Structure, secretion, and bacterial specificity of an endogenous lectin from cystic fibrosis lung.

Endogenous heparin-binding lectin purified from postmortem lung samples of two cystic fibrosis (CF) patients was compared to lectin derived from normal tissue with respect to structure, carbohydrate specificity, interaction with alginate derived from CF isolates of Pseudomonas aeruginosa, and secretion within the lung. Lectin was purified from extracts of lung tissue by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. Lectin purified from either CF lung or control tissue ran as two peptides of approximately 16,000 and 13,000 molecular weight on electrophoresis in sodium dodecyl sulfate. The lectins displayed similar carbohydrate specificity and interacted in much the same way with bacterial alginate. An increase in lectin secretion was seen in CF lungs affecting the bronchial epithelial cells and the mucosal glands. The data suggest that the major changes seen in endogenous heparin-binding lectin in CF are related to the quantity and distribution of lectin secretion.

Physical, chemical and immunological properties of the bacterioferritins of Escherichia coli, Pseudomonas aeruginosa and Azotobacter vinelandii.

The 70-amino-acid-residue N-terminal sequence of the bacterioferritin (BFR) of Azotobacter vinelandii was determined and shown to be highly similar to the N-terminal sequences of the Escherichia coli and Nitrobacter winogradskyi bacterioferritins. Electrophoretic and immunological analyses further indicate that the bacterioferritins of E. coli, A. vinelandii and Pseudomonas aeruginosa are closely related. A novel, two-subunit assembly state that predominates over the 24-subunit form of BFR at low pH was demonstrated. The results indicate that the bacterioferritins form a family of proteins that are distinct from the ferritins of plants and animals.

Functional analysis of exotoxin A-related protein of Pseudomonas aeruginosa lacking residues 225-412.

The crystal structure of the exotoxin A (ETA) of Pseudomonas aeruginosa showed that this protein is folded into three distinct domains. Domain I (Ia and Ib), the amino-terminal domain, is the receptor-binding domain of ETA and domain III, the carboxy-terminal domain, is responsible for the ADP-ribosyl transferase activity of the toxin. To elucidate the function(s) of domains 1b and II in the intoxication process and to define the region of the domain III necessary for ADP-ribosylating activity, a defined deletion in the structural gene of P. aeruginosa ETA encompassing residues 225-412 was constructed and an ETA-related product DeID, (from which all of domains II and Ib were deleted) was expressed. The ETA-related protein did not penetrate sensitive cells, but retained the same specific activity to ADP-ribosylate elongation factor-2 as wild-type toxin. This suggests that domain II is necessary to allow toxin internalization by sensitive cells and that the absence of domain Ib does not interfere with enzymic activity. The domain strictly involved in ADP-ribosylation activity encompasses residues 412-613.

X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa.

The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods. The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer. The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 109.74 A, b = 99.15 A, c = 47.82 A for H35Q, a = 57.82 A, b = 81.06 A, c = 110.03 A for H35L. In each crystal form, there are four molecules in the asymmetric unit. They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L. The final crystallographic R-value is 16.3% for 20.747 reflections to a resolution of 2.1 A for H35Q and 17.0% for 32,548 reflections to 1.9 A for H35L. The crystal structures reported here represent the first crystallographically refined structures for azurin from P. aeruginosa. The structure is very similar to that of azurin from Alcaligenes denitrificans. The copper atom is located about 7 A below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion. The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer.

In vivo and in vitro effect of Cantastim, an immunomodulatory agent extracted from a highly pathogenic Pseudomonas aeruginosa strain.

Cantastim is a biological product consisting of distinct molecules of phospholipids obtained from a peculiar Pseudomonas aeruginosa strain. It enhanced the IgM and especially the IgG responses of mice pretreated with Cantastim and then intraperitoneally inoculated with T-dependent antigens such as sheep erythrocytes; restored cellular immunocompetence as evidenced by an increase in the in vitro proliferative responses to T-cell mitogens and allogeneic stimuli of murine splenocytes from in vivo pretreated mice activated NK cytotoxicity, and inhibited in vivo growth of the Ehrlich ascites tumor. In vitro it stimulated the mitogenesis of mouse lymphocytes but did not exert such stimulatory effect on guinea pig or human lymphocytes. All these findings allow to define Cantastim as an immunomodulating agent influencing mainly the cell-mediated immune response.

Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose.

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.

Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels.

A silver staining method used routinely for detecting bacterial lipopolysaccharide (LPS) in sodium dodecyl sulfate-polyacrylamide gels (C. Tsai and E. Frasch, Anal. Biochem. 119:115-119, 1982) appeared to be inappropriate for visualizing certain LPS preparations. It did not stain S-form fractions of polyagglutinable Pseudomonas aeruginosa LPS or several partly deacylated (alkali-treated) S-form LPS after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, these LPS preparations could be detected by anti-LPS sera after electroblotting onto nitrocellulose, thereby confirming their integrity and presence in the polyacrylamide gel. This is because LPS fractions containing a low number of fatty acids are washed out of the gel during the initial fixing step (40% ethanol-4% acetic acid, overnight). By omitting this fixing step, which was originally developed for detecting proteins, and by increasing the LPS oxidation time (from 5 to 20 min), we restored the ability to detect LPS fractions that otherwise would not be stained. These modifications did not affect the detection of other S- and R-form LPSs. Thus, differences in the number of fatty acids present in polyagglutinable P. aeruginosa LPS may result in a selective loss of fatty acid-deficient S-form LPS in these apparent R-form LPS preparations. This modified procedure provides a fast, simple, and sensitive way to analyze LPS in polyacrylamide gels despite the number of acyl groups present.

A new and rapid method for the assay of bacterial fatty acids using high resolution capillary gas chromatography and trimethylsulfonium hydroxide.

Gas-liquid chromatography of cellular fatty acids is a useful tool for the identification of bacteria. Derivatization of bacterial fatty acids to methyl esters by conventional techniques is usually time-consuming and complicated. A new one-step technique using trimethyl-sulfonium hydroxide allows the direct formation of fatty acid methyl esters within 1-2 min. Some random examples of profiles demonstrate that straight, branched, saturated, unsaturated, hydroxy and cyclopropyl fatty acids match conventional preparations well. The method is a very sensitive one, since only a few colonies are sufficient for preparation of fatty acid methyl esters.

Sequential 1H NMR assignments of iron(II) cytochrome c551 from Pseudomonas aeruginosa.

Sequence-specific 1H NMR resonance assignments for all but the C-terminal Lys 82 are reported for iron(II) cytochrome c551 from Pseudomonas aeruginosa at 25 degrees C and pH = 6.8. Spin systems were identified by using TOCSY and DQF-COSY spectra in 2H2O and 1H2O. Sequential assignments were made by using NOESY connectivities between adjacent amide, alpha, and beta protons. Resonances from several amino acids including His 16, Gly 24, Ile 48, and Met 61 experience strong ring-current shifts due to their placement near the heme. All heme protons, including the previously unassigned propionates, have been identified. Preliminary analysis of sequential and medium-range NOEs provides evidence for substantial amounts of helix in the solution structure. Long-range NOEs indicate that the folds in solution and crystal structures are similar. For one aromatic side chain (Tyr 27) that is close to the heme group we found a transition from hindered ring rotation at low temperature to rapid rotation at high temperature.

Comparison of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa.

Laboratory-derived mucoid variants of Pseudomonas aeruginosa were selected by plating the standard PAO1 laboratory strain with bacteriophage. These mucoid variants formed two distinct groups of strains on the basis of phage typing. The first group had the same phage-typing pattern as the parent PAO1 strain, while the second group had a distinctly different phage-typing pattern. One strain from each group was assessed along with the parent PAO1 strain for its outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles by sodium dodecyl sulfate-gel electrophoresis followed by appropriate staining. The mucoid derivatives were found to differ from the parent PAO1 nonmucoid strain in having lost a high-molecular-weight LPS species. Furthermore, the reversion of the mucoid strains to the nonmucoid phenotype was accompanied by a return of the missing high-molecular-weight LPS species. No observable difference between the mucoid derivatives and the parent nonmucoid strain was noted in the OMP profiles. The opposite was found in the case of four isolates of mucoid P. aeruginosa from patients with cystic fibrosis. Two OMP bands (of approximately 55 and 25 kilodaltons) were present in the mucoid isolates but missing in their sister nonmucoid strains. In the case of the cystic fibrosis isolates, no difference in the LPS profiles within mucoid-nonmucoid pairs was noted.

Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa.

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N-terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365-404). As a result, the polypeptides contained a large portion (13-34 amino acids) of domain Ib linked to the adjacent C-terminal domain III (amino acids 405-613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S-fragment; residues 392-613; 24201 Da; pI 4.7) possessed the same level of ADP-ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37-fold higher NAD-glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP-ribosyltransferase activity of both exotoxin A and the S-fragment, but not the NAD-glycohydrolase activity of the S-fragment. Antibodies against the S-fragment neutralized the ADP-ribosyltransferase activity of exotoxin A. These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392-404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF-2) and show that antibodies recognize the EF-2-binding site but not the NAD(+)-binding site.

Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa.

Purified flagella from two strains of 32P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.

Photophysics of metalloazurins.

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.

[Evaluation of microbial air contamination in dental areas]. / Valutazione della contaminazione microbica dell'aria in strutture odontoiatriche.

The Authors carried out a survey in dental wards in order to evaluate the qualitative and quantitative microbial contamination level of the air, which is considered a crucial habitat for the transmission of many infectious diseases. A high microbial counts were found in all of the environments tested; the microorganisms isolated belonged to pathogenous species such as Staphylococcus aureus, Staphylococcus haemoliticus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus var. lwoffi. It seems necessary, therefore, to carry out preventive interventions directed to the protection of the personnel of dental care and, in particular, of the patients.

An outer membrane protein characteristic of mucoid strains of Pseudomonas aeruginosa.

SDS-polyacrylamide gel electrophoresis of outer membrane (OM) proteins of different mucoid strains of P. aeruginosa revealed a protein of about 54 kDa that was absent in nonmucoid strains. This 54 kDa protein was expressed under iron-restricted and iron sufficient growth conditions. Electrophoretic mobility of the 54 kDa protein was modified by the solubilization temperature as well as by the addition of lipopolysaccharide and alginate prior to electrophoresis. Treatment of OMs with octylglucoside/KCl or SDS completely extracted the 54 kDa protein at low temperatures. The possible role of this protein in biosynthesis and/or excretion of bacterial alginate is discussed.

Instability of in vitro resistance to imipenem in Pseudomonas aeruginosa.

In 3 from 19 clinical isolates of Pseudomonas aeruginosa imipenem-resistant subpopulations could be detected in vitro. In comparison to their wild strains these imipenem-resistant cells were lacking an outer-membrane-protein of 50 kD. In the subpopulation derived from Pseudomonas aeruginosa 76 resistance to imipenem was found to be instable. It could be lost within a short period of time after subcultivation in the absence of imipenem. Cells with restored sensitivity to imipenem possess the outer membrane protein of 50 kD as the wild strain does.

Adherence of Pseudomonas aeruginosa to respiratory epithelium and the effect of leucocyte elastase.

The tracheobronchial secretions from patients with cystic fibrosis often contain high amounts of free proteases. To evaluate whether human leucocyte elastase (HLE) can favour the persistence of bacterial airways infection, we exposed the frog palate mucosa to HLE and then to radiolabelled Pseudomonas aeruginosa and followed the sequence of events by scanning electronmicroscopy. In response to HLE there was a marked outpouring of mucus and a desquamation of the epithelium. P. aeruginosa was shown to adhere to recently secreted granules of mucus and to the exposed submucosal underlying connective tissues. For the eight different bacterial strains studied, a significative adherence to HLE-injured mucosa was observed only in strains that possessed internal haemagglutinating activity. Neither the presence of fimbriae, nor of the mucoid exopolysaccharide, nor of the bacterial surface haemagglutinating activity could be related to adherence of P. aeruginosa to the injured mucosa. These results support the hypothesis that HLE enhances bacterial infection of the respiratory mucosa both by inducing mucus hypersecretion and by exposing receptors to the microbial adhesins. It is also suggested that P. aeruginosa internal lectins may be implicated in adherence to host tissues.

Resistance to ciprofloxacin appearing during therapy.

The development of resistance to ciprofloxacin in nine clinical isolates of Pseudomonas aeruginosa was investigated. Isolates had increases in minimal inhibitory concentrations (MICs) from 0.25 to 16 micrograms/ml. The isolates also became resistant to ofloxacin and norfloxacin, but did not show increases in MICs to aminoglycosides, antipseudomonas penicillins, or cephalosporins. One isolate from a patient with endocarditis showed a reduction in a 43-kD outer membrane protein and simultaneous increase in the imipenem MIC. This isolate also showed impaired uptake of ciprofloxacin. Respiratory isolates from cystic fibrosis patients did not show loss of outer membrane protein. MICs were lowered by ethylene diaminetetra-acetic acid, suggesting changes in lipopolysaccharide. Resistant isolates were synergistically inhibited by combinations of ciprofloxacin plus tobramycin or ceftazidime, but MICs remained beyond the achievable serum level.