NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a beta-turn

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [McInnes, C., et al. (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al. (1995) Biochemistry 34, 16255-16268] reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.     

Pseudoverdin, a compound related to the pyoverdin chromophore from a Pseudomonas aeruginosa strain incapable to produce pyoverdins

From a genetically manipulated strain of Pseudomonas aeruginosa ATCC 15692 (PAO1 strain) a compound named pseudoverdin, 3-formylamino-6,7-dihydroxycoumarin, was obtained which is related to the typical pyoverdin chromophore and thus allows to shed some light on the biogenesis of the latter.     

En bloc transduction of imipenem resistance with other resistance determinants from a clinical strain of Pseudomonas aeruginosa

Immunization of BALB/c mice with sonicates of M. smegmatis and kansasii and fusion of splenocytes of the immunized mice with cells of syngeneic myeloma P3X63Ag8653 yielded hybrid clones synthetizing antibodies to these antigens. The selection of hybrid clones and analysis of the antibodies specificity were performed at ELISA using the immunization antigens and antigens from other mycobacteria and E. coli. To define immunoglobulin class of the antibodies the authors used antiglobulin sera of class A, M, G. The monoclonal antibodies belonged to IgG. Molecular mass of polypeptides carrying the epitope against which the antibodies were directed was measured at immunoblotting. Two antibodies reacted only with M. smegmatis, 38 and 10 kD proteins. The other 2 cross-reacted with other mycobacteria and were directed against epitopes on polypeptides varying in molecular mass.     

Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion

The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin-like Xcp proteins was investigated using bifunctional crosslinking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT-containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PIlC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N-terminus or PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be crosslinked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine-tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.     

Pseudomonas aeruginosa as a cause of infectious diarrhoea

Pseudomonas aeruginosa is not generally considered a cause of infectious diarrhoea. However, it was the predominant organism isolated from the faeces of 23 unrelated, hospital outpatients investigated in the course of a year for persistent (> 1 week duration) diarrhoea. To investigate the possible aetiological role of P. aeruginosa, these patient histories were reviewed and a selection of their faecal isolates were investigated in vitro (n > or = 10) and in vivo (n = 2) for virulence. The patients had a mean age of 60 years, were receiving antibiotics and/or had an underlying illness. Extensive microbiological investigations identified no other potential or recognized enteropathogen in the faeces of 20 of these patients. More than 40% of the isolates tested were able to adhere to HEp-2 cells and exhibited twitching motility (type IV pili), properties indicative of their ability to colonize the human intestine. Cytotoxic activity was demonstrated in bacterium-free cell supernatants of over 80% of isolates; supernatants of four isolates tested in infant mice were weakly enterotoxigenic. Two isolates intragastrically inoculated into clindamycin pre-treated rats established persistent infections and induced signs and symptoms of enteritis. Overall these findings suggest that P. aeruginosa can cause diarrhoea particularly in immunodeficient individuals.     

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.     

Surveillance of Pseudomonas aeruginosa in intensive care units: clusters of nosocomial cross-infection and encounter of a multiple-antibiotic resistant strain

Fiber length has been implicated as a determinant of fiber toxicity. Fibers of narrowly defined length can be generated by dielectrophoretic classifiers. Since the quantities of fibers produced are very small, we developed a rat alveolar macrophage microculture system to study the toxicity of these samples. The objective of this study was to examine the role of fiber length on the cytotoxicity of Manville code 100 (JM-100) fibers. Rat alveolar macrophages were cultured with 0-500 microg/ml of 5 lengths of JM-100 fibers on 96-well plates. After 18 h, well supernatants were removed and lactate dehydrogenase (LDH) activity was measured to assess cell damage. Chemiluminescence (CL), an assessment of macrophage function, was measured by adding lucigenin with or without zymosan, a particulate stimulus, to appropriate wells. For each fiber length the effects were concentration dependent: CL declined and LDH rose with increasing fiber concentration. Comparing the effects of different lengths showed the greatest toxicity from a relatively long fiber sample (mean length = 17 microm). Microscopic examination of the interaction of fibers with macrophages revealed multiple macrophages attached along the length of the long fibers. This suggests that frustrated, or incomplete, phagocytosis may be a factor in the increased toxicity of longer fibers. Overall the results demonstrate that length is an important determinant of toxicity for JM-100 fibers.     

In vitro enzyme activation and folded stability of Pseudomonas aeruginosa exotoxin A and its C-terminal peptide

Pseudomonas aeruginosa exotoxin A(ETA) and its C-terminal, enzymatically active fragment (PE40, 375 residues) were studied by high-performance size-exclusion chromatography, steady-state and stopped-flow fluorescence spectroscopy, and circular dichroism spectroscopy. Both proteins have been overexpressed and purified by high-performance liquid chromatography. The effect of various activation conditions (pH, urea, and DTT) on enzymatic activity was studied. Upon enzymatic activation, structural changes induced within both proteins' structures were monitored, and these changes were correlated with concomitant alterations in the catalytic activity of the proteins. The pH optimum of enzymatic activity for both ETA and PE40 was between 7.0 and 8.0, decreasing to nearly zero at acidic (pH 5.0) and basic (pH 11-12) values. Analysis of the pH titration data revealed the presence of two distinct pKa values which implicate a His residue(s) (likely His-440 and -426) and a Tyr or Lys residue (possibly Tyr-481). The identity and possible role of an active site Lys residue is not known. Additionally, a significant increase in the Stokes radii of both proteins was detected when the pH was lowered from 8.0 to 6.0. The enzymatic activity of PE40 was not affected by urea or DTT, and its Stokes radius decreased monotonically with increasing urea concentration in the presence of DTT. In contrast, the enzymatic activity of ETA peaked when the protein was preincubated with 4.0 M urea, and this coincided with a large transition (increase) in the protein's Stokes radius between 3 and 5 M urea. Furthermore, loss of helical secondary structure of both PE40 and ETA commenced at approximately 2 M urea and progressively diminished at higher denaturant concentrations. The unfolding of both proteins in urea (and DTT) was reversible, and the free energies of unfolding were determined by both circular dichroism and fluorescence spectroscopy and were found to be 13.7 +/- 2.9 and 9.8 +/- 3.4 kJ/mol, respectively, for ETA and were 17.8 +/- 6.8 and 7.5 +/- 3.6 kJ/mol, respectively, for PE40. The refolding rate of PE40 was relatively rapid [t 1/2(1) = 27 s, t 1/2(2) = 624 s], which was in stark contrast to the refolding rate of ETA (t 1/2 = several hours). The relative refolding rates of PE40 and ETA help to explain the mechanism of in vitro enzyme activation and assay.     

Copper accumulation by a strain of Pseudomonas putida

A study on the copper accumulation by resting cells of copper-resistant bacteria, isolated from activated sludge and electroplating effluent, was conducted. The best selected strain, identified as Pseudomonas putida II-11, retained copper ions, Cu(II), as high as 6.5% of its dry weight. Bacterial cells grown in the sulphate-limiting medium had the highest copper removal capacity [RC, mg of Cu(II)/g of dry cells], while the presence of glucose or sodium azide did not affect Cu(II) RC of the bacterial cells. A possible mechanism of Cu(II) accumulation by this bacterium is suggested.     

Something's rotten: a nosocomial outbreak of malodorous Pseudomonas aeruginosa

From July 1994 through November 1996, a phenotypically unique strain of Pseudomonas aeruginosa producing a pungent, "rotten-potato" odor and a positive lysine decarboxylase reaction was isolated from 39 patients at UCLA Medical Center (Los Angeles). Most cases (95%) were in intensive care units and had clinical infections (72%). Most isolates (74%) were recovered from cultures of respiratory secretions. To determine risk factors for acquisition of the organism, 23 cases were compared with 23 randomly selected controls matched by service and isolate date. Multivariate analysis revealed that isolation of malodorous P. aeruginosa was associated with mechanical ventilation of > 24 hours' duration (odds ratio [OR] = 9.4; P = .001) and transfer from an outside hospital (OR = 5.7; P = .04). DNA from outbreak strains hybridized to P. aeruginosa-specific toxin A and phospholipase C gene probes and all outbreak isolates tested were found to be identical by use of pulsed-field gel electrophoresis. An unusual phenotypic characteristic of the strain led to the recognition of a nosocomial outbreak of P. aeruginosa infection associated with mechanical ventilation.     

Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa

We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2-oxa-1, 3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370-24374). This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent Km of 139 microM, and a Vmax of 5.3 micromol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.     

Report of Pseudomonas aeruginosa sinusitis in a patient with AIDS

Pseudomonas aeruginosa can cause infections in AIDS patients who frequently do not have the usual predisposing conditions such as neutropenia and intravenous drug use. The infections due to P. aeruginosa may be difficult to treat in AIDS patients. Sinusitis has been increasingly recognized as a complication in patients with AIDS. This article describes a case of recurrent P. aeruginosa sinusitis which, until recently, was rarely reported in AIDS.