Reaction of antibody in sera from cystic fibrosis patients with nontoxic forms ofPseudomonas aeruginosa exotoxin A (original) (raw)
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Structure and function relationship of Pseudomonas exotoxin A
Journal of Biological Chemistry, 1989
We have raised antisera against Pseudomonas exotoxin A (PE) and domains Ia and I11 to study the structure-function relationships of PE. Anti-PE antibody (AbPE) was shown to abolish the ADP-ribosylation activity of PE. However, neither antidomain Ia antibody nor antidomain I11 antibody inhibited the ADP-ribosylation activity of PE. This suggests that the inhibition of ADP-ribosylation by AbPE results from the binding of AbPE to the region between domains Ia and 111. Since the binding of AbPE to PE did not inhibit NAD hydrolysis in the absence of elongation factor 2, the inhibitory effect of AbPE on ADP-ribosylation may be due to steric hindrance rather than a direct action on the catalytic function. Thus, the interface between domain Ia and I11 may be the site of entry of elongation factor 2 during ADP-ribosylation. The antibodies were also used to study both the inhibitory effects of PE on protein synthesis and its cytotoxic activity. Either AbPE or antidomain Ia antibody, but not antidomain I11 antibody, was able to reverse the inhibition of protein synthesis by PE and to block its cytotoxicity. In addition, rabbits immunized with domain I s acquired tolerance against 100 pg of PE injected subcutaneously. These results suggest that domain Ia is the cell-binding domain of PE and may be used for vaccination against PE-mediated diseases. Pseudomonas exotoxin A (PE)' is the most toxic component of the cellular and extracellular fractions of Pseudomonas aeruginosa and has been identified to play a major role in the pathogenicity of P. aeruginosa infections (1-3). The toxic effect of PE results from the inhibition of protein synthesis in eukaryotic cells by ADP-ribosylation of EF-2 (4, 5). Recently, structural and functional studies of PE have yielded new insights into its mechanism of action. The three-dimensional structure of P E has been determined by x-ray crystallography (6). Pseudomonas exotoxin A contains three structurally distinct domains: domain I (domain Ia, residues 1-254 and domain Ib, residues 365-404); domain I1 (residues 253-364); and domain I11 (residues 405-613). Based on the threedimensional structure and the use of recombinant DNA methods, domain Ia, and domain I11 with a portion of domain Ib have been identified as the cell binding and ADP-ribosylation domains, respectively (7). In this study, we have used immu
Processing of Pseudomonas aeruginosa Exotoxin A Is Dispensable for Cell Intoxication
Infection and Immunity, 2009
Exotoxin A is a major virulence factor of Pseudomonas aeruginosa . This toxin binds to a specific receptor on animal cells, allowing endocytosis of the toxin. Once in endosomes, the exotoxin can be processed by furin to generate a C-terminal toxin fragment that lacks the receptor binding domain and is retrogradely transported to the endoplasmic reticulum for retrotranslocation to the cytosol through the Sec61 channel. The toxin then blocks protein synthesis by ADP ribosylation of elongation factor 2, thereby triggering cell death. A shorter intracellular route has also been described for this toxin. It involves direct translocation of the entire toxin from endosomes to the cytosol and therefore does not rely on furin-mediated cleavage. To examine the implications of endosomal translocation in the intoxication process, we investigated whether the toxin required furin-mediated processing in order to kill cells. We used three different approaches. We first fused to the N terminus of th...
Cloning and expression of the immunogenic moiety of Pseudomonas aeruginosa exotoxin A
Introduction: Pseudomonas aeruginosa, as an opportunistic microorganism, is a major cause of nosocomial infections worldwide. Exotoxin A (ETA) is an extracellular enzyme that is produced by most clinical strains of P. aeruginosa. Although the pathogenesis of the diseases due to Pseudomonas are complex, clinical and experimental data linking ETA with the morbid and lethal consequences of Pseudomonas infection are accumulating. Materials and methods: An immunogenic 490–bp DNA segment including translocation domain plus 1b domain of the ETA from P. aeruginosa strain PAO1 were reproduced by PCR. The PCR product was cloned in E.coli DH5α and expressed in E.coli BL21 using recombinant pET28a vector. The cloned polypeptide was found to have an electrophoretic mobility in sodium dodecyle sulfate- polyacrylamide gels (SDS -PAGE) of 18kDa. Results: PCR and colony PCR results approved the cloning of immunogenic moiety of exotoxin A. Analysis of the location of cloned polypeptide by SDS- PAGE electrophoresis revealed that it was exported by E.coli into the bacterial periplasmic space. Discussion and conclusion: Since the whole toxin is not necessary for enhancing the immune responses and this recombinant polypeptide has antigenic qualities, so it may serve as a useful vaccine to prevent Pseudomonas infections. Key words: Pseudomonas aeruginosa PAO1, Cloning, Exotoxin A, Immunogenic moiet
Journal of Bacteriology, 2000
Pseudomonas aeruginosa is a gram-negative bacterium that secretes many proteins into the extracellular medium via the Xcp machinery. This pathway, conserved in gram-negative bacteria, is called the type II pathway. The exoproteins contain information in their amino acid sequence to allow targeting to their secretion machinery. This information may be present within a conformational motif. The nature of this signal has been examined for P. aeruginosa exotoxin A (PE). Previous studies failed to identify a common minimal motif required for Xcp-dependent recognition and secretion of PE. One study identified a motif at the N terminus of the protein, whereas another one found additional information at the C terminus. In this study, we assess the role of the central PE domain II composed of six α-helices (A to F). The secretion behavior of PE derivatives, individually deleted for each helix, was analyzed. Helix E deletion has a drastic effect on secretion of PE, which accumulates within th...
Avicenna Journal of Clinical Microbiology and Infection, 2017
Background Pseudomonas aeruginosa is a gram-negative and an opportunistic pathogen which grows in minimal nutritional requirements and a wide range of temperature. It can grow on most surfaces, especially moist surfaces such as medical devices and skin (1,2). P. aeruginosa in weakened and immunosuppressed patients, who are with third-degree burns, cystic fibrosis (CF), wounds, indwelling catheter, and prolonged duration of ventilation, can cause nosocomial diseases (3). Multiple factors are involved in pathogenicity of P. aeruginosa. The type III secretion system (TTSS) which has been known to be a major virulence, is determined in pathogenesis of acute infection, bacteremia, sepsis, and subsequent mortality. The TTSS allows the injection of toxins into the cytosol of target eukaryotic cells, where they destroy host cell defense and signaling systems and subsequently rapid call necrosis or modulating the actin cytoskeleton (4-6). Four effector proteins have been identified: ExoU, a phospholipase which has been characterized as a major virulence factor in acute lung injury, ExoY, which is an adenylate cyclase, and ExoS as well as ExoT which are bifunctional proteins (5,6). ExoU and ExoS are variably present and are important in pathogenesis, whereas almost all of the isolates encode ExoT and ExoY and have a minor effect on virulence (4,5). Previous studies have shown that production of ExoU was correlated with increasing virulence (7). Similarly, in other studies, it was found that infected patients with TTSS + isolates show more severe infections and the mortality rate of this patients in first 30 days of infection is high (3,8). The existence of many agents in P. aeruginosa leads to intrinsic resistance to many antimicrobials including bacterium's outer membrane barrier, the presence of multi-drug efflux transporters, and endogenous antimicrobial inactivation. All of these agents, as well as inappropriate chemotherapy and lagging in antibiotic discovery caused "antibiotic resistance crisis" (6,9). Previous studies demonstrated
Infection and Immunity, 1978
Evidence is presented which suggests that both the proteases and the exotoxin produced by Pseudomonas aeruginosa multiplying in situ in a burned mouse model are virulence factors. A 50% decrease in functional elongation factor 2 (EF-2) was seen 16 h postinfection in the liver of mice infected with the toxigenic, protease-producing P. aeruginosa strain M-2; at the time of death EF-2 was depleted by 80%. This correlates with a reduction in the level of protein synthesis in the liver of infected animals. Treatment with specific antitoxin extended the mean time to death and blocked depletion of EF-2. Administration of gentamicin 24 h after infection caused rapid clearance of bacteria and extended the mean time to death, but all animals treated with either antitoxin or gentamicin eventually died. In contrast, treatment with both antitoxin and gentamicin provided virtually complete protection. Infection of mice with P. aeruginosa WR5 (protease-producing, nontoxigenic) or with P. aeruginosa PA103 (toxigenic, slow protease producer) required several logs more bacteria and did not result in the same extensive depletion in EF-2 content. When challenge with PA103 was supplemented by injection of purified Pseudomonas protease, the mean time to death was shortened and significant reduction in liver EF-2 was observed. It is suggested that both toxin and proteases are required for the full expression of virulence in Pseudomonas infections.
Mouse liver contains a Pseudomonas aeruginosa exotoxin A-binding protein
Infection and Immunity, 1991
The opportunistic pathogen Pseudomonas aeruginosa produces several potential virulence factors, including the ADP-ribosylating toxin, exotoxin A (PE). Studies using a burned mouse model have shown that PE consistently inhibits protein synthesis and depletes elongation factor 2 in mouse liver and variably in other organs. One reason for toxin sensitivity could be the presence of a PE receptor on the surface of cells. Therefore we examined detergent extracts of mouse tissues for the presence of toxin-binding proteins. Proteins which specifically bind PE were present in extracts from liver, kidney, lung, spleen, and heart. Because liver appears to be a prominent target for the toxin in a burned animal, we choose to isolate the PE-binding protein from mouse liver and compare this protein to the recently characterized toxin-binding protein from toxin-sensitive mouse LM fibroblasts. The toxin-binding proteins from both sources have a molecular mass of approximately 350 kDa, share similar ...
Ecto-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exoenzyme S
Infection and immunity, 1997
Pseudomonas aeruginosa produces two ADP-ribosyltransferases, exotoxin A and exoenzyme S (ExoS). Although the physiological target protein remains to be defined, ExoS has been shown to ADP-ribosylate several eukaryotic proteins in vitro, including vimentin and members of the family of low-molecular-weight GTP-binding proteins. Recently, ExoS ADP-ribosyltransferase activity has been detected in the pleural fluid of rabbits infected with P. aeruginosa. This observation prompted an examination of the potential for ExoS to function as an ecto-ADP-ribosyltransferase. We have observed that ExoS preferentially ADP-ribosylated two extracellular serum proteins with molecular masses of 150 and 27 kDa. The ADP-ribosylation of these serum proteins by ExoS was stimulated by, but not dependent upon, exogenous FAS (for factor activating exoenzyme S), which indicated that serum contained endogenous FAS activity. Biochemical analysis showed that the 150-kDa ADP-ribosylated protein was immunoglobulin ...