Induction of murine cytolytic T lymphocytes by Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A (PA), a potent protein synthesis inhibitor, was found to be a weak T-cell mitogen for murine splenocytes. Maximal stimulation of [3H]thymidine incorporation was obtained with 10 to 100 ng of toxin per ml following a 4-day induction. PA was also shown to be a polyclonal activator of cytolytic T lymphocytes (CTL), effective against concanavalin A-treated target cells. The effective PA dose for CTL induction was the same as that for mitogenic stimulation, only with a prolonged priming time (7 days). In contrast to other mitogens, PA could not reactivate memory CTL into secondary CTL. The stimulation of CTL by subcytotoxic doses of PA may be relevant to its modulatory effect on the immunocellular system.     

Alteration of murine immune response by Pseudomonas aeruginosa exotoxin A.

Pseudomonas exotoxin A has been implicated as a possible virulence factor in Pseudomonas infections. This toxin has a direct cytotoxic effect on a number of cell types, including macrophages and their precursors, and therefore may affect other cells of the immune system. NFR/N(H-2q) (+/nu or nu/nu) mice were immunized with either T-dependent or T-independent antigens along with various doses of exotoxin A. The immune response was then assayed by a modification of the Jerne plaque assay. Exotoxin A induced a dose-dependent suppression of the in vitro and in vivo immune responses to T-dependent and T-independent antigens in immunocompetent +/nu mice. However, in NFR/N nu/nu mice, suppression of the immune response to the T-independent antigen trinitrophenylated-Ficoll was not observed. Instead, a marked enhancement of the response was observed at doses of 100 and 10 ng of exotoxin A. Removal of T-cells with anti-Thy 1.2 antiserum plus complement before antigen and exotoxin A stimulation in +/nu mice results in abrogation of the suppression. These data suggest that Pseudomonas exotoxin A exerts an effect on both B- and T-lymphocyte populations to modulate the immune response and that this activity may be one facet of the pathogenic effects of this toxin.     

Toxicity of Pseudomonas aeruginosa exotoxin A for human macrophages.

Pseudomonas aeruginosa exotoxin A was toxi in vitro for human peripheral blood macrophages. Cytotoxicity, manifested by morphological evidence of cell death and inhibition of [3H]thymidine uptake, followed exposure to as little as 10 ng of exotoxin per ml for 1 h. In addition, phagocytosis of heat-killed Candida albicans by macrophages exposed to sublethal concentrations of exotoxin was impaired. This cytotoxicity was neutralizable with antiexotoxin serum.     

Temperature-dependent inactivating factor of Pseudomonas aeruginosa exotoxin A.

The adenosine diphosphate ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A(PA toxin) was found to be rapidly destroyed by heating at 45 to 60C but not by heating at 70 to 90C (for at least 30 min). This phenomenon has been previously described for other bacterial toxins (staphylococcal alpha-toxin and Vibrio parahaemolyticus hemolysin) and is termed an Arrhenius effect. In contrast, the Arrhenius effect was not seen when the PA toxin was heat-treated as above and tested for cell toxicity or mouse lethality. Although the PA toxin treated at 70C for 30 min retained a significant proportion (is greater than 70%) of its adenosine diphosphate ribosyl transferase activity, the cell toxicity and mouse lethality of the toxin were virtually abolished. A temperature-dependent inactivating factor that has proteolytic activity and is co-purified with the PA toxin was shown to be responsible for the Arrhenius effect. PA toxin separated from the factor by conventional disc gel electrophoresis or PA toxin preparations lacking the factor did not show the Arrhenius effect.     

Purification of Pseudomonas aeruginosa exotoxin by affinity chromatography.

Pseudomonas aeruginosa exotoxin A was purified by affinity chromatography from culture supernatants by elution of toxin from antitoxin immunoglobulin G-Sepharose 4B with 3 M NaSCN. The purity, toxicity, and enzymatic activity of exotoxin obtained were comparable to those of toxin purified by previously reported multiple-step procedures.     

Enzyme-linked immunosorbent assay for Pseudomonas aeruginosa exotoxin A.

An enzyme-linked immunosorbent assay (ELISA) is described for Pseudomonas aeruginosa exotoxin A. A double antibody sandwich method was used, employing polyvinyl microtiter plates as the solid phase, a primary coat of monospecific rabbit antitoxin serum, an outer layer composed of a horseradish peroxidase-sheep antitoxin immunoglobulin G conjugate, and an ortho-phenylene-diamine substrate. Absorbance (optical density) of hydrolyzed end product was read spectrophotometrically at 492 nm. ELISA detected as little as 30 pg (0.3 ng/ml) of purified toxin, and absorbance was linear over a 20-fold or greater concentration range. Toxin was demonstrated in culture filtrates from 42 of 48 (88%) consecutive clinical P. aeruginosa isolates compared with 37 of 48 (77%) positive by hemagglutination inhibition. Results of the two assays correlated closely (r = 0.82, P less than 0.001). Specificity was confirmed by neutralizability of ELISA activity with monospecific antitoxin. ELISA was thus a sensitive, specific, and quantifiable technique for the assay of P. aeruginosa exotoxin A in both purified and crude culture materials.     

Production of exotoxin A by Pseudomonas aeruginosa in a chemically defined medium.

A defined medium was developed in which easily measured quantities of exotoxin A (PE) were produced by Pseudomonas aeruginosa PA-103. The medium contained three L-amino acids (arginine, aspartic acid, and alanine), basal and trace salts including 14 mM K2HPO4, 14 mM glucose, and 140 mM glycerol. The concentrations of amino acids which yielded most satisfactory results were 6 mM alanine, 13 mM aspartic acid, and 16 mM arginine. The identity of PE in the culture supernatant fluid was demonstrated by adenosine diphosphate-ribosyl transferase activity and by immunodiffusion with sheep antitoxin elicited with purified PE and with PE produced in Trypticase soy broth dialysate and pure PE as controls. PE production was also demonstrated by mouse lethality and passive hemagglutination. As compared to Trypticase soy broth dialysate, P. aeruginosa produced 25 to 50% PE in the defined medium. Different strains of P. aeruginosa produced PE in the defined medium in proportions relative to those in Trypticase soy broth dialysate.     

A macromolecular structure produced by Pseudomonas aeruginosa is recognized by antibody to exotoxin A.

Organized particulate structures (rods) identified in purified preparations of exotoxin A from culture supernatants of Pseudomonas aeruginosa PA103 were found to be immunochemically cross-reactive with exotoxin A. The rods were visualized by electron microscopy after negative staining as hollow tubes or sheaths (45 by 15 nm). Purified rods were not toxic and not enzymatically active in the ADP-ribosylation assay. Antigenic cross-reactivity between exotoxin A and rods was demonstrated by using monoclonal antibodies directed against either rods or a toxoid of exotoxin A. Hybridoma clones derived from mice immunized with rods or toxoid reacted with both antigens in the enzyme-linked immunosorbent assay. Rods could be dissociated by boiling and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three subunit polypeptides with molecular weights of 70,000, 45,000, and 27,000. Two of the three subunit polypeptides reacted both with antirod and antitoxin monoclonal antibodies after electrophoretic transfer of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins to nitrocellulose filters. The results indicate that rods and exotoxin A share common antigenic determinants.     

Effect of Pseudomonas aeruginosa exotoxin A on endotoxin-induced tumour necrosis factor production in murine lung

The ability of several Pseudomonas aeruginosa exo-enzymes, including exotoxin A (ETA), to induce inflammation and their influence on endotoxin-induced tumour necrosis factor (TNF) production in murine lung were evaluated. Intratracheal administration of lipopolysaccharide (LPS; 0.1-10 microg/mouse), 2(-1) LD50 of P. aeruginosa alkaline protease (7.5 microg/mouse) and elastase (1.2 microg/mouse) elevated total cell number and the percentage of neutrophils in broncho-alveolar lavage fluid (BALF), whereas ETA (0.1 microg/mouse) did not. LPS induced TNF production in BALF in a dose-dependent manner, whereas the P. aeruginosa exo-enzymes did not. When ETA was inoculated into the respiratory tract before LPS, production of TNF in BALF was significantly suppressed in a dose-dependent manner. ETA also suppressed TNF production by alveolar macrophages (AMs) stimulated with LPS in vitro. Flow cytometric analysis showed that ETA markedly reduced the expression of CD14 and CD11c/CD18 on the surface of AMs. ETA also depressed partially the expression of TNF-alpha mRNA in AMs. These findings suggest that ETA regulates TNF production in murine lung by suppressing LPS receptor expression, mRNA expression and protein synthesis and/or secretion of TNF.     

Toxoid of Pseudomonas aeruginosa exotoxin A generated by deletion of an active-site residue.

Glutamic acid-553 of Pseudomonas aeruginosa exotoxin A (ETA), identified previously as an active-site residue, was deleted by oligonucleotide-directed mutagenesis of the cloned toxin gene in Escherichia coli. The purified mutant toxin was stable, fully immunoreactive, and capable of blocking toxin receptors. ADP-ribosyltransferase and cytotoxic activities were at least 10(6)-fold lower than those of wild-type ETA, and injection of mice with 50 micrograms (equivalent to 400 lethal doses of ETA) produced no ill effects. The mutant toxin elicited high levels of neutralizing anti-ETA antibodies in mice, which protected against a challenge with 100 micrograms of authentic ETA (greater than 600 lethal doses). The mutant protein has the attributes of a toxoid and may be useful as a component of vaccines for individuals at risk for infection by P. aeruginosa.     

Structure-activity relationships of an exotoxin of Pseudomonas aeruginosa.

The relation of the structure of Pseudomonas aeruginosa exotoxin A (PA toxin) to its enzymatic activity (adenosine 5'-diphosphate-ribosyl transferase) in vitro and to its toxicity in vivo was examined. PA toxin is produced as a single polypeptide chain with a molecular weight of about 71,500. PA toxin is produced by Pseudomonas as a toxic proenzyme that lacks enzymatic activity. Adenosine 5'-diphosphate-ribosyl transferase activity is expressed when the molecule is denatured and reduced or when its is cleaved by Pseudomonas proteases to yield an enzymatically active 27,000-dalton fragment (fragment a). A 45,000-dalton protein is tentatively identified as the enzymatically inactive fragment b of PA toxin. Enzymatically active forms of the toxin lack toxicity for mouse L-cells or mouse lethality. Thus, it is concluded that the native toxin proenzyme is required for toxicity and that a structural rearrangement must precede its intracellular activity.     

Production and characterization of monoclonal antibodies to exotoxin A from Pseudomonas aeruginosa

Hybridomas secreting monoclonal antibodies specific for exotoxin A from Pseudomonas aeruginosa strain PA103 were derived from the fusion of spleen cells from mice immunized with: (i) purified exotoxin A, (ii) Formalin-treated exotoxin A, (iii) exotoxin A covalently coupled to Sepharose 4B, or (iv) P. aeruginosa-infected mice. All hybridomas were screened and selected by using an enzyme-linked immuno-adsorbent assay. All antibody isotypes were represented (immunoglobulins G, A, and M) as determined by enzyme-linked immunoadsorbent assay. The most productive fusions resulted from immunization with antigens coupled to an insoluble matrix, such as Sepharose 4B, or by infection of mice. Several hybridomas were selected and cloned by limiting dilution. The specificity of the monoclonal antibodies for exotoxin A was demonstrated by indirect immunoprecipitation of 125I-labeled exotoxin A followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and by the immunoblotting technique. The protective ability of certain monoclonal antibodies was demonstrated in vitro by toxin neutralization in tissue culture and in vivo by prolonged survival time in the burned mouse infection model, after passive immunization. One monoclonal antitoxin displayed specificity for PA103-derived exotoxin yet failed to react with exotoxin purified from PAO-PR1 or PAO1, suggesting that structural differences exist between these exotoxins.     

Induction of interferon synthesis and cytotoxicity by murine peritoneal macrophages exposed to glycoprotein ligands.

Thioglycollate-induced murine C57BL/6 and C3H/HeN peritoneal macrophages synthesized interferon-beta (IFN-beta) in response to exposure to glycoproteins such as horseradish peroxidase (HRP) or mannosyl or fucosyl bovine serum albumin (BSAman of BSAfuc, respectively), but not glucosylated or galactosylated BSA (BSAglu or BSAgal, respectively). These results suggest participation of the mannosyl-fucosyl receptor (MFR) in this response. IFN synthesis was augmented by culturing macrophages in L cell-conditioned medium prior to exposure to these substances. Macrophages obtained from lipopolysaccharide (LPS)-resistant C3H/HeJ mice did not produce IFN in response to HRP. Furthermore, IFN-induction by HRP was blocked by polymyxin B. In addition, exposure of macrophages to HRP or BSAman induced cytotoxicity against NIH 3T12 cells. Cytotoxicity was not inhibited by the presence of anti-IFN-alpha/beta. In contrast to IFN induction, however, macrophages activation was LPS-independent, since this activity was demonstrated in macrophages from C3H/Hej mice. The carbohydrate specificity of these responses suggests that the MFR or an another scavenger receptor may be involved in the responses to these substances, and that cytotoxicity and IFN-induction by glycoproteins follow unique pathways.     

A murine model of chronic mucosal colonization by Pseudomonas aeruginosa.

Chronic mucosal colonization by Pseudomonas aeruginosa is an integral part of the pathologic process associated with disease due to infection with this organism. We have adapted the streptomycin-treated murine model of chronic mucosal colonization by enteric pathogens to study colonization by P. aeruginosa. Mice first received 1 mg of streptomycin per ml of drinking water for 2 to 5 days and then ingested 10(7) CFU of P. aeruginosa per ml of drinking water for a minimum of 5 days. The result of this regimen was chronic mucosal colonization with P. aeruginosa for up to 10 weeks, which was determined by fecal cultures and confirmed by culture of the intestines after killing of the experimental animals. Bacterial counts were highest in the cecum and colon, with some evidence for extraintestinal bacterial translocation as well. Use of P. aeruginosa mutants deficient in the production of colonization factors such as pili and those dependent on the rpoN gene product resulted in a lower level of chronic colonization. Immune responses to type-specific lipopolysaccharide, pili, and flagellar antigens were measured, and increases in both serum and intestinal antibodies were usually elicited when a strain elaborated a given antigen. This model represents an easy method of routinely achieving chronic mucosal colonization by P. aeruginosa and should prove useful for the study of both bacterial virulence factors and host responses associated with this infectious process.     

Variables which affect suppression of the immune response induced by Pseudomonas aeruginosa exotoxin A.

Pseudomonas exotoxin A has been shown previously to induce suppression of the murine immune response. In the present study, various parameters were examined which may have an effect on immunosuppression. The addition of 10(-4) ng of exotoxin A induced suppression of the immune response to trinitrophenylated Ficoll from days 3 to 10, while 10 ng of toxin exerted no suppressive effect over the same examination periods. When the toxin was administered 1 or 2 days before antigen stimulation, suppression of the response was observed with both 10 and 10(-4) ng. Priming splenocytes with toxin either in vivo or in vitro for 1 or 2 days suppressed the response of fresh cultured splenocytes to antigenic stimulation. Heated toxin, photoaffinity-labeled toxin, or preincubation of the toxin with rabbit anti-exotoxin A antiserum eliminated the toxin-induced suppression. These results suggest that Pseudomonas exotoxin A can generate multiple biological effects.     

Induction of tumoricidal activity of human and murine peritoneal macrophages by antitumor chemotherapy

Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. M. Navashin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 9, pp. 330–332, September, 1989.     

Comparative immunochemistry of two fragments from domains Ib and III of Pseudomonas aeruginosa exotoxin A.

Two rabbit polyclonal antisera have been produced by immunization with two fragments corresponding to sequences 392 to 404 and 392 to 613 of Pseudomonas aeruginosa exotoxin A. Both antisera inhibit the ADP-ribosyltransferase activity of exotoxin A but do not inhibit its NAD-glycohydrolase activity. In addition, only the second antiserum was capable of neutralizing exotoxin A cytotoxicity in cell culture and in vivo. Consequently, the common sequence 392 to 404 of the two fragments is not a neutralizing epitope and such an epitope should reside within residues 405 to 613 of exotoxin A. The sequence 392 to 404 was shown to be hidden in the native molecule, and the results suggest that this sequence is most likely in close proximity to residues involved in eukaryotic elongation factor 2 binding.     

Induction of serine and threonine protein phosphorylation by endotoxin-associated protein in murine resident peritoneal macrophages.

Endotoxin-associated protein (EP) from Salmonella typhi activated murine resident peritoneal macrophages to produce prostaglandin E2 (PGE2). Cells from both endotoxin nonresponder (C3H/HeJ) and the endotoxin responder (C3H/OuJ) mouse strains were activated by EP. This EP-induced prostaglandin E2 production was blocked by the protein kinase C (PKC) inhibitor H-7 as well as the tyrosine kinase inhibitor genistein, suggesting the involvement of both serine and threonine phosphorylation and tyrosine phosphorylation pathways in the activation of resident peritoneal macrophages by EP. Immunoblot analysis using antiphosphoserine and antiphosphothreonine antibodies showed that EP induced the serine and threonine phosphorylation of a 14-kDa protein (p14). This phosphorylation was not induced by phorbol myristic acid or by lipopolysaccharide endotoxin. Inhibitors of PKC, PKA, and PKG did not block the phosphorylation of p14. However, the tyrosine kinase inhibitor piceatannol blocked p14 serine and threonine phosphorylation, suggesting that this phosphorylation is dependent upon and preceded by a tyrosine phosphorylation step.     

Age-related susceptibility of mice to ocular challenge with Pseudomonas aeruginosa exotoxin A.

We studied the responses of mice to ocular challenge with purified exotoxin A from Pseudomonas aeruginosa in 5-, 10-, 16-, 21-, and 30-day-old animals. In the absence of trauma, injection of 3 to 6 microgram of exotoxin per mouse beneath the fused eyelids of 5-day-old Swiss-Webster mice resulted in death of all animals within 24 h. Administration of 1.5, 0.75, and 0.375 microgram of exotoxin per mouse resulted in 24-h mortality rates of 50, 22, and 20%, respectively. Additional deaths were recorded throughout the next 4 days. Similar lethality results were obtained with 10-day-old animals that received equivalent amounts of exotoxin beneath the fused eyelid and in these experiments, the 72-h 50% lethal dose was 0.49 microgram of exotoxin per mouse. Mice that were 16 and 21 days old, whose eyelids were open, each received from 0.375 to 15 microgram of exotoxin topically applied to the surface of a wounded cornea. Cataracts were observed within 1 week in both groups, and none of the animals that received the higher concentrations of toxin died. Young adult (30-day-old) animals also received from 1.8 to 15 microgram of exotoxin topically on the surfaces of wounded corneas. Corneal swelling and slight opacity were observed at 24 h and within 1 month; 80% of these mice had cataracts of the ocular lens.