culture liquor, followed by column chromatography on phosphonic acid cellulose and Bio-Gel P2. The staphylolytic enzyme exhibited maximal activity at 37 C in 0.01 m sodium phosphate (pH 8.5) and was stable at 37 C in the pH range of 7.5 to 9.5. The inhibition and stabilization of the enzyme by various organic and inorganic materials was investigated. Spheroplasts of S. aureus were formed by treating viable cells with the staphylolytic enzyme in 1 m sucrose or human serum.     

Isolation and characterization of a lysine-specific protease from Pseudomonas aeruginosa

We report here a procedure which results in the purification of an extracellular protease (designated Ps-1) from Pseudomonas aeruginosa. This enzyme cleaves fibrinogen so that the modified molecules form microcrystals and large single crystals. Precise knowledge of the Ps-1 cleavage sites is essential for the interpretation of the structural information provided by these crystals (Weisel, J. W., Stauffacher, C. V., Bullitt, E., and Cohen, C. (1985) Science 230, 1388-1391). Ps-1 is a single-chain polypeptide of Mr 30,000 which appears to function as a monomer. The pH optimum is 8-9. The activity of the protease is not decreased by inhibitors of thiol, carboxyl, or metallo proteases; the abolishment of activity by N alpha-p-tosyl-L-lysine chloromethyl ketone and the partial inhibition obtained with serine-reactive inhibitors suggests that Ps-1 may be a serine protease with an unusual active-site conformation. Studies with synthetic peptide substrates show that Ps-1 exhibits one of the most restricted specificities known for an endoproteinase: only peptide, ester, and amide bonds containing the carbonyl group of lysine are hydrolyzed. The limited specificity of Ps-1 should make it useful for other applications requiring the selective cleavage of proteins, such as sequence analysis and the isolation of domains.     

Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides

The structure of the lipid A component of lipopolysaccharides isolated from two wild-type strains (Fisher 2 and 7) and one rough mutant (PAC 605) of Pseudomonas aeruginosa was investigated using chemical analysis, methylation analysis, combined gas-liquid chromatography/mass spectrometry, laser-desorption mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of a pyranosidic beta 1,6-linked D-glucosamine disaccharide [beta-D-GlcpN-(1----6)-D-GlcpN], phosphorylated in positions 4' and 1. Position 6' of the beta-D-GlcpN-(1----6)-D-GlcpN disaccharide was identified as the attachment site of the core oligosaccharide and the hydroxyl group at C-4 was not substituted. Lipid A of the three P. aeruginosa strains expressed heterogeneity with regard to the degree of acylation: a hexaacyl as well as a pentaacyl component were structurally characterized. The hexaacyl lipid A contains two amide-bound 3-O-acylated (R)-3-hydroxydodecanoic acid groups [12:0(3-OH)] at positions 2 and 2' of the GlcN dissacharide and two ester-bound (R)-3-hydroxydecanoic acid groups [10:0(3-OH)] at positions 3 and 3'. The pentaacyl species, which represents the major lipid A component, lacks one 10:0(3-OH) residue, the hydroxyl group in position 3 of the reducing GlcN residue being free. In both hexa- and pentaacyl lipid A the 3-hydroxyl group of the two amide-linked 12:0(3-OH) residues are acylated by either dodecanoic (12:0) or (S)-2-hydroxydodecanoic acid [12:0(2-OH)], the lipid A species with two 12:0(2-OH) residues, however, being absent. The presence of only five acyl residues in the major lipid A fraction may account for the low endotoxic activity observed with P. aeruginosa lipopolysaccharide.     

Identification and characterization of porins in Pseudomonas aeruginosa.

Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane is protein F (OprF), which produces channels wider than those produced by Escherichia coli porins. In contrast, Yoshihara and Nakae ((1989) J. Biol. Chem. 264, 6297-6301) reported that protein F has no pore-forming activity as measured by the flux of L-arabinose, and that the channels in P. aeruginosa outer membrane, being produced by proteins C, "D," and "E," are much narrower than E. coli porin channels. In this study, we followed the protein purification scheme of Yoshihara and Nakae as closely as possible, and found that protein F had a specific activity for pore formation similar to that of proteins D1, D2, and E2. Furthermore, proteoliposome reconstitution assays showed conclusively that the channels formed by protein F, as well as by unfractionated outer membranes, allowed the diffusion of a tetrasaccharide, stachyose, at a significant rate, indicating that these channels are much larger than E. coli porin channels. It appears likely that in the study of Yoshihara and Nakae protein F was inadvertently inactivated during purification. We further suggest a hypothesis that resolves the apparent conflict between the presence of large diameter channels and the low permeability of the outer membrane in P. aeruginosa.