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At least 19 recordsLinked to original sources

PubMed1990

Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography.

An improved high-performance liquid chromatography separation was developed to detect and quantify beta-exotoxin production in Bacillus thuringiensis culture supernatants. Exotoxin production was assigned to a plasmid in five strains, from three subspecies (B. thuringiensis subsp. thuringiensis serotype 1, B. thuringiensis subsp. tolworthi serotype 9, and B. thuringiensis subsp. darmstadiensis serotype 10). A new exotoxin, called type II beta-exotoxin in this report, was discovered in B. thuringiensis subsp. morrisoni serotype 8ab, purified, and partially characterized. This material is more specific than type I beta-exotoxin and is very active against the Colorado potato beetle, Leptinotarsa decemlineata.

Bacillus thuringiensis↗

PubMed1985

Corynebacterium pseudotuberculosis exotoxin: fatal hemolytic anemia induced in gnotobiotic neonatal small ruminants by parenteral administration of preparations containing exotoxin.

Inoculation of live Corynebacterium pseudotuberculosis, culture supernatant, ammonium sulfate-fractionated crude exotoxin, or chromatographically purified exotoxin preparations into gnotobiotic small ruminants (n = 13) caused death of the ruminants within 48 hours. Characteristic changes observed in animals living greater than or equal to 2 hours after inoculation included hemorrhage and edema at the site of injection, severe hemolytic anemia and hemoglobinuria, dark red fluid in body cavities, lung edema, and icterus. The crude exotoxin preparation caused a syndrome of acute shock in 2 lambs that died within 15 minutes after inoculation. Clinical and pathologic responses of animals inoculated with culture supernatant and purified toxin were similar. Histopathologic evidence indicated that the exotoxin caused necrotic changes in the proximal convoluted tubules of the kidneys. Inoculation with live organisms caused multiple foci of suppurative inflammation in skeletal muscle and adjacent adipose tissue, whereas such changes were not observed in animals administered exotoxin preparations. Although C pseudotuberculosis exotoxin induced a hemolytic anemia in the experimental animals, it did not lead to in vitro lysis of ovine, caprine, or bovine erythrocytes, unless they had been sensitized with Rhodococcus (Corynebacterium) equi filtrate. The toxic sphingomyelin-specific phospholipase D from C pseudotuberculosis had a molecular weight of 31,000 daltons and an isoelectric point of approximately 9.6. The elution profile of exotoxin on a carboxymethyl Sephadex column was studied and the majority of the enzymatic activity was eluted by a NaCl gradient (0.25M to 0.7M) with a maximum at 0.35M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Hemolytic↗

PubMed1994

Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A.

In Pseudomonas aeruginosa, production of exotoxin A, an ADP-ribosyltransferase, is a complex and highly regulated process. Two positively acting regulatory genes, regA and regB, have been cloned and characterized. To identify additional exotoxin A regulatory genes, we have characterized four N-methyl-N'-nitro-N-nitrosoguanidine-generated mutants of P. aeruginosa PA103 which are deficient in exotoxin A production. These mutants (PA103-8, PA103-15, PA103-16, and PA103-19) do not accumulate intracellular exotoxin A and are not complemented by the cloned toxA or regAB genes. This observation indicates that the lesion(s) in the mutants is probably in an exotoxin A regulatory gene(s) and is not in the genes for secretion of exotoxin A or in the toxA or regAB genes. To assess the effect of the putative regulatory mutations on the toxA and regAB genes, we compared the activity of the toxA and regAB promoters in the mutant and parental strains using plasmids containing the genes for beta-galactosidase or chloramphenicol acetyltransferase under the control of either the toxA or the regAB promoter. The toxA promoter-beta-galactosidase fusion plasmid could not be maintained in PA103-8. beta-Galactosidase expression driven by the toxA promoter was absent in the mutant PA103-19 and occurred at a low level, which was not repressed by iron in mutants PA103-15 and PA103-16. The regAB genes are temporally controlled by two promoters, P1 and P2. In all four mutants, regAB P1 promoter activity was reduced; however, expression under the control of the regAB P2 promoter was normal. These observations suggest the existence of one or more regulatory genes which directly affect expression of both the toxA and the regAB P1 promoters.

ADP Ribose Transferases↗

PubMed1991

A Pseudomonas aeruginosa alginate-exotoxin A conjugate that elicits anti-alginate and exotoxin A-neutralizing antibodies.

Pseudomonas aeruginosa alginate was covalently coupled to exotoxin A by reductive amination using adipic acid dihydrazide as spacer. The conjugate was composed of 25% alginate and 75% exotoxin A and possessed an average molecular mass higher than 700 kDa as determined by polyacrylamide gel electrophoresis. The conjugate had virtually no ADP-ribosyltransferase activity and a reduced cytotoxicity for TSA8 murine cells, derived from Friend erythroleukemia cells, as indicated by a greater than 50-fold increased LD50. Anti-conjugate antibodies recognized exotoxin A and alginate. A booster injection resulted in markedly increased antibody ELISA titers to both exotoxin A and alginate. The antibodies neutralized the exotoxin A toxicity.

ADP Ribose Transferases↗

PubMed1989

Relative strength of the mitogenic and interleukin-2-production-inducing activities of staphylococcal exotoxins presumed to be causative exotoxins of toxic shock syndrome: toxic shock syndrome toxin-1 and enterotoxins A, B and C to murine and human T cells.

Several observations suggest that staphylococcal enterotoxins A, B and C (SEA, SEB and SEC, respectively), in addition to toxic shock syndrome toxin-1 (TSST-1), are causative exotoxins of toxic shock syndrome (TSS). Based on the view that polyclonal T cell activation with the causative exotoxins, resulting in over-production of lymphokines, is involved in the development of the pathological changes observed in TSS, we investigated the activities of these four exotoxins to induce proliferation and interleukin 2 production in murine and human lymphocytes by using in vitro culture systems. The results showed that all these exotoxins are strong polyclonal inducers of proliferation and interleukin 2 production in human T cells, whereas TSST-1 and SEA are strong and SEB and SEC are weak polyclonal inducers in murine T cells. These results suggest that SEA, SEB and SEC, in addition to TSST-1, are possibly involved as causative exotoxins in the development of the pathological changes observed in TSS.

Adult↗

PubMed1990

Inhibition of antibody response to Pseudomonas exotoxin and an immunotoxin containing Pseudomonas exotoxin by 15-deoxyspergualin in mice.

Immunotoxins are potent cell-killing agents that may be useful in the treatment of cancer. The early production of neutralizing antibodies to immunotoxins is one of the major limiting factors for their use in humans. 15-Deoxyspergualin (DSG), a derivative of spergualin, which is a metabolite of Bacillus laterosporus, has been found to have immunosuppressive activity in rodents, dogs, and primates. We examined the suppressive activity of DSG on the antibody response to Pseudomonas exotoxin in mice by enzyme-linked immunosorbent assay. Male BDF1 mice were immunized with a single dose of a nontoxic mutant of Pseudomonas exotoxin (40 micrograms) and then treated with i.p. injections of DSF at a dose of 10 mg/kg for 3 days. Although antibodies to Pseudomonas exotoxin were observed within 7 days in the control group, there was complete suppression of antibody production in the DSG-treated group. Immunosuppression has also been observed in animals immunized with multiple doses (10 mg x 7 d) of Pseudomonas exotoxin and treated with DSG at a dose of 5 mg/kg for 21 days. Similar immunosuppression was observed in mice given multiple doses of the immunotoxin, anti-Tac-LysPE40. We conclude that the immunosuppressive activity of DSG may be useful in increasing the duration of immunotoxin treatment.

ADP Ribose Transferases↗

PubMed1984

Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with Pseudomonas exotoxin.

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40-fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF-PE). Synergistic effects of verapamil and EGF-PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti-TFR-PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF-PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I-EGF by KB cells, but it significantly delays the disappearance of internalized 125I-EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I-EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF-PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF-PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF-PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.

ADP Ribose Transferases↗

PubMed2000

Mice immunized with DNA encoding a modified Pseudomonas aeruginosa exotoxin A develop protective immunity against exotoxin intoxication.

A recombinant plasmid, which contains the Pseudomonas aeruginosa exotoxin A (PE) gene with a C-terminal deletion, was inserted into expression vector pSecTag Xpress. The expression of this bacterial exotoxin in an animal cell was first demonstrated in 3T3 cell by transient transfection and western blot assay. Recombinant plasmid DNA was then injected intramuscularly to BALB/c mice, anti-PE specific antibodies were found in all animals vaccinated with plasmid containing the PE gene and with 'detoxicated' recombinant PE protein. Mice vaccinated with DNA were protected from the intoxication of lethal dosage of P. aeruginosa exotoxin A. Our results indicated that mice vaccinated with DNA encoding the PE gene could express PE protein in vivo, induced specific immune response, and provided sufficient protective immunity that safeguarded mice from the injection of lethal dosage of PE toxin.

ADP Ribose Transferases↗

PubMed1995

The crystal structure of Pseudomonas aeruginosa exotoxin domain III with nicotinamide and AMP: conformational differences with the intact exotoxin.

Domain III of Pseudomonas aeruginosa exotoxin A catalyses the transfer of ADP-ribose from NAD to a modified histidine residue of elongation factor 2 in eukaryotic cells, thus inactivating elongation factor 2. This domain III is inactive in the intact toxin but is active in the isolated form. We report here the 2.5-A crystal structure of this isolated domain crystallized in the presence of NAD and compare it with the corresponding structure in the intact Pseudomonas aeruginosa exotoxin A. We observe a significant conformational difference in the active site region from Arg-458 to Asp-463. Contacts with part of domain II in the intact toxin prevent the adoption of the isolated domain conformation and provide a structural explanation for the observed inactivity. Additional electron density in the active site region corresponds to separate AMP and nicotinamide and indicates that the NAD has been hydrolyzed. The structure has been compared with the catalytic domain of the diphtheria toxin, which was crystallized with ApUp.

ADP Ribose Transferases↗

PubMed1992

The effects of transferrin receptor antibody, transferrin receptor antibody bound to Pseudomonas exotoxin and transforming growth factor-alpha bound to Pseudomonas exotoxin on human tenon's capsule fibroblast proliferation.

Pharmacological agents which modulate the wound healing process by the inhibition of proliferation of fibroblasts may improve the success of glaucoma filtration surgery. Since cell proliferation is essential to the wound healing process, we targeted the surface receptors that are associated with proliferating cells. We present the effects of three such agents-purified mouse anti-human transferrin receptor monoclonal antibody 42/6 (anti-TfR-42/6), anti-transferrin monoclonal antibody bound to a Pseudomonas exotoxin (anti-TfR-PE40) and transforming growth factor-alpha Pseudomonas exotoxin (TGF-alpha-PE40)--on human fibroblasts from Tenon's capsule. The inhibition of human subconjunctival fibroblast proliferation by anti-TfR-42/6 (with a concentration up to 25 micrograms/ml) and by anti-TfR-PE40 and TGF-alpha-PE40 (both with a concentration range of 5000-0.00001 micrograms/ml) was determined by colorimetric (OD), and cell counting (CC) assays over a 9-day period. Neither anti-TfR-42/6 nor anti-TfR-PE40 had an antiproliferative effect on the fibroblasts. TGF-alpha-PE40 demonstrated an antiproliferative effect in a dose response manner. The mean 50% inhibitory dose (ID50) by OD was 32.91 micrograms/ml, while the ID50 by CC was 27.88 micrograms/ml. EGF was used as a negative control for TGF-alpha-PE40 toxin. The inhibitory effect of the toxin conjugate was completely blocked by the addition of 1000 micrograms/ml of EGF. These in vitro studies show that TGF-alpha-PE40 may be useful in modulating the proliferation of human ocular fibroblasts; they also give some indication of drug dosages for future in vivo testing.

ADP Ribose Transferases↗

PubMed1994

Similar cytokine induction profiles of a novel streptococcal exotoxin, MF, and pyrogenic exotoxins A and B.

The cytokine production induced by a newly discovered streptococcal exotoxin, MF, and the pyrogenic exotoxins SpeA and SpeB was determined by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) obtained from healthy blood donors. The induction and kinetics of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1 receptor antagonist, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, gamma interferon, tumor necrosis factor alpha (TNF-alpha), TNF-beta, and granulocyte-macrophage colony-stimulating factor were studied at the single-cell level by use of cytokine-specific monoclonal antibodies and intracellular immunofluorescent juxtanuclear staining. The cytokine-producing cells, with the exception of IL-1-expressing cells, had a characteristic morphology generated by the accumulation of cytokines in the Golgi organelle. MF, SpeA, and SpeB induced a massive gamma interferon and TNF-beta response in 10 to 16% of the PBMCs after 48 to 96 h of cell stimulation. In contrast, IL-2 and TNF-alpha production was detected in only 1 to 3% of the PBMCs. The induction of a lymphocyte TH2 phenotype response, including production of IL-3, IL-4, IL-5, and IL-10, was weak. However, the monokines, IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, and IL-8, were consistently found and gradually produced, peaking at 24 h in approximately 5 to 8% of the PBMCs. MF showed extensive cytokine- and proliferation-inducing capacities equal to those of SpeA and SpeB, which suggests that MF is also a superantigen. A marked interindividual variation could be noted both in the proliferative response and in the cytokine induction of lymphocytes isolated from different individuals, which may be one explanation for the varying clinical severity noticed during group A streptococcal infections.

Bacterial Proteins↗

PubMed2001

Pyrogenicity and cytokine-inducing properties of Streptococcus pyogenes superantigens: comparative study of streptococcal mitogenic exotoxin Z and pyrogenic exotoxin A.

Streptococcal mitogenic exotoxin Z (SMEZ), a superantigen derived from Streptococcus pyogenes, provoked expansion of human lymphocytes expressing the Vbeta 2, 4, 7 and 8 motifs of T-cell receptor. SMEZ was pyrogenic in rabbits and stimulated the expression of the T-cell activation markers CD69 and cutaneous lymphocyte-associated antigen. A variety of cytokines was released by human mononuclear leukocytes stimulated with SMEZ, which was 10-fold more active than streptococcal pyrogenic exotoxin A. Th2-derived cytokines were elicited only by superantigens and not by streptococcal cells.

Animals↗

PubMed1991

Staphylococcal exotoxin activation of T cells. Role of exotoxin-MHC class II binding affinity and class II isotype.

Staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 bind directly to class II molecules of the MHC and stimulate T cells based predominantly on the V beta segment used by the TCR. We investigated the relationship between the class II binding affinities of four of these exotoxins, SEA, SEB, SEC1, and toxic shock syndrome toxin-1 and their T cell signaling capabilities. Although the toxins stimulated T cells at concentrations that ranged over more than two orders of magnitude, their affinities for class II (DR1) differed by less than sixfold. The affinities of the toxins predicted their capacity to stimulate resting T cells to proliferate. The binding affinities of the toxins for class II molecules indicated that at concentrations required for T cell stimulation, as few as 0.1% of the class II molecules are complexed with toxin. Finally, the isotype of class II molecules affected the ability of the toxins to bind and use these MHC Ag to stimulate T cells. These data thus demonstrate that of the staphylococcal exotoxins studied, both their potency as T cell mitogens and their ability to function in the presence of single class II isotypes can be attributed in part to their characteristic abilities to bind class II molecules.

Animals↗

PubMed1999

[A new exanthematous disease in newborn infants caused by exotoxins producing Staphylococcus aureus; exotoxins production of the isolates and serum levels of antitoxin antibody in the patients and umbilical cord blood].

Recently, in Japan newly neonatal exanthematous disease was elucidated to be caused by staphyloccocal supcrantigcnic exotoxins, mainly TSST-1. We studied exotoxins producibility of 43 strains of S. aureus isolated from neonates with exanthematous disease and examined antibody titers to staphylococcal enterotoxin A, B, C (SEA, SEB, SEC) and toxic shock syndrome toxin 1 (TSST-1) of the patients and control (umbilical cord blood from term infants). The results were as follows 1.34 of 43 strains (79%) isolated from the patients were SEC and TSST-1 producing MRSA, 5 strains (12%) were SEB, SEC, and TSST-1 producing MRSA, 1 strain (2%) was SEB and TSST-1 producing MRSA, 2 strains (12%) were SEB producing MSSA and did not produce TSST-1. The 1 strain (2%) was MSSA which produced SEC and TSST-1. 2. 16 neonates with exanthematous disease, who showed typical clinical signs and laboratry findings of thrombocytopenia, with SEC and TSST-1 producing MRSA isolates had significantly low anti-TSST-1 antibody titers at onset (p < 0.05), compared with the control (umbilical cord blood from term infants): TSST-1 appeared to the causative agent for the disease. In two neonates with exanthematous disease, with SEB- and non- TSST-1-producing MSSA isolates, anti-SEB antibody titers were low at onset, so SEB appeared to the causative agent for the disease. 3. In Japan, low anti-TSST-1 antibody titers were found in the umbilical blood samples from about 70% of term infants; and low anti-SEB or anti-SEC antibody titers were found in samples from only about 10% of them, that is, a number of term infants had anti-SEB and anti-SEC antibodies. The majority of S. aureus isolated from neonates with exanthematous disease were enterotoxin- and TSST-1-producing MRSAs. The results of our study by measuring antitoxin antibody titers suggested that SEB and SEC might not be pathogenically responsible, but TSST-1 was considered to be responsible for the majority of exanthematous disease. Prevalence of TSST-1-producing MRSA in the neonatal and premature baby ward is the main cause for the high incidence of this disease in Japan, whereas the low antibody titer to TSST-1 in the mother, in comparison with the anti-enterotoxin antibody titers, may also be a predisposing factor.

Antibodies, Bacterial↗

PubMed1997

Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins.

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609. Two minor epitopes were identified as well: amino acids 501-510 and 582-589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.

ADP Ribose Transferases↗

PubMedDate not supplied

A nontoxic Pseudomonas exotoxin A induces active immunity and passive protective antibody against Pseudomonas exotoxin A intoxication.

Pseudomonas exotoxin A (PE) is one of the most potent cytotoxic agents produced by Pseudomonas aeruginosa. In this study, we examined the possibility of using PE with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(Delta576-613), for active immunization against PE-mediated disease. We first examined the toxic effects of PE and PE(Delta576-613) on 5- and 9-week-old ICR mice. The results show that the subcutaneous administration of PE(Delta576-613) at a dose of 250 microg was still nontoxic to 5- and 9-week-old ICR mice, while native PE was lethal at a dose of 0.5 and 1 microg, respectively. PE(Delta576-613) was then used to immunize ICR mice. The minimum dose of PE(Delta576-613) that could effectively induce anti-PE antibodies in 5- and 9-week-old ICR mice was found to be 250 ng. However, immunization with 250 ng PE(Delta576-613) failed to protect the immunized mice from a lethal dose of PE. The effective immunization dose of PE(Delta576-613) that could protect mice against a 2 microg PE challenge was found to be 15 microg. In addition, sera obtained from PE(Delta576-613)-immunized ICR mice were able to neutralize PE intoxication and effectively protect mice from PE. Thus, PE(Delta576-613) may be used as an alternative route to new PE vaccine development.

3T3 Cells↗

PubMedDate not supplied

Basic fibroblast growth factor-Pseudomonas exotoxin chimeric proteins; comparison with acidic fibroblast growth factor-Pseudomonas exotoxin.

We have constructed growth factor-toxin chimeric molecules composed of basic fibroblast growth factor (bFGF) and two different binding mutant forms of Pseudomonas exotoxin termed bFGF-PE40 and bFGF-PE4E KDEL. The chimeric molecules were expressed in Escherichia coli and localized to both inclusion bodies and the spheroplast cytoplasm. The bFGF-toxin fusion protein that was isolated and purified from inclusion bodies was 3-fold more active in inhibiting protein synthesis than that purified from spheroplast cytoplasm. Immunoreactivity of purified bFGF-toxin fusion protein to anti-bFGF antibodies was similar to that of native bFGF, as determined by ELISA analysis. A variety of carcinoma cell lines were sensitive to bFGF-PE40 and bFGF-PE4E KDEL, including H3396 (breast), Hep G2 (hepatocellular), and A431 (epidermoid). The concentration of chimeric toxin that inhibited protein synthesis by 50% (EC50) was 110, 70, and 18 ng/mL for bFGF-PE40 and 15, 1, and 18 ng/mL for bFGF-PE4E KDEL. In comparison with fusion-toxins composed of acidic fibroblast growth factor (aFGF) and either PE40 or PE4EKDEL, bFGF-PE40 and bFGF-PE4E KDEL were similarly cytotoxic on most cell lines tested. Human aortic smooth muscle cells were sensitive to both bFGF and aFGF toxin fusion proteins. However, human aortic endothelial cells were sensitive to the bFGF-toxins but were resistant to both aFGF-toxin forms. Time course studies showed that bFGF-PE40 needed a 4-6-h exposure to target cells for peak inhibition of protein synthesis on both MCF-7 and A431 cells, while aFGF-PE40 was almost fully active within a 2-h incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP Ribose Transferases↗

PubMed1992

Binding of monoclonal antibody specific for domain Ia/II of Pseudomonas aeruginosa exotoxin A at pH 4 strongly neutralizes exotoxin A-induced cytotoxicity in cell culture and in vivo.

Mouse monoclonal antibodies (MAbs) against Pseudomonas aeruginosa exotoxin A (Ex-A) were established, and 4 of 20 MAbs were extensively studied for analysis of the structure-function relationship of Ex-A. IN vivo experiments demonstrated that MAb Ex-3C7 protected mice either injected with Ex-A or infected with Ex-A-producing P. aeruginosa from death caused by Ex-A at the highest rate, followed by MAbs Ex-4F2 and Ex-8H5, in that order. MAb Ex-2A10 failed to rescue the mice. MAb Ex-3C7 (immunoglobulin G1 [IgG1]) inhibited incorporation of Ex-A into target cells and strongly neutralized cytotoxicity in cell culture but did not inhibit an enzymatic activity of Ex-A, ADP-ribosyltransferase, at all. The MAb also bound Ex-A, even at a low pH of 4, and recognized amino acid residues 241 to 297 (domain Ia/II), suggesting that MAb Ex-3C7 can interfere with the conformational change and/or processing of Ex-A by keeping a complex of Ex-A and antibody stable at low pH in the phagolysosome. MAb Ex-4F2 (IgG1), which recognizes residues 550 to 590 (domain III), strongly inhibited Ex-A incorporation and neutralized cytotoxicity in cell culture but only weakly inhibited ADP-ribosyltransferase. MAb Ex-8H5 (IgG1), which recognizes residues 591 to 613 (domain III), also inhibited cytotoxicity in cell culture, but weakly. In contrast to the above three MAbs, MAb Ex-2A10 (IgG2b) greatly inhibited ADP-ribosyltransferase but showed no inhibition of Ex-A incorporation and no neutralizing activity against cell toxicity. A line of evidence indicates that (i) domain Ia/II plays an important role in the pathogenesis of Ex-A and (ii) MAbs that inhibit an intracellular postbinding process, such as conformational change, processing, and translocation of Ex-A in target cells, can display potent inhibitory activity against cytotoxicity in vivo, as well as in cell culture, and would be a good candidate for therapy of pseudomonal infections.

ADP Ribose Transferases↗