Analysis of sequences required for the cytotoxic action of a chimeric toxin composed of Pseudomonas exotoxin and transforming growth factor alpha

Chimeric toxins composed of transforming growth factor alpha fused to mutant forms of Pseudomonas exotoxin bind to the EGF receptor and kill cells bearing these receptors. In early experiments, the binding domain of Pseudomonas exotoxin was deleted and replaced with TGF alpha to make TGF alpha-PE40. This chimeric toxin required proteolytic processing within the target cell to be converted to its active form (Siegall et al. (1989) FASEB J. 3, 2647-2652). Subsequently, recombinant toxins that do not require proteolytic processing were constructed by inserting TGF alpha near the carboxyl terminus of domain III and deleting toxin residues up to the processing site at position 280. In addition, the carboxyl terminus of this toxin was converted from REDLK to KDEL to increase its activity. Recombinant toxins of this type, termed PE37/TGF alpha/KDEL, are about 100-fold more potent than TGF alpha-PE40. To determine if other sequences can be removed from such chimeric toxins to make a smaller molecule that can penetrate tissues better, we have carried out a deletion analysis of sequences present within domains II and Ib. We find that all of domain Ib and a portion of domain II can be deleted without significant loss of cytotoxic activity, but larger deletions extending further into domain II lose cytotoxic activity. We also find that inserting a small linking peptide (Gly)4Ser between residual sequences in domain II and domain III, in molecules with diminished cytotoxic activity, enhances cytotoxicity suggesting that one role of domain Ib is to prevent undesirable interactions between domains II and III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human glioma cells overexpress receptors for interleukin 13 and are extremely sensitive to a novel chimeric protein composed of interleukin 13 and pseudomonas exotoxin

Recently, we have demonstrated that a spectrum of human adenocarcinoma cell lines express binding sites for interleukin 13 (IL-13). These cells are killed by a chimeric protein composed of human (h) IL-13 and a derivative of Pseudomonas exotoxin, PE38QQR (Debinski et al., J. Biol. Chem., 270: 16775-16780, 1995). The cell killing was hIL-13- and hIL-4-specific, indicating that a common binding site for the two cytokines is present in several solid tumor cell lines. Herein, we report that an array of established glioma cell lines is killed by very low concentrations of hIL-13-PE38QQR, often reaching <1 ng/ml (<20 pM). Glioma cells express up to 30,000 molecules of IL-13 receptor/cell which has intermediate affinity toward hIL-13. hIL-13-PE38QQR is more active (up to 3 logs difference in cytotoxic activities) than are the corresponding chimeric toxins containing hIL-4 or hIL-6. The cytotoxic action of hIL-13-PE38QQR is blocked by an excess of hIL-13 on all cell lines studied, and it is not neutralized by hIL-4 on some of these cells. Our results show that human brain cancers richly express receptors for IL-13. Furthermore, the interaction detected previously between receptors for IL-13 and IL-4 on solid tumors cell lines is of a qualitatively different character in U-251 MG and U-373 MG glioma cells. The receptor for IL-13 may represent a new marker of brain cancers and an attractive target for anticancer therapies.     

Generation of neutralizing antipeptide antibodies to the enzymatic domain of Pseudomonas aeruginosa exotoxin A

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.     

Acute hepatotoxicity of Pseudomonas aeruginosa exotoxin A in mice depends on T cells and TNF

The most potent virulence factor of Pseudomonas aeruginosa, its exotoxin A (PEA), inhibits protein synthesis, especially in the liver, and is a weak T cell mitogen. This study was performed to correlate hepatotoxic and possible immunostimulatory features of PEA in vivo. Injection of PEA to mice caused hepatocyte apoptosis, an increase in plasma transaminase activities, and the release of TNF, IFN-gamma, IL-2, and IL-6 into the circulation. Most strikingly, liver damage depended on T cells. Athymic nude mice or mice depleted of T cells by anti-Thy1.2 mAb pretreatment failed to develop acute hepatic failure, and survival was significantly prolonged following T cell depletion. Neutralization of TNF or lack of TNF receptors prevented liver injury. In the liver, TNF was produced by Kupffer cells before hepatocellular death occurred. After T cell depletion, Kupffer cells failed to produce TNF. Transaminase release was significantly reduced in perforin knockout mice, and it was even elevated in lpr/lpr mice. These results demonstrate that PEA induces liver damage not only by protein synthesis inhibition but also by TNF- and perforin-dependent, Fas-independent, apoptotic signals.     

Overexpression and large-scale purification of recombinant hamster polymorphic arylamine N-acetyltransferase as a dihydrofolate reductase fusion protein

N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics. Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology. This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site to allow liberation of the rNAT. Typically, however, only 0.5-1.5 mg of the recombinant NAT's could be readily purified in a single isolation sequence by immunoaffinity chromatography. Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant. Unfortunately, the affinity chromatography strategy did not work satisfactorily. Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered. The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX. Ion-exchange chromatography was investigated as an alternative purification method. An initial DEAE anion-exchange column resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin and reapplied to a DEAE anion-exchange column. The second DEAE column resulted in not only the separation of rNAT2-70D from FLAG-L54F DHFR, but also the purification of rNAT2-70D to near homogeneity. Application of the nearly homogeneous rNAT2-70D to a gel-filtration column resulted in recovery of homogeneous protein. The ion-exchange method of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture.     

In vitro enzyme activation and folded stability of Pseudomonas aeruginosa exotoxin A and its C-terminal peptide

Pseudomonas aeruginosa exotoxin A(ETA) and its C-terminal, enzymatically active fragment (PE40, 375 residues) were studied by high-performance size-exclusion chromatography, steady-state and stopped-flow fluorescence spectroscopy, and circular dichroism spectroscopy. Both proteins have been overexpressed and purified by high-performance liquid chromatography. The effect of various activation conditions (pH, urea, and DTT) on enzymatic activity was studied. Upon enzymatic activation, structural changes induced within both proteins' structures were monitored, and these changes were correlated with concomitant alterations in the catalytic activity of the proteins. The pH optimum of enzymatic activity for both ETA and PE40 was between 7.0 and 8.0, decreasing to nearly zero at acidic (pH 5.0) and basic (pH 11-12) values. Analysis of the pH titration data revealed the presence of two distinct pKa values which implicate a His residue(s) (likely His-440 and -426) and a Tyr or Lys residue (possibly Tyr-481). The identity and possible role of an active site Lys residue is not known. Additionally, a significant increase in the Stokes radii of both proteins was detected when the pH was lowered from 8.0 to 6.0. The enzymatic activity of PE40 was not affected by urea or DTT, and its Stokes radius decreased monotonically with increasing urea concentration in the presence of DTT. In contrast, the enzymatic activity of ETA peaked when the protein was preincubated with 4.0 M urea, and this coincided with a large transition (increase) in the protein's Stokes radius between 3 and 5 M urea. Furthermore, loss of helical secondary structure of both PE40 and ETA commenced at approximately 2 M urea and progressively diminished at higher denaturant concentrations. The unfolding of both proteins in urea (and DTT) was reversible, and the free energies of unfolding were determined by both circular dichroism and fluorescence spectroscopy and were found to be 13.7 +/- 2.9 and 9.8 +/- 3.4 kJ/mol, respectively, for ETA and were 17.8 +/- 6.8 and 7.5 +/- 3.6 kJ/mol, respectively, for PE40. The refolding rate of PE40 was relatively rapid [t 1/2(1) = 27 s, t 1/2(2) = 624 s], which was in stark contrast to the refolding rate of ETA (t 1/2 = several hours). The relative refolding rates of PE40 and ETA help to explain the mechanism of in vitro enzyme activation and assay.     

Evaluation of protein A and protein G as indicator system in an ELISA for detecting antibodies in mink to Pseudomonas aeruginosa

A modified, indirect enzyme-linked immunosorbent assay (ELISA) was developed and applied in the detection of mink antibodies to Pseudomonas aeruginosa. In this assay, peroxidase conjugated protein A and protein G were evaluated as indicator systems for detecting antigen-antibody complexes. It was found that protein A has a strong affinity for mink immunoglobulins. In contrast, protein G showed no such affinity. The affinity of protein A for mink immunoglobulins was further demonstrated by immunoprecipitation assays.     

Pseudomonas aeruginosa as a cause of infectious diarrhoea

Pseudomonas aeruginosa is not generally considered a cause of infectious diarrhoea. However, it was the predominant organism isolated from the faeces of 23 unrelated, hospital outpatients investigated in the course of a year for persistent (> 1 week duration) diarrhoea. To investigate the possible aetiological role of P. aeruginosa, these patient histories were reviewed and a selection of their faecal isolates were investigated in vitro (n > or = 10) and in vivo (n = 2) for virulence. The patients had a mean age of 60 years, were receiving antibiotics and/or had an underlying illness. Extensive microbiological investigations identified no other potential or recognized enteropathogen in the faeces of 20 of these patients. More than 40% of the isolates tested were able to adhere to HEp-2 cells and exhibited twitching motility (type IV pili), properties indicative of their ability to colonize the human intestine. Cytotoxic activity was demonstrated in bacterium-free cell supernatants of over 80% of isolates; supernatants of four isolates tested in infant mice were weakly enterotoxigenic. Two isolates intragastrically inoculated into clindamycin pre-treated rats established persistent infections and induced signs and symptoms of enteritis. Overall these findings suggest that P. aeruginosa can cause diarrhoea particularly in immunodeficient individuals.     

Protection against exotoxin A (ETA) and Pseudomonas aeruginosa infection in mice with ETA-specific antipeptide antibodies

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious and sometimes fatal infections in the compromised host, especially in patients with major trauma or thermal injuries. Exotoxin A (ETA) is the major and most lethal virulence factor produced by this ubiquitous microorganism. In a recent study (H. S. Elzaim, A. K. Chopra, J. W. Peterson, R. Goodheart, and J. P. Heggers, Infect. Immun. 66:2170-2179, 1998), we identified two major epitopes, one within the translocation domain (amino acid [aa] residues 289 to 333) of ETA and another within the enzymatic domain (aa 610 to 638), by using a panel of antipeptide antibodies. Synthetic peptides representing these two epitopes induced ETA-specific antibodies which were able to abrogate the cytotoxic activity of ETA, as measured by incorporation of [3H]leucine into 3T3 fibroblasts. In the present study, these antibodies were tested for the ability to provide protection against ETA and infection with a toxin-producing strain of P. aeruginosa in a mouse model. Antibodies to either of the synthetic peptides conferred protection against ETA. Also, when used for immunization, both peptides induced active immunity to ETA in mice. Antibodies to the peptide representing a region within the enzymatic domain of ETA, in combination with the antibiotic amikacin, enhanced the survival of mice infected with a toxin-producing strain of P. aeruginosa. Thus, antipeptide antibodies specific for ETA might be paired with antibiotic treatment for passive immunization of patients suffering from P. aeruginosa infection.     

Detection of Pseudomonas aeruginosa in bronchial and tracheal aspirates by PCR by amplification of the exotoxin A gene

For the first time, a PCR test based on the amplification of the Exotoxin A was evaluated for its ability to rapidly detect Pseudomonas aeruginosa in tracheal and bronchial aspirates from mechanically ventilated patients. The reaction is based on the amplification of a 396 bp region within the Exotoxin A gene. The results show that this PCR-method is suitable for the detection of Pseudomonas aeruginosa even in clinical samples such as aspirates. Among the 380 clinical samples tested in this way, 57 were found to be positive while only 36 were positive using routine culture. In conclusion, these results suggest that the PCR-method mentioned above can be used to provide a specific, rapid, simple, and highly sensitive detection of Pseudomonas aeruginosa in clinical samples.     

Expression and activity of a recombinant chimeric protein composed of pokeweed antiviral protein and of human interleukin-2

Liv.100 is an improvised herbal formulation of Liv.52. Liv.52 is an important component of the ayurvedic system of medicine. This report highlights on the protective effect of Liv.52 and Liv.100 against in vitro peroxidation induced by hydrogen peroxide in rat liver homogenate. Addition of the two herbal formulations reduced the peroxidation effect of hydrogen peroxide in the dose- and time-dependent manner. The protective effect of the drugs is attributed to the enhanced supply of reduced glutathione that inhibit the deleterious process of lipid peroxidation. The results suggest on the antioxidant potential of Liv.52 and Liv.100.     

Evaluation of antimicrobial and lipopolysaccharide-neutralizing effects of a synthetic CAP18 fragment against Pseudomonas aeruginosa in a mouse model

CAP18 (cationic antimicrobial protein; 18 kDa) is a neutrophil-derived protein that can bind to and inhibit various activities of lipopolysaccharide (LPS). The 37 C-terminal amino acids of CAP18 make up the LPS-binding domain. A truncated 32-amino-acid C-terminal fragment of CAP18 had potent activity against Pseudomonas aeruginosa in vitro. We studied the antimicrobial and LPS-neutralizing effects of this synthetic truncated CAP18 peptide (CAP18106-137) on lung injury in mice infected with cytotoxic P. aeruginosa. To determine its maximal effect, the CAP18106-137 peptide was mixed with bacteria just prior to tracheal instillation, and lung injury was evaluated by determining the amount of leakage of an alveolar protein tracer (125I-albumin) into the circulation and by the quantification of lung edema. The lung injury caused by the instillation of 5 x 10(5) CFU of P. aeruginosa was significantly reduced by the concomitant instillation of CAP18106-137. However, the administration of CAP18106-137 alone, without bacteria, induced lung edema, suggesting that it has some toxicity. Also, the peptide did not significantly reduce the number of bacteria that had been simultaneously instilled, nor did it significantly improve the survival of the infected mice. The addition of CAP18106-137 to aztreonam along with the bacteria did decrease the level of antibiotic-induced release of inflammatory mediators including tumor necrosis factor alpha, interleukin-6, and nitric oxide and also improved the survival of the mice. Therefore, more investigations are needed to confirm the toxicities and the therapeutic benefits of CAP18106-137 as an adjunctive therapy to antibiotics in the treatment of infections caused by gram-negative bacteria.     

GSP-dependent protein secretion in gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa

Bacteria have evolved several secretory pathways to release proteins into the extracellular medium. In Gram-negative bacteria, the exoproteins cross a cell envelope composed of two successive hydrophobic barriers, the cytoplasmic and outer membranes. In some cases, the protein is translocated in a single step across the cell envelope, directly from the cytoplasm to the extracellular medium. In other cases, outer membrane translocation involves an extension of the signal peptide-dependent pathway for translocation across the cytoplasmic membrane via the Sec machinery. By analogy with the so-called general export pathway (GEP), this latter route, including two separate steps across the inner and the outer membrane, was designated as the general secretory pathway (GSP) and is widely conserved among Gram-negative bacteria. In their great majority, exoproteins use the main terminal branch (MTB) of the GSP, namely the Xcp machinery in Pseudomonas aeruginosa, to reach the extracellular medium. In this review, we will use the P. aeruginosa Xcp system as a basis to discuss multiple aspects of the GSP mechanism, including machinery assembly, exoprotein recognition, energy requirement and pore formation for driving through the outer membrane.     

A new procedure for improving the predictiveness of CoMFA models and its application to a set of dihydrofolate reductase inhibitors

A new automated procedure to improve the predictive quality of CoMFA models for both training and test sets is described. A model of greater consistency is generated by performing small reorientations of the underlying molecules for which too low activities are calculated. In order to predict activities of test compounds, the most similar molecules in the previously optimized model are identified and used as a basis for the prediction. This method has been applied to two independent sets of dihydrofolate reductase inhibitors (80 compounds each, serving as training sets), resulting in a significant increase of the cross-validated r2 value. For both models, the predictive r2 value for a test set consisting of 70 compounds was improved substantially.     

In vivo effect of growth-inhibitor produced by Pseudomonas aeruginosa isolates and anti-pseudomonal drugs on model infection due to Pseudomonas aeruginosa

Pseudomonas aeruginosa Nos. 1 and 5, each co-existing growth-inhibitor-producing and -nonproducing cells, were used in this study. An equal number of both cells (each 10(8) CFU/mouse) was challenged intraperitoneally to mice, and these cells in the heart blood and kidneys of mice were determined. Furthermore, the effect of piperacillin, ceftazidime and sisomicin on the cell distribution in mice was studied in the model infection due to P. aeruginosa Nos. 1 and 5. As a control experiment both cells of P. aeruginosa No. 1 were each challenged intraperitoneally at a dose of 10(8) CFU/mouse to mice of two groups, but there were no marked differences between the two types in cell counts of the heart blood or kidneys 9 hours after challenge. When a concomitant challenge of both cells (each 10(8) CFU/mouse) was performed in mice, the number of growth-inhibitor-producing cells of the heart blood and kidneys was about 100 times greater than that of the non-producing cells. These in vivo results were well comparable to the previous in vitro results and indicated that the inhibitor affected the invasion of the non-producing bacteria in the body in the model infection due to P. aeruginosa isolates consisting of the two types of cells. Similar results were obtained in mice with the model infection due to P. aeruginosa No. 5. Anti-pseudomonal drugs such as piperacillin (50 mg/mouse) and ceftazidime (50 mg/mouse) and sisomicin (1 mg/mouse) were given intramuscularly to mice infected concomitantly with both cells of P. aeruginosa No. 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyphosphate kinase as a nucleoside diphosphate kinase in Escherichia coli and Pseudomonas aeruginosa

Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or over-abundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of polyphosphate kinase (PPK) to use inorganic polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E. coli; the activity was absent from the membrane fractions obtained from E. coli mutants lacking the ppk gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2-60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5' tetraphosphate.     

Chimeric influenza viruses incorporating epitopes of outer membrane protein F as a vaccine against pulmonary infection with Pseudomonas aeruginosa

Peptide 10 (NATAEGRAINRRVE, residues 305-318 of mature protein F) is one of two linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa both of which have been shown to elicit whole cell-reactive antibodies and to afford protection in animal models against P. aeruginosa infection. Influenza A virus was chosen as a vector to present this epitope in a human-compatible vaccine. Various lengths of the peptide 10 epitope ranging from a 5-mer (GRAIN), 7-mer (AINRRVE), 8-mer (TAEGRAIN), 9-mer (GRAINRRVE), 11-mer (AEGRAINRRVE) to a 12-mer (TAEGRAINRRVE) were attempted to be presented into the antigenic B-site of the hemagglutinin (HA) of live recombinant influenza virus. Using PCR, DNA sequences encoding these various peptide 10 lengths were inserted into the HA gene of influenza A/WSN/33 virus. By using a reverse-genetics transfection system, RNA transcribed in vitro from these chimeric HA genes was reassorted into infectious virus. To date chimeric viruses have been rescued and purified containing the peptide 10 5-mer, 7-mer, 8-mer, and 11-mer. RT-PCR and sequencing have confirmed the presence of P. aeruginosa sequences in the HA RNA segment of each chimeric virus. Each of the four chimeric viruses produced to date was used to immunize mice to determine the ability of each chimeric virus to elicit antibodies reactive with whole cells of P. aeruginosa. The immunization protocol consisted of a series of three intranasal inoculations, followed by two intramuscular injections of the chimeric virus. The chimeric virus incorporating the 11-mer elicited IgG antibodies that reacted with various immunotype strains of P. aeruginosa in a whole cell ELISA at titers of 80 to 2,560, whereas the chimeric virus incorporating the 8-mer elicited whole cell-reactive IgG antibodies at titers of 320 to 2,560. These data suggest that these two chimeric viruses may have vaccine efficacy against P. aeruginosa infection. These studies may result in the development of a chimeric influenza virus-protein F vaccine which would prove to be suitable for use in children with cystic fibrosis for the prevention of pulmonary colonization of these children with P. aeruginosa.