Immunological study of cellular components of Pseudomonas aeruginosa. IV. Fractionation of Pseudomonas aeruginosa mucus by diafiltration and biological properties of isolated fractions]

In fractionation of Pseudomonas aeruginosa mucus (strains No. 8 and 1463) by means of diafiltration on the system of membranes Diaflo XM-300, XM-100A, PM-30, and PM-10 there was obtained a successive series of fractions differing by the molecular weight and chemical composition. According to the results of gel chromatography fractions with the mol wt of 100000 dalton and over apparently represented protein-polysaccharide components of mucus in the form of complexes; fractions with the mol wt of 30000 dalton and lower contained a considerable amount of free protein along with the protein-polysaccharide complex. The fractions obtained differed by biological properties: fractions with the mol wt of 100000 dalton and over were toxic for mice and possessed weak antigenic properties in the precipitation in agar test and immunoelectrophoresis; fractions with the mol wt lower than 30000 dalton expressed in the mentioned test distinct antigenic properties and proved to be practically nontoxic for mice. Thus the use of diafiltration method permitted to separate the antigenic, weakly toxic component of Ps. aeruginosa mucus from the toxic factor with weak antigenic properties.     

Immunochemical analysis and protective properties of components of Pseudomonas aeruginosa mucus with different molecular weights

P. aeruginosa slime has been separated into fractions XM-300 (3 X 10(5) daltons and more), XM-100 (1 X 10(5) = 3 X 10(5) daltons), PM-30 (3 X 10(4) = 1 X 10(5) daltons) and PM-10, (1 X 10(4) = 3 X 10(4) daltons) by ultrafiltration. The high-molecular slime components (3 X 10(4) daltons and more) have been found to be serologically more active than the low-molecular components (1 X 10(4) = 3 X 10(4) daltons). As shown in experiments on mice, both high-molecular toxic and low-molecular nontoxic slime components have protective activity, but the high-molecular components are more active than the low-molecular ones. The slime components stimulate the formation of immunity to homologous and partially heterologous P. aeruginosa strains in mice. Antigenic relationship between the slime components (especially the high-molecular ones) and the corresponding lipopolysaccharides has been noted.     

Immunologic study of the cellular components of bacillus pyocyaneus. III. Immunochemical analysis, toxicity and protective properties of water-soluble antigenic complexes]

Aqueous extracts of two Ps. aeruginosa strains killed with acetone were subjected to fractionation by preparative ultracentrifugation and gel-chromatography. Toxic activity of the extract was found to be connected with the high-molecular, possibly glycoprotein components reacting with the corresponding antiserum in the immunoprecipitation test, and protecting 30--40% of rats against the generalized infection with Pseudomonas aeruginosa. This protective activity is apparently connected with the protein components (molecular weight--20000--60000 dalton), nontoxic for mice, not reacting with the corresponding antiserum in the immunoprecipitation test, and protecting 60 to 80% of rats against Ps. aeruginosa infecsion. Thus, as a result of preparative ultracentrifugation and gel-chromatography it was postible to divide the toxic and the nontoxic protective components of Ps. aeruginosa.     

Immunologic study of the cellular components of bacillus pyocyaneus. V. Antigenic analysis of slime and its fractions]

Various slime fractions were obtained from newly isolated mucoid strains of P. aeruginosa by the method of ultrafiltration or differential centrifugation with subsequent gel chromatography. Purified slime was found to react with a broader spectrum of typing O sera than the corresponding cell wall lipopolysaccharides. Erythrocytic diagnostic preparations produced on the basis of slime antigens allowed to reveale the presence of circulating antibodies against P. aeruginosa. The slime components with molecular weight of 30,000--100,000 daltons and greater contained common antigenic determinants, and the slime components with molecular weight of 10,000--30,000 daltons contained both specific antigenic determinants and those also common to the high molecular components.     

Ferrisiderophore reductases of Pseudomonas. Purification, properties and cellular location of the Pseudomonas aeruginosa ferripyoverdine reductase.

Purification of the ferripyoverdine reductase from Pseudomonas aeruginosa, strain PAO1, lead to the isolation of a soluble protein of M(r) 27,000-28,000, as determined by HPLC sieving filtration and by denaturating gel electrophoresis. In the presence of NADH as the reductant, ferripyoverdine as the iron substrate, ferrozine as an iron(II)-trapping agent and FMN, this protein displayed an iron-reductase activity which resulted in the formation of ferrozine-iron(II) complex, providing that the enzymic assay was run under strict anaerobiosis. FMN was absolutely required for the activity to occur, but the lack of a visible spectrum and the lack of fluorescence for the protein in solution suggested that ferripyoverdine reductase is not a flavin-containing protein and that covalently bound FMN is not a prerequisite for the enzymatic reaction. A search of ferripyoverdine reductase by immunological detection amongst the different cellular compartments of P. aeruginosa lead to the conclusion that the soluble enzyme, which represented more than 95% of the total cellular enzyme, is not located in the periplasm but specifically in the cytoplasm. A strongly immunoreacting material, corresponding to a protein with identical M(r) as the ferripyoverdine reductase of P. aeruginosa PAO1, was detected in all the eighteen fluorescent pseudomonad strains belonging to the P. aeruginosa, P. fluorescens, P. putida and P. chlororaphis species, as well as in P. stutzeri, a non-fluorescent species, suggesting that the enzyme acting as a ferripyoverdine reductase in P. aeruginosa PAO1 is ubiquitous among the Pseudomonas.     

Antigenic properties of Pseudomonas aeruginosa anatoxin and the protective action of antitoxic anti-Pseudomonas aeruginosa serum

The antigenic properties of P. aeruginosa toxoid, prepared with the use of casein culture medium, were not inferior to those of the toxoid obtained in Martin broth. In experiments on white mice antisera obtained by the immunization of rabbits with the toxoid prepared on the basis of casein culture medium showed sharply pronounced protective properties against P. aeruginosa homologous and heterologous strains, as well as toxigenic reference strain PA-103.     

Pseudomonas aeruginosa aspartate transcarbamoylase. Characterization of its catalytic and regulatory properties

Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.     

Protective cross activity of the extracellular mucus of Pseudomonas aeruginosa

Extracellular slime was isolated from 15 P. aeruginosa typing strains of different O-serotypes (immunotypes). The isolated slime, partially purified by ethanol precipitation, was later referred to as crude slime. Glycolipoprotein was obtained from crude slime and lipopolysaccharide (LPS) was obtained from acetone-dried microbial cells by the method of aqueous-phenol extraction. All these antigenic preparations were studied in the active mouse cross-protection tests: immunized mice were challenged with 7 strains of different immunotypes, strain No. 170 019 or toxigenic strain PA-103. In experiments on mice the slime of different P. aeruginosa serotypes (immunotypes) was found to stimulate immunity to intraperitoneal infection with P. aeruginosa, both homologous or heterologous in respect to their immunotype, including toxigenic strains. Slime glycoprotein also stimulated active cross-immunity in mice, but the level of this immunity was higher than that of immunity stimulated by crude slime. LPS showed mostly weak protective activity in experiments on mice.     

Preparation of Pseudomonas aeruginosa recombinant outer-membrane protein F and the study of its antigenic properties

In Escherichia coli M15, the gene of P. aeruginosa recombinant outer-membrane protein F (OprF) was cloned. OprF, chromatographically purified on Ni-agarose and containing an additional sequence of 6 histidines on the N-end, was obtained. The purified OprF specifically reacted with rabbit serum, hyperimmune to P. aeruginosa, and in the mice injected with this protein specific IgG antibodies were synthesized. The optimum concentrations of P. aeruginosa OprF were selected for further tests of its protective properties from infection induced by P. aeruginosa.     

Immunological characteristics of the cellular components of Brucellae

Low-molecular glucopeptide stimulated protection against brucellosis without the accumulation of complete antibodies and the increase of skin sensitivity. Water-insoluble lipopolysaccharide, though inducing the intensive synthesis of circulating antibodies and moderately pronounced delayed hypersensitivity, did not stimulate protection against brucellosis. This suggests that the cellular mechanisms and among them the system of mononuclear phagocytes, play the leading role in immunity to brucellosis.     

The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties

Special analysis of the tryptophan residue localization in the structure of the macromolecule of Pseudomonas aeruginosa azurin made it possible to prove many explanations in the existing literature of the extraordinary fluorescence properties of this protein, to choose between various contradictory conclusions and in some cases even to make new interpretations of the known experimental data. It has been revealed that the microenvironment of the tryptophan residue is in principle formed by non-polar hydrocarbon groups. The density of the microenvironment is not very high and there are cavities around the ring. The conformation of the side chain of the tryptophan residue is unstrained. These results have been analysed in connection with available data on the unique short-wave fluorescence spectrum position and the existence of the high-frequency indole ring mobility with significant amplitude. Judging by the distance between tryptophan and tyrosine residues and their mutual orientation, the conclusion was made that there is no energy transfer from Tyr 72 to tryptophan and that the efficiency of the energy transfer from Tyr 108 to tryptophan is about 0.5. The mechanism of the dramatic increase in fluorescence efficiency when the copper atom is removed has been discussed with due regard to the fact that the 'blue' copper centre is displaced from the indole ring by more than 10 A.     

Isolation and study of the properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine. An evaluation of the biological properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine

P. aeruginosa killed polyvalent corpuscular vaccine, tested in a trial on 42 volunteer donors, is safe, low reactogenic and possesses pronounced immunogenicity, as the injection of the vaccine induced a rise in the titer of antibodies to P. aeruginosa up to 1:1280 in 95% of the immunized donors. To obtain specific antibodies in the blood plasma of the donors in an amount sufficient for the protective activity of the plasma becoming manifest, it is expedient to use the vaccination schedule providing for subcutaneous injections of 0.5, 0.5 and 1.0 ml at intervals of 7 days. Intense immunity thus induced in the donors lasts for 3-4 months. The hyperimmune plasma obtained from the donors has an antibody titer of at least 1:320 and shows a 90-100% protective effect in mice infected intraperitoneally with P. aeruginosa. The preliminary results of the clinical trial of anti-P. aeruginosa plasma have demonstrated its efficiency as a part of the complex treatment of patients with purulent septic complications of P. aeruginosa etiology.     

Induction of bacteriolytic enzyme from pyocinogenic Pseudomonas aeruginosa and its enzymatic properties.

Mitomycin C induced a pyocinogenic Pseudomonas aeruginosa P15 to produce a bacteriolytic enzyme, PR1-lysozyme, together with pyocin R1. No significant accumulation of the enzyme was observed inside the induced cells. The enzyme was partially purified by acrinol treatment and Amberlie CG-50 column chromatography. The mode of action of the enzyme on the host bacterial cells as well as on Micrococcus lysodeikticus cells or peptidoglycan isolated from Salmonella typhimurium, was compared with that of hen egg-white lysozyme or phage lambda-lysozyme. It is suggested that PR1-lysozyme should be classified as a glycosidase, rather than an amidase or an endopeptidase.     

Cell-free Pseudomonas vaccine. III. Immunochemical analysis of purified Pseudomonas aeruginosa protein antigens and protective properties of antisera against these antigens

The immunochemical analysis of isolated and purified antigens A and B obtained from P. aeruginosa, strains 868 (serogroup O3 according to Lányi or immunotype 3/7 according to Fisher) and 170015 (serogroup O7 or immunotype 2), was carried out. Rabbit antisera to proteins A and B, as well as to the initial aqueous extracts and partially purified aqueous extracts, were obtained. Cross activity between the protein antigens of different strains was established by the methods of immunodiffusion and two-dimensional immunoelectrophoresis. Isolated proteins A and B contained both common and specific antigenic determinants detected by the method of two-dimensional immunoelectrophoresis. The immunization of rabbits with proteins A and B was found to stimulate the synthesis of protective, probably species-specific, antibodies.     

The properties of the Pseudomonas aeruginosa bacteriophage phiPMG1

The properties of the isolated Pseudomonas aeruginosa bacteriophage phiPMG1 include the lytic infection cycle, and the formation of a broad halo (semi-transparent zone) around the plaques. We consider phiPMG1 as a potential member of therapeutic cocktails of live phages, and as a source of peptidoglycan and lipopolysaccharide degrading enzymes. Partial sequencing of phiPMG1 genome has revealed high similarity with known temperate P. aeruginosa phage D3. An open reading frame encoding lytic transglycosilase was identified in the genome. This enzyme PMG MUR was obtained in recombinant form, and its activity and substrate specificity has been studied.     

Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity.

Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm. In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively. The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH. Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin. The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure. Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding. The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication.