Exotoxin production by clinical isolates of pseudomonas aeruginosa

Pollack, Matthew; Taylor, Nancy S.; Callahan, L T

doi:10.1128/iai.15.3.776-780.1977

Cited by 80 publications

(30 citation statements)

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“…Exotoxin A (ETA), a cytotoxic virulence factor produced by Pseudomonas aeruginosa (Liu, 1966;1974;Iglewski and Sadoff, 1979), ADP-ribosylates host cell EF-2, halting protein synthesis and causing cell death (Iglewski and Kabat, 1975;Chung and Collier, 1977). ETA is produced by 80-90% of clinical isolates of P. aeruginosa (Bjorn et al, 1977;Pollack et al, 1977). In bacteraemias, infection by an ETA-producing strain is associated with increased mortality (Cross et al, 1980).…”

Section: Introductionmentioning

confidence: 99%

Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa

Raivio

Hoffer

Prince

et al. 1996

Molecular Microbiology

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RegA is a transcriptional activator that controls exotoxin A (ETA) production in Pseudomonas aeruginosa. To date, functional assays performed with the purified protein have not clearly defined the molecular mechanism of action of RegA. In this study, we sought to identify important coding regions of regA by analysing the sequences around linker insertion mutations in regA that affected toxA transcription. First, we constructed a strain with the regAB locus deleted from the chromosome, PA103 delta regAB::Gm. toxA transcription was obliterated in strain PA103 delta regAB::Gm, demonstrating that the regAB locus is essential for ETA production. Next, we constructed a series of 6 bp linker insertion mutations distributed throughout regA. These regA linker insertion mutants were sequenced and screened in PA103 delta regAB::Gm for their effects on regulation of ETA production. Six linker insertion mutations occurring between amino acids (aa) 53 and 163 of RegA were isolated that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus. One of these linker insertion mutations (pTR53), resulted in a lack of iron-regulated ETA production and occurred directly upstream from a predicted transmembrane alpha-helix. The other five linker mutations (pTR88, pTR124, pTR132, pTR132-2 and pTR163) occurred within or flanked a region of RegA between aa 87-142 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR. These data suggest the presence of a previously unidentified transcriptional activation domain in RegA between aa 87-142 and implicate the predicted transmembrane alpha-helix in the N-terminus as being involved in sensory transduction.

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Section: Introductionmentioning

confidence: 99%

Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa

Raivio

Hoffer

Prince

et al. 1996

Molecular Microbiology

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“…The cytotoxic activity of ETA is attributed to the enzymatic domain, which inhibits protein synthesis through ADP-ribosylation of eukaryotic elongation factor 2 (eEF-2) in a manner similar to that of diphtheria toxin (19). When cultured in vitro, 80 to 90% of all P. aeruginosa clinical isolates produce ETA (34), and over 90% of all P. aeruginosa strains harbor the chromosomal gene for ETA (42). ETA is believed to be the most toxic virulence factor produced by P. aeruginosa (24), and its cytotoxic activity extends to a wide variety of mammalian cells (25).…”

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confidence: 99%

Generation of Neutralizing Antipeptide Antibodies to the Enzymatic Domain of Pseudomonas aeruginosa Exotoxin A

et al. 1998

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Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas aeruginosa. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P. aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.on July 3, 2020 by guest http://iai.asm.org/ Downloaded from MATERIALS AND METHODS Synthetic peptides. Specific amino acid sequences within ETA were selected for production of antibodies. Amino acid sequence selection for synthetic peptide synthesis was based on the analysis of hydrophilicity (Kyte-Doolittle), antigenic index (Jameson-Wolf), and surface probability (Emini) ( Fig. 1 and Table 1). Peptides were synthesized by the Synthetic Antigen Laboratory at the University of Texas, M. D. Anderson Cancer Center, Houston. Individual peptides were 12 to 45 aa long. Peptides, supplied as lyophilized powder, were reconstituted with distilled water to a stock solution of 10 mg/ml. A dilute solution of each peptide was conjugated to keyhole limpet hemocyanin (KLH) (Pierce, Rockford, Ill.) according to the manufacturer's recommendations. Because of the relatively large size of peptides 9 (45 aa) and 11 (29 aa), and based on our stud...

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“…ETA is one of the most cytotoxic of these virulence factors (23,30,32). The gene encoding ETA, toxA, is found in 90 to 95% of strains studied (3,40). Infection by an ETA-producing strain is associated with a poor clinical outcome (4).…”

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confidence: 99%

Association between transcript levels of the Pseudomonas aeruginosa regA, regB, and toxA genes in sputa of cystic fibrosis patients

et al. 1994

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In this study, we examined the regulation of exotoxin A (ETA) production by Pseudomonas aeruginosa during chronic lung infections of cystic fibrosis (CF) patients. We used a recently developed technique termed population transcript accumulation in hybridization studies with RNA extracted from sputa. With this technique, we demonstrated that the structural gene for ETA, toxA, as well as two genes encoding positive regulators of ETA synthesis, regA and regB, were expressed in the lungs of CF patients infected with P. aeruginosa. These genes were always expressed together, never alone or in pairs, suggesting coincident expression and a possible regulatory role for regA and regB in this environment. Fluctuations in the levels of the three gene products were observed among samples, consistent with a regulatory phenomenon. The level of regB RNA detected never exceeded that ofregA, although the ratio of regA RNA to regB RNA detected did change between samples. These observations are in agreement with in vitro observations which have shown that regB is located 3' to regA in an operon which is expressed from two independently regulated promoters located upstream of regA. The presence of high levels of toxA, regA, and regB RNAs in some sputum samples prompted us to look for hyperproducing-toxin strains in the sputa of CF patients. In vitro, one such strain, 4384, had a transcript accumulation pattern for toxA, regA, and regB similar to that of a laboratory hyperproducer of ETA, strain PA103. These observations suggest that regA and regB are involved in the regulation of ETA production in strains of P. aeruginosa infecting the lungs of CF patients and that some of these strains may regulate ETA production in a manner similar to that of the hyperproducing-ETA strain PA103.

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Exotoxin production by clinical isolates of pseudomonas aeruginosa

Cited by 80 publications

References 14 publications

Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa

Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa

Generation of Neutralizing Antipeptide Antibodies to the Enzymatic Domain of Pseudomonas aeruginosa Exotoxin A

Association between transcript levels of the Pseudomonas aeruginosa regA, regB, and toxA genes in sputa of cystic fibrosis patients

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