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Synthetic cells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, it is shown for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules, up to 2 × 10 7 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, are synthesized inside the particles. Incubating synthetic cells, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during infection natriuretic peptides, particularly CNP, could enhance the virulence of PAO1. This activity is relayed by Vfr and PtxR activation, and a general diagram of the virulence activation cascade involving AHL, HCN and exotoxin A is proposed.

Using strepavidin as a scaffold, we have assembled a composite immunotoxin that consists of recombinant Pseudomonas exotoxin A subunit (PE38) and recombinant 25-D1.16 Fab fragment which recognizes the SIINFEKL (pOV8) peptide from ovalbumin in association with H-2K(b) MHC class I protein. The composite immunotoxin exercises cytotoxicity against H-2K(b+) cells sensitized with pOV8 peptide but not with irrelevant peptide. Specific binding of the immunotoxin to H-2K(b+) cells infected with recombinant rabies virus (RV) expressing pOV8 epitope (RV-pOV8) resulted in the suppression of the production of virus particles by the infected cells. This strategy allows readily produce different immunotoxins with desired specificity by combining various targeting and toxin molecules. The results provide a proof of concept that composite immunotoxins can be utilized as novel immunotherapeutics to stop virus spread in the acute phase of the infection allowing winning time for the development of protective immune response., (2009 Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa exotoxin A (ETA) production depends on the virulence-factor regulator Vfr. Recent evidence indicates that the P. aeruginosa iron-starvation sigma factor PvdS also enhances ETA production through the ETA-regulatory gene regA. Mutants defective in vfr, regA and pvdS, plasmids that overexpress these genes individually and lacZ transcriptional/translational fusion plasmids were utilized to examine the relationship between vfr, regA and pvdS in regulating P. aeruginosa ETA production. ETA concentration and regA expression were reduced significantly in PAODeltavfr, but pvdS expression was not affected. Overexpression of Vfr produced a limited increase in ETA production in PAODeltapvdS, but not PAODeltaregA. Additionally, overexpression of either RegA or PvdS did not enhance ETA production in PAODeltavfr. RT-PCR analysis showed that iron did not affect the accumulation of vfr mRNA in PAO1. These results suggest that: (i) Vfr enhances toxA expression in PAO1 both directly and indirectly through regA, but not through pvdS; (ii) vfr expression is not regulated by iron; and (iii) both Vfr and PvdS cooperate in the presence of RegA to achieve a maximum level of toxA expression.

The objective of the experiment is to explore the purification and production of immunotoxin. The chimeric toxin, which is composed of 40 peptides of interleukin 10 (from amino acids 18 to 57) fused to a mutant form of Pseudomonas extoxin (PE) devoid of its native cell recognition domain. Two kinds of prokaryotic expression vector containing the chimeric toxin IL-10(18-57)-PE40 were constructed respectively. After induction of IPTG for 3 hours, IL-10(18-57)-PE40 was expressed highly in cytoplasmic fraction in Rosettablue(DE3), and was directed to periplasmic space as soluble form in E. coli BL21(DE3)pLysS . Western -blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PE40. With the application of salting out of (NH4)2SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography, the purity of IL-10(18-57)-PE40 was about 96%. The cytotoxicity assay, Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10(18-57) based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.

Within certain infection sites, such as the lung of cystic fibrosis patients, Pseudomonas aeruginosa grows statically under either decreased oxygen tension or anaerobic conditions, a situation that is likely to influence the production of virulence factors. The goal of this study was to determine the effect of static growth under microaerobic (decreased oxygen) and anaerobic conditions on the expression of the P. aeruginosa exotoxin A (ETA) gene toxA and its positive regulator ptxR. Using toxA-lacZ and ptxR-lacZ fusion plasmids, the level of toxA and ptxR expression was measured throughout the growth cycle of strain PAO1, which was grown in either iron-deficient or iron-sufficient medium under four different conditions: 20%-SH (aerobic, shaking), 20%-ST (aerobic, static), 10%-ST (microaerobic, static) and 0%-ST (anaerobic, static). In iron-deficient medium, toxA expression was higher under 20%-ST and 10%-ST than under 20%-SH. However, the highest level of toxA expression occurred under 0%-ST. Analysis of ETA protein using sandwich ELISA revealed that at time points between 8 and 24 h of the growth curve, PAO1 produced higher levels of ETA under 0%-ST than under 20%-SH. In iron-sufficient medium, toxA expression was significantly repressed under all conditions. Additional analyses using PAO1 strains that carry lacZ fusions with the toxA regulatory genes regA and pvdS revealed that the expression of regA and pvdS is reduced rather than increased at 0%-ST. ptxR expression under different conditions paralleled that of toxA expression, except that it was repressed by iron under 20 %-SH only. Between 6 and 24 h of growth, and under all conditions, the level of dissolved oxygen (DO) within the PAO1 cultures was sharply reduced. These results suggest that (1) the combined effect of static growth and anaerobic conditions produce a significant increase in toxA and ptxR expression in PAO1; (2) this effect appears to be unique to toxA and ptxR, since the level of regA and pvdS expression was reduced under the same conditions; (3) neither static growth nor anaerobic conditions interfere with the repression of toxA expression by iron, although static growth deregulates ptxR expression with respect to iron; and (4) the enhanced expression of toxA and ptxR is not related to the reduced levels of DO in PAO1 cultures.

Objective: To construct the interleukin-18-PE38 fusion gene expression vector and explore the expression of the fusion gene in the chondrocyte., Methods: The recombinant eukaryotic expression vector PsecTag2B-IL-18-PE38 was constructed by inserting interleukin-18-PE38 fusion gene into eukaryotic expression vector PsecTag2B with molecular cloning technique. It was confirmed by restrictive enzymes (EcoR I) digestion assay and PCR. The vector was transfected into primary chrondrocyte by liposome protocol, and the transient expression was identified by fluorescence immunocytochemical assay., Results: Restrictive enzymes digestion analysis and PCR revealed that the interleukin-18-PE38 fusion gene was cloned into the eukaryotic expression vector PsecTag2B successfully. Immunofluorescence photograph of fluorescence immunocytochemical method confirmed that the fusion gene can be expressed in the cytomembrane and cytoplasm., Conclusion: The results confirmed that PsecTag2B-IL-18-PE38 fusion gene can be expressed in the chondrocyte, which could serve as a foundation for the study on rheumatoid arthritis therapy.

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.

Polymerase chain reaction (PCR)-based detection and transcription of the gene encoding a potent virulence factor, the exotoxin A, were done on 32 isolates of Pseudomonas aeruginosa belonging to 23 genotypes. These isolates were obtained from 22 patients who were admitted to the emergency room in a medical center during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa. Patients showed symptoms that ranged from mild to severe. PCR amplification of a 396-bp fragment of the gene encoding the exotoxin A was done on extracted DNA. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was performed on extracted RNA to detect exotoxin A gene mRNA transcripts. Quantitation of RT-PCR amplicons from P. aeruginosa isolates associated with mild and severe symptoms was determined by end-point titration of c-DNA and scanning of amplicons with the Storm Gel and Blot Imaging System. Data have shown that all of the 32 isolates of P. aeruginosa carry the exotoxin A gene, and all isolates with the exception of two had the exotoxin A transcription demonstrated by the production of a 396-bp amplicon from RT-PCR-amplified RNA. The remaining two isolates amplified fragments that were slightly smaller than the expected size. Additional studies are needed to characterize these two mRNA transcripts. Transcription levels of exotoxin A gene associated with severe symptoms were significantly more elevated than those associated with mild to moderate symptoms. Studies are under way to determine expression of P. aeruginosa exotoxin A by detecting quantitatively levels of the translated exotoxin A protein produced by isolates associated with severe and mild to moderate symptoms.

The ExoS regulon of Pseudomonas aeruginosa encodes diverse type III secreted effector proteins which have been shown to exert cytotoxic effects in cell culture experiments. However, little information exists about the environmental conditions and stimuli for upregulation of the ExoS regulon. Translational reporter fusion proteins of exoenzyme (Exo) S, ExoT and ExoU, as well as the type II secreted exotoxin A (ETA) to the green fluorescent protein (GFP), were constructed in order to compare exoprotein production under diverse growth conditions. Reporter protein activity was recorded by FACS-analysis and by conventional and confocal laser scanning microscopy. Low ion concentration induced co-ordinated upregulation of ExoS, ExoT and ExoU with a maximum effect at 37 degrees C. A dose-dependent upregulation was seen with human serum or increasing NaCl concentrations. A type III secretion-negative pcrD mutant of P. aeruginosa showed a weak ExoS response to environmental stimuli, compared with the parental strain, suggesting a negative regulatory mechanism. Co-culture with the mammalian cell lines J774A.1 or HeLa led to rapid upregulation of ExoS, ExoT and ExoU synthesis. These data suggest that the ExoS regulon of P. aeruginosa can be triggered by a variety of environmental signals as well as by cell contact with eukaryotic cells., (Copyright 2000 Academic Press.)

The role of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in the lung infections of cystic fibrosis (CF) patients is not well understood. Transcript-accumulation studies of bacterial populations in sputum reveal high levels of transcription of toxA, which encodes ETA, in some patients with CF. However, in general, tissue damage in the lungs of patients with CF does not seem to be consistent with a high level of expression of active ETA. To address this discrepancy the authors analysed the production and activity of ETA produced by a number of P. aeruginosa CF isolates. One CF isolate, strain 4384, transcribed toxA at levels similar to the hypertoxigenic strain PA103 but produced an ETA with reduced ADP-ribosyltransferase (ADPRT) activity. Complementation in trans of strain 4384 with the wild-type toxA and a mixed toxin experiment suggested the absence of inhibitory accessory factors within this strain. The toxA gene from strain 4384 was cloned and sequenced, revealing only three mutations in the gene, all within the enzymic domain. The first mutation changed Ser-410 to Asn. The second mutation was located within an alpha-helix, altering Ala-476 to Glu. The third mutation, Ser-515 to Gly, was found at the protein surface. To date, Ser-410, Ala-476 and Ser-515 have not been reported to play a role in the ADPRT activity of ETA. However, it may be the combination of these mutations that reduces the enzymic activity of ETA produced by strain 4384. Expression of 4384 toxA and wild-type toxA in an isogenic strain revealed that 4384 ETA had 10-fold less ADPRT activity than wild-type ETA. ETA purified from strain 4384 also demonstrated 10-fold less ADPRT activity as compared to wild-type ETA. Cytotoxicity assays of purified ETA from strain 4384 indicated that the cytotoxicity of 4384 ETA is not reduced; it may be slightly more toxic than wild-type ETA. Analysis of five other CF isolates revealed a similar reduction in ADPRT activity to that seen in strain 4384. Sequence analysis of the enzymic domain of toxA from the five CF strains identified a number of mutations that could account for the reduction in ADPRT activity. These results suggest that some CF isolates produce an ETA with reduced enzymic activity and this may partially explain the pathogenesis of chronic lung infections of CF due to P. aeruginosa.

The expression of the toxA and regA genes in Pseudomonas aeruginosa is negatively regulated by iron at the transcriptional level. We have previously described ptxR, an exotoxin A regulatory gene which appears to enhance toxA expression through regA. In this study, we have tried to determine if ptxR expression correlates with its effect on toxA and regA expression throughout the growth cycle of P. aeruginosa strain PAO1. This was done using Northern blot hybridization experiments (with toxA, regA, and ptxR probes), and ptxR transcriptional fusion studies. To avoid problems related to the presence of multiple copies of ptxR in PAO1, we have constructed a PAO1 strain (PAO1-XR) that carries only two ptxR genes in its chromosome. Our results showed that when PAO1-XR was grown in iron-limited conditions, the increase in exotoxin A activity and the accumulation of toxA mRNA appeared at about mid- to late-exponential phase. A similar increase in the accumulation of regA mRNA was detected. Both regA transcripts, T1 and T2, were enhanced in PAO1-XR. In iron-sufficient medium, neither toxA nor regA mRNA was detected at any time point in the growth cycle of PAO1-XR. In contrast, the accumulation of ptxR mRNA was detected throughout the growth cycle of PAO1-XR under both iron-deficient and iron-sufficient conditions. The presence of iron in the growth medium also had no effect on the level of beta-galactosidase activity produced by a ptxR-lacZ fusion in PAO1. These results suggest that (i) the enhancement in toxA expression by ptxR correlates with the enhancement in regA expression; (ii) ptxR affects the expression of the regA P1 and P2 promoters; (iii) ptxR expression precedes its effect on toxA and regA expression; and (iv) unlike toxA and regA, the overall expression of ptxR throughout the growth cycle of PAO1 is not negatively regulated by iron.

We have previously characterized several Pseudomonas aeruginosa isolates that were obtained from patients with tracheal, urinary tract, or wound infections (A. H. Hamood, J. A. Griswold, and C. M. Duhan, 1996, J. Surg. Res. 61: 425). Analysis of additional isolates showed that regardless of the isolation site, some isolates produced significantly higher or significantly lower levels of either exotoxin A or exoenzyme S proteins. These variations did not correlate with the mucoid phenotype of the isolates. One aim of this study was to determine if the variations in the level of exotoxin A or exoenzyme S are due to DNA rearrangements within either the toxA or the exoS gene. This was accomplished by Southern blot hybridization experiments using a toxA internal probe, a toxA upstream probe, or an exoS internal probe. Hybridization with the toxA internal probe produced a 0.8-kb hybridizing fragment, whereas hybridization with the exoS internal probe produced either a 2.0- or a 2.3-kb hybridizing fragment. Hybridization with the toxA upstream probe, however, produced hybridizing fragments of varying sizes, regardless of their isolation site. Isolates that showed a similar hybridization fragment with either the toxA upstream probe or the exoS internal probe produced variable levels of exotoxin A or exoenzyme S. These results suggest that: [1] specific location within the host has no effect on either the mucoid phenotype of the isolate or the level of exotoxin A or exoenzyme S produced by the isolates; [2] although restriction polymorphism exists within the toxA upstream region, both the toxA and the exoS structural genes are relatively conserved; and [3] variations in the level of exoenzyme S and exotoxin A produced by different isolates do not correlate with either the observed heterogeneity within the toxA upstream region or the mucoid phenotype of the isolates., (Copyright 1999 Academic Press.)

Glutamate enhances the yield of exotoxin A (ETA), which is induced by iron limitation, from Pseudomonas aeruginosa. We tested the possibility that glutamate affects growth during iron restriction. We confirmed that iron limitation caused early entry into stationary phase but had no effect on the exponential growth rate. We showed that glutamate, as well as citrate and isocitrate, partially overcame this growth limitation. Glutamate had no effect on toxA (ETA-encoding) transcription, which implies that glutamate primarily increases the number of toxin-producing cells. In contrast, citrate and isocitrate diminished toxA transcription. Since glutamate, citrate, and isocitrate stimulated growth, we suspected a block in the citric acid cycle. Iron limitation reduced the activity of the iron-containing aconitase 12-fold but had no effect on isocitrate dehydrogenase activity, which was assayed as a control. There is a reciprocal relationship between aconitase activity and ETA synthesis, and this correlation does not appear to be coincidental because aconitase-specific effectors affect ETA synthesis. We tested whether a metabolic block is sufficient to induce ETA synthesis, but an aconitase-specific inhibitor diminished ETA production, which argues against this possibility. Finally, we present preliminary evidence that iron limitation may reversibly and posttranslationally inactivate aconitase in vivo. In summary, the environmental factors that stimulate ETA synthesis are related: glutamate bypasses an iron limitation-dependent metabolic block that causes entry into stationary phase. We speculate that one or more of the aconitases in P. aeruginosa may contribute to the control of virulence factor synthesis.

The role of Pseudomonas aeruginosa quorum-sensing systems in the lung infections associated with cystic fibrosis (CF) has not been examined. The purpose of this study was to determine if genes regulated by the LasR-LasI quorum-sensing system were coordinately regulated by the P. aeruginosa populations during the lung infections associated with CF. We also wanted to ascertain if there was a relationship between the expression of lasR, a transcriptional regulator, and some P. aeruginosa virulence factors during these infections. We extracted RNAs from the bacterial populations of 131 sputa taken from 23 CF patients. These RNAs were blotted and hybridized with probes to P. aeruginosa lasA, lasB, and toxA. The hybridization signals from each probe were ranked, and the rankings were analyzed by a Spearman rank correlation to determine if there was an association between the population transcript accumulations for the three genes. The correlations between the transcript accumulation patterns of pairs of the genes suggested that lasA, lasB, and toxA might be coordinately regulated during CF lung infections. To determine if this coordinate regulation might be due to regulation by LasR, we probed RNAs, extracted from 84 sputa, with the lasR, lasA, lasB, toxA, and algD probes. Statistical analysis indicated that lasR transcript accumulation correlated to lasA, lasB, toxA, and algD transcript accumulations. These results indicated that lasR may at least partially regulate or be coordinately regulated with lasA, lasB, toxA, and algD during the lung infections associated with CF. These results also suggested that the LasR-LasI quorum-sensing system may control the expression of at least some virulence factors in the lungs of patients with CF.

Pseudomonas aeruginosa causes a chronic infection in the lungs of individuals with cystic fibrosis. The P. aeruginosa isolates from these infections, when grown under laboratory conditions, characteristically are mucoid and produce low levels of the more destructive virulence factors, such as exotoxin A and the proteases. We wanted to determine if during the chronic lung infections associated with CF, the expression of alginate was inversely correlated to the expression of exotoxin A, elastase, and the LasA protease. We measured the transcript accumulation of algD, a marker of alginate, toxA, the structural gene for exotoxin A, lasB, the structural gene for elastase, and lasA, the structural gene for LasA protease, from the sputum bacterial populations of 23 patients. In the 131 samples tested, we frequently detected transcripts from the four genes. When a Spearman rank correlation analysis was done on the samples, we found no correlation between algD transcript accumulation and toxA transcript accumulation. This result suggested that toxA was regulated independently of algD. Curiously, we found a positive correlation between algD transcript accumulation and both lasB and lasA transcript accumulation levels. This correlation may not indicate a direct association between algD and either lasA or lasB. More likely, it indicates a common regulatory element in a cascade of regulators or a common environmental cue that triggers transcription.

The potential pathogenicity of a microorganism is a major concern for Health Canada evaluators, who will be processing new biotechnology products under the Canadian Environmental Protection Act. Potential pathogenicity is generally predicted by the results of animal pathogenicity studies. In an attempt to define surrogate data for an animal model, this study was initiated. Pseudomonas aeruginosa isolates from clinical and environmental sources were screened for their pilus type, serotype, lipopolysaccharide type, ability to evade host responses, and production of toxin A, exoenzyme S, elastase, phospholipase C, and total protease. The 50% lethal dose (LD50) of the same isolates was determined in the neutropenic mouse model of infection. An attempted correlation was drawn between each (or combinations) of the virulence determinants and the LD50. Stepwise linear regression showed that the presence of high levels of exoenzyme S in association with elastase or phospholipase C, or to a minor extent toxin A, was correlated with low numbers of bacteria required to elicit an LD50. No correlation between any of the other factors examined and virulence was detected. The data suggest that an in vitro high level of exoenzyme S production could be used as surrogate information for neutropenic mouse modelling; however, the levels of all of the extracellular enzymes should be considered when making such an assessment.

Electrophysiological changes were studied in the albino rat following instillation of Pseudomonas aeruginosa exotoxin A into the middle ear cavity through the tympanic membrane. Hearing threshold was measured by a burst-elicited, frequency-specific auditory brainstem response (ABR) technique prior to exposure, then 24 and 48 h, 5 days, 2 and 4 weeks after the toxin instillation. A single dose (1 microgram/20 microliters) of Pseudomonas aeruginosa exotoxin A raised the ABR threshold over the whole frequency range, by 5-25 dB, particularly in the high tones. All threshold shifts were of combined conductive and cochlear type, reversible, with deterioration starting at 24-48 h and recovery at 2-4 weeks. Effusion of serous fluid occurred at 24 or 48 h, resulting in conductive hearing loss. Latency/intensity curves revealed a cochlear component in addition to conductive hearing loss. Morphological examination by SEM showed slight and inconsistent derangement of OHCs. It is concluded that Pseudomonas aeruginosa exotoxin A causes middle ear inflammation, facilitating penetration to the inner ear and that this toxin also reversibly affects cochlear function.

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.

The receptor for human interleukin-9 (hIL-9) might be a target for selective immunotherapy. It is expressed on a variety of malignant cells, including Hodgkin's lymphoma, non-Hodgkin lymphoma and acute myeloid leukemia (AML). We therefore constructed a new chimeric toxin by fusing hIL-9-cDNA to modified Pseudomonas aeruginosa exotoxin A (ETA'). The binding properties of the new recombinant protein, rhIL-9-ETA', were assessed on different cell lines expressing the hIL-9 receptor. The antitumor potency of rhIL-9-ETA' was evaluated against the Hodgkin-derived cell lines L540Cy, KM-H2 and L1236, the Burkitt lymphoma cell line Daudi, the erythroleukemia cell line K562, and the mastocytoma cell line P815-hIL9R, transfected with hIL-9 receptor cDNA. Recombinant hIL-9-ETA' exhibited potent specific cytotoxic effects against P815-hIL9R, K562 and L1236 cells, inhibiting protein synthesis by 50% (IC50) at concentrations of 0.05, 0.58 and 3 micrograms/ml respectively. The cytotoxic effect was abrogated after addition of polyclonal antibodies against the human IL-9. rhIL-9-ETA' might be of potential use against hIL-9R-expressing malignancies.

Because the ferric uptake regulator (fur) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene. Two parental strains were used in this study. Three mutants were isolated from the widely used strain, PAO1. One of these, CS (cold sensitive), has a mutation in the 5' non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86-->R; mutant A4, H86-->Y. The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P. aeruginosa. Fur from the C6 mutant has an A10-->G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position. All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media. The CS mutant constitutively expressed siderophores at 25 degrees C. At 42 degrees C siderophore biosynthesis was iron repressed as in the parental strain PAO1. The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively. The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1. Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions. Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50-100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays. By contrast in the CS and C6 mutants, while iron-dependent repression os ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment. RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA. The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS) that positively regulates the expression of siderophores and ETA. Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter region of the pvdS gene, but it retained some weak binding activity in gel mobility shift assays. The data presented in this study not only furnish some additional insights into the structure-function relationships of Fur, but also afford novel perspectives of virulence factors in P. aeruginosa under environmental conditions that have not previously been considered.

Expanded bed chromatography was used for the recovery and purification of modified Pseudomonas aeruginosa exotoxin A. The exotoxin accumulates in the periplasmic space of E. coli BL21 (lambda DE3), was released from the cells by osmotic shock and captured by applying the open cell suspension directly to an anion exchanger (STREAMLINE DEAE) using an expanded bed (STREAMLINE) column. Processing of 4.5 kg of E. coli using the expanded bed process was 3 times faster and did not require clarification of the bacterial extract, in comparison with the conventional purification method. Also, the recovered protein solution was 3 times more concentrated and the yield slightly higher.

Exotoxin A production in Pseudomonas aeruginosa is a complicated and highly regulated process that involves several genes. In this report, we describe the isolation of a new toxA regulatory gene (ptxR) which affects exotoxin A production in P. aeruginosa. In an iron-deficient medium, the presence of a plasmid carrying ptxR in P. aeruginosa PAO1 resulted in a four-to fivefold increase in exotoxin A synthesis. No effect was observed on the levels of elastase, phospholipase C, exoenzyme S, and alkaline protease. Using subcloning and complementation experiments, ptxR was localized to a 2.1 kb Kpnl-Bg/II fragment. Nucleotide sequence analysis revealed the presence of an open reading frame which encodes a 34.97 kDa protein (PtxR). The size of the predicted PtxR compares closely with the 34 kDa PtxR that was synthesized in Escherichia coli using the T7 expression system. The deduced amino acid sequence of PtxR is homologous to that of several members of the LysR family of transcriptional activators. The amino-terminus region of PtxR contains a putative helix-turn-helix DNA-binding motif. Specific ptxR-deletion mutants in P. aeruginosa strains PAO1 and PA103 were constructed. In comparison with their parent strains, both mutants showed a significant reduction in the level of exotoxin A activity. However, upon extensive subculturing, the level of exotoxin A produced by the PAO1::ptxR mutant was similar to that of PAO1. Transcriptional studies, using both toxA-lacZ fusion and RNA analysis, confirmed that ptxR increases toxA and regA transcription. These results suggest that ptxR regulates (through regA) exotoxin A production at the transcriptional level.

Cleavage of p220, a component of the initiation factor eIF-4F, has been correlated with the inhibition of host translation during poliovirus infection. To obtain p220 cleavage in the absence of any other poliovirus gene products, hybrid proteins containing Pseudomonas aeruginosa exotoxin A and poliovirus protease 2Apro have been constructed. The addition of the hybrid molecules to cultured cells did not lead to substantial p220 cleavage. However, the simultaneous presence of the hybrid toxin with replicationally inactive chicken adenovirus particles results in efficient cleavage of p220 in the intact cells. Under these conditions, cellular translation continues unabated for several hours, arguing against a direct requirement for intact p220 in each round of the initiation of translation of cellular mRNAs.

This study was conducted to determine the effect of the local environment within the host on the ability of P. aeruginosa to produce different extracellular virulence factors (elastase, phospholipase C, toxin A, and exoenzyme S). A total of 105 P. aeruginosa isolates was obtained from patients with tracheal, urinary tract, and wound infections. Quantitative analysis of the virulence factors was done by growing the isolates in vitro in different defined media. Single colonies of each isolate were inoculated from the primary isolation plates into the defined medium. All four virulence factors were produced by most isolates. However, depending on the location of their isolation, the isolates varied in the level of virulence factors they produced. High levels of elastase and phospholipase C were produced by most isolates obtained from trachea, urinary tract, and wounds. A significantly higher level of toxin A was produced by wound isolates, while a significantly higher level of exoenzyme S was produced by wound and urinary tract isolates. Some P. aeruginosa strains were frequently isolated from the same site of infection (persistent infection isolates). Comparative analysis of virulence factors produced by these isolates showed that, regardless of the isolation site, subsequent isolates produced higher levels of exoenzyme S. These results suggest that: (1) elastase, phospholipase C, toxin A, and exoenzyme S are produced by P. aeruginosa isolates from different sites of infection; (2) the production of higher levels of elastase and phospholipase C is important in all types of infections, while the production of toxin A and exoenzyme S is important in wound infection; (3) persistent infection with P.aeruginosa may enhance exoenzyme S production.

B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells. In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1. The light chain (VL) domain is connected to the VH domain by a disulfide bond. This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation. Furthermore, it is more cytotoxic to antigen-positive cell lines. B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed. B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg). By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.

A number of Gram-negative bacteria, including Pseudomonas aeruginosa, actively secrete a subset of periplasmic proteins into their surrounding medium. The presence of a putative extracellular targeting signal within one such protein, exotoxin A, was investigated. A series of exotoxin A truncates, fused to beta-lactamase, was constructed. Hybrid proteins, which carry at their N- termini 120, 255, 355 or the entire 613 residues of the mature exotoxin A, were stable and were secreted into the extracellular medium. Hybrid proteins which carry residues 1-30 and 1-60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period. A hybrid with beta-lactamase was constructed which carried only the N-terminal residues 1-3 and region 60-120 of exotoxin A. It was also secreted into the culture medium, suggesting that a specific 60 amino acid domain contains the necessary targeting information for translocation of exotoxin A across the outer membrane. The secretion of the hybrid proteins is independent of the passenger protein, since a similar exotoxin A-murine interleukin 4 hybrid protein was also secreted. The extracellular targeting signal between amino acids 60 and 120 is rich in anti-parallel beta-sheets. It has been shown previously to be involved in the interaction of the exotoxin A with the receptors of the eukaryotic cells. In the three- dimensional view, the targeting region is on the toxin surface where it is easily accessible to the components of the extracellular secretion machinery.

Using PCR and oligonucleotide-directed mutagenesis, recombinant plasmids which express IL2-PE fusion genes, e.g., IL2-PE40, IL2-PE40 KDEL, IL2-PE66(4Glu), and IL2-PE66(4Glu) KDEL, were constructed and expressed in E. coli at a high level. The fusion proteins were 20-30% of the total soluble bacterial proteins, existing as inclusion bodies in the host cell. In addition, there are single EcoRI, PstI, and SmaI restriction sites at the 5', 3' terminals and the linking region of the fusion genes, respectively, in the constructed expression plasmids so that these vectors could be conveniently converted to other expression vectors containing other cytokines or toxins.

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains which are responsible for cell binding (domain Ia, amino acids 1-252), translocation into the cytosol (domain II, amino acids 253-364) and ADP-ribosylation activity (domain III, amino acids 405-613). We have previously described (Prior, T. I., FitzGerald, D. J., and Pastan, I. (1992) Biochem. 31, 3555-3559) a molecule composed of amino acids 1-412 of PE and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase (Bar), and shown that this protein (PE1-412-Bar) is toxic to cells due to its ribonuclease activity, which had been delivered to the cytosol. We have now used this model to evaluate the role played by the carboxyl end of domain II (amino acids 347-364), domain Ib, and the amino end of domain III (amino acids 405-412) in the translocation event. Toxins completely lacking domain III, termed PE1-380-Bar, or both domains Ib and III, termed PE1-364-Bar, were equally cytotoxic to a murine fibroblast cell line (L929) as was PE1-412-Bar. Extending the deletion to include part of the E-helix and all of the F-helix of domain II (amino acids 347-364) resulted in a toxin (PE1-346-Bar) that was 10-fold less toxic. Previously tested on only murine cell lines, we demonstrate that barnase toxins are cytotoxic also to a variety of human cell lines. Cytotoxicity was assessed by measuring inhibition of DNA synthesis. Surprisingly, PE1-380-Bar is not lethal when injected into mice, either intraperitoneally or intravenously, at 9 nmol, which is 2200-fold more than the amount required for killing by PE (4 pmol). In cell culture these barnase-containing toxins are 100-fold less toxic to murine fibroblast cells than PE. Barnase toxin has a greater survival time in the blood of mice than PE, with a half-life of 102 min. We conclude that domain II is sufficient to transport proteins into the cytosol. Further, since domain Ia can be replaced with other cell targeting moieties, we propose that barnase-toxins should be evaluated for utility in targeted cancer therapy.

The heregulins (HRGs) are a family of growth factors that bind direction to erbB3 and erbB4 and induce tyrosine phosphorylation of erbB2 via receptor heterodimerization. Since erbB2, erbB3, and erbB4 (erbB2-4) are often overexpressed in human breast cancer cells, we produced recombinant HRGs and a HRG-based ligand toxin to investigate the signaling events triggered by HRGs and the ability of these ligands to specifically target such cells. Recombinant HRG beta 2 stimulated the tyrosine phosphorylation of erbB2-4 in ZR-75-1 human breast cancer cells. This was accompanied by the tyrosine phosphorylation of Shc and the formation of complexes between Shc and the adapter protein Grb2. Complexes were also detected between Shc and erbB2-4. However, GRb2 was detected in erbB2 and erbB4 but not erbB3 immunoprecipitates. Thus, these receptors exhibit mechanistic differences in their coupling to Ras signaling, and HRG beta 2 administration triggers multiple inputs into the Ras signaling pathway, involving receptor-Grb2, receptor-Shc, and Shc-Grb2 complexes. HRG beta 2 addition also stimulated the association of erbB3 with phosphatidylinositol-3-kinase. In accordance with the activation of key mitogenic signaling pathways, HRG beta 2 stimulated the proliferation of MCF-7 and T-47D human breast cancer cells. Moreover, when tested for the ability to stimulate cell cycle re-entry of T-47D cells arrested under serum-free conditions, HRG beta 2 was more effective than insulin, previously the most potent mitogen identified using this system. Finally, a HRG beta 2 PE40 ligand toxin was constructed and found to exhibit cytotoxic activity against human breast cancer cells overexpressing erbB3 alone or in combination with erbB4 and/or erbB2.

The effects of exotoxin A (EXA) from Pseudomonas aeruginosa on polymorphonuclear leucocytes (PMNLs) were studied in a mouse model and in vitro. P. aeruginosa PA103, which produced EXA, was 20 times more virulent for normal mice than was its EXA-deficient mutant, PA103-29. EXA was detected in the plasma of mice infected with P. aeruginosa PA103, and its presence correlated with increasing numbers of bacteria in the blood and internal organs. A monoclonal antibody (MAb) against EXA prevented the death of the mice if it was given simultaneously with, or 2 h before, infection with strain PA103. The number of PMNLs in murine blood decreased by 50% within 30 min of intravenous injection of EXA, but this decrease was prevented by simultaneous or prior injection of MAb to the toxin. EXA inhibited in-vitro phagocytosis and killing of P. aeruginosa by human and murine PMNLs and decreased the number of the PMNLs by between 60 and 68%. Collectively, these results not only confirm that EXA is toxic in vivo, but also suggest that this toxin accelerates the growth of virulent P. aeruginosa in mice.

Procedures have been devised for producing high yields of purified recombinant PE40, a protein important for the development of the anti-AIDS therapeutic, sCD4-PE40. PE40 is a truncated form of the bacterial toxin, Pseudomonas exotoxin A; it lacks the cell-binding domain, but retains domains II and III that are involved in membrane translocation and inhibition of protein synthesis in eukaryotic cells. Expression vectors in Escherichia coli encoding PE40 synthesized the product as a soluble protein under control of the T7 promoter. The expression capabilities of transformants of E. coli BL21(DE3) were highly unstable. Expression levels (secreted and total) were evaluated in shake flasks and at the 10-1 scale at 27 degrees C and 37 degrees C, and following induction by IPTG or lactose. The cell-free media from the batch process was applied directly to a Cibacron blue F3GA-chromatographic medium and PE40 was eluted by nicotinamide in high yield and purity. This purification strategy was based on the structural similarity of the blue dye to NAD, a natural substrate for domain III of PE40. Green and red dye-ligand chromatography steps removed nicotinamide as well as minor residual E. coli proteins from PE40. Reversed-phase liquid chromatography peptide maps of purified PE40 were characterized by electrospray ionization mass spectrometry to determine the sequence and verify disulfide bonding.

The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.

To express recombinant forms of Pseudomonas aeruginosa exotoxin A in high yield, we have developed a nontoxigenic strain of P. aeruginosa derived from the hypertoxigenic strain PA103. The nontoxigenic strain, designated PA103A, was produced by the excision marker rescue technique to replace the toxA structural gene in PA103 with an insertionally inactivated toxA gene. The PA103A strain (ToxA-) was used subsequently as the host strain for the expression and production of several recombinant versions of exotoxin A, and the results were compared with exotoxin A production in other P. aeruginosa and Escherichia coli strains. Use of the PA103A strain transformed with the high-copy-number pRO1614 plasmid bearing various toxA alleles resulted in final purification yields of exotoxin A averaging 23 mg/liter of culture. By comparison, exotoxin A production in other expression systems and host strains yields approximately 1/4 to 1/10 as much toxin.

Secretion of Pseudomonas aeruginosa elastase, exotoxin A, and alkaline protease in sputum during bronchopulmonary exacerbations was examined in 18 cystic fibrosis patients chronically infected with this microorganism. The patients were studied during one or several exacerbation periods necessitating hospitalizations of 12 to 20 days. In all cases, P. aeruginosa was present in bronchial secretions at admission and was not eradicated after treatment. The P. aeruginosa density decreased significantly after antibiotic therapy but remained greater than 10(6) CFU/g of sputum in most cases. Significant amounts of P. aeruginosa exoproteins were measured in total homogenized bronchial secretions by immunoenzymatic assays. The detection of higher levels of exoproteins at admission, the significant decrease after treatment, and the absence of exoproteins during intercrisis phases constituted arguments for a renewal of virulence of P. aeruginosa during exacerbations. Nevertheless, the concomitant changes in bacteria load and the triggering of the inflammatory process and immune complex formation could also contribute to pulmonary exacerbations.

We have constructed, expressed, and purified a fusion protein, HAR-TX beta 2, consisting of heregulin-beta 2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40. The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone. The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin. HAR-TX beta 2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range. However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at > 2 micrograms/ml. Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX beta 2. All lines that express HER4 were killed by HAR-TX beta 2, while none lacking HER4 were affected. HAR-TX beta 2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity. Thus HAR-TX beta 2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells.

The Pseudomonas aeruginosa (Pa) structural gene (toxA), which encodes the exotoxin A protein has been shown to be regulated at the transcriptional level by a protein designated ToxR (also known as RegA). We have previously reported that ToxR directly enhances toxA transcription in vitro; however, in the absence of ToxR, Pa RNA polymerase (RNAP) transcribes toxA with low efficiency. In the present study, we have examined the ability of ToxR to initiate toxA transcription using the heterologous Escherichia coli (Ec) RNAP and found that ToxR can function with Ec RNAP to efficiently transcribe toxA both in vitro and in vivo. Antibodies produced against the sigma 70 subunit of Ec RNAP inhibit ToxR-mediated enhancement of toxA transcription, suggesting that the RNAP holoenzyme (E sigma 70) is required for transcriptional activation of toxA. We further demonstrate that ToxR is required for open-complex formation at the toxA promoter. By selectively deleting toxA upstream sequences, we have localized at 214-bp region containing both the toxA promoter and a putative ToxR-binding site sufficient for ToxR-mediated transcription of toxA.

The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. We cloned a gene, designated vfr for virulence factor regulator, that restored ETA and protease production to parental levels in these mutants. In addition, transcription from the regA P1 promoter was restored. In Escherichia coli, when vfr was overexpressed from a phage T7 promoter, a protein with an apparent molecular mass of 28.5 kDa was produced. Analysis of the deduced amino acid sequence of vfr revealed that the expected protein is 67% identical and 91% similar over a 202-amino-acid overlap to the E. coli cyclic AMP receptor protein (CAP or Crp). The cloned vfr gene complemented the beta-galactosidase- and tryptophanase-deficient phenotypes of E. coli RZ1331, a crp deletion mutant. However, the E. coli crp gene under the control of the tac promoter did not complement the ETA-deficient or protease-deficient phenotype of PA103-15 or PA103-16. The ability of vfr to restore both ETA and protease production to these mutants suggests that vfr is a global regulator of virulence factor expression in P. aeruginosa.

Unlike Escherichia coli, living cells of Pseudomonas aeruginosa can complete the fatty acylation of lipid A when the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo) is inhibited (R. C. Goldman, C. C. Doran, S. K. Kadam, and J. O. Capobianco, J. Biol. Chem. 263:5217-5233, 1988). In this study, we demonstrate the presence of a novel enzyme in extracts of P. aeruginosa that can transfer lauroyl-acyl carrier protein (ACP) to a tetraacyl disaccharide-1,4'-bis-phosphate precursor of lipid A (termed lipid IVA) that accumulates in Kdo-deficient mutants of E. coli. Comparable E. coli extracts cannot transfer laurate from lauroyl-ACP to lipid IVA, only to (Kdo)2-lipid IVA (K. A. Brozek, and C. R. H. Raetz, J. Biol. Chem. 265:15410-15417, 1990). P. aeruginosa extracts do not utilize myristoyl- or R-3-hydroxymyristoyl-ACP instead of lauroyl-ACP to acylate lipid IVA. Laurate incorporation in P. aeruginosa extracts is dependent upon time, protein concentration, and the presence of Triton X-100 but is inhibited by lauroyl-coenzyme A. P. aeruginosa extracts transfer only one laurate to lipid IVA, whereas E. coli extracts can transfer two laurates to (Kdo)2-lipid IVA. These results demonstrate that incorporation of laurate into lipid A does not require prior attachment of Kdo in all gram-negative bacteria.

phi CTX is a temperate phage of Pseudomonas aeruginosa harbouring the ctx gene that encodes cytotoxin (CTX). We identified phi CTX as an R pyocin-related phage, by serological and molecular analysis, based on the findings that the infectivity of the phage was inhibited with the antisera directed R pyocins and R pyocin-related phages and that the phi CTX genome showed DNA homology to the genome of PS17 (a representative of the R pyocin-related phages) as well as to the pyocin R2 genes. Another new CTX-converting, R pyocin-related phage named PS21 was isolated from a CTX-producing strain of P. aeruginosa, suggesting the distribution of the ctx gene by certain members of R pyocin-related phage family.

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 of regA, 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.

The expression of exotoxin A (ExoA) from Pseudomonas aeruginosa is influenced by iron and is under the control of the regulatory gene regA. To test whether regA plays a role in the expression of other iron-regulated proteins a RegA- mutant was constructed by insertional mutagenesis. The polypeptide pattern of this mutant (PA103R) was compared with the parental strain (PA103) and a trans-complemented strain PA103R(pREX18) after growth of the strains in conditions containing low or high concentrations of iron. An iron-regulated 42 kDa protein (RRP) was identified and purified from the culture supernatant of PA103 and PA103R(pREX18) which was missing in PA103R. Database analysis of the N-terminal sequence of this regA-regulated protein (RRP) revealed no similarity to other proteins. Preliminary investigations into the function of RRP revealed that it has no proteolytic or cytotoxic activity. Using two-dimensional electrophoretic analysis of whole cells, a technique which allowed separation of over 600 polypeptides, we were unable to identify any other iron-regulated protein whose expression was regulated by regA.

The inhibitory effects of azithromycin (AZM), a new 15-membered macrolide antibiotic, on the production of exotoxin A, total protease, elastase, and phospholipase C by Pseudomonas aeruginosa were determined, and the virulence-suppressing effects of AZM were compared with those of erythromycin (EM), roxithromycin (RXM), and rokitamycin (RKM). The effect of exposure of P. aeruginosa PA103 or B16 in cultures to sub-MICs of these macrolide antibiotics on the production of exoenzymes was determined. AZM suppressed the in vitro production of extracellular and intracellular exotoxin A by P. aeruginosa PA103 more than did EM, even at a concentration of only 2 micrograms/ml. At concentrations of between 4 and 32 micrograms/ml, AZM also inhibited total protease, elastase, and phospholipase C production by P. aeruginosa B16 more than did EM, RXM, and RKM. AZM was effective in suppressing exotoxin A and total protease production through 24 h of incubation in the presence of drug at sub-MICs, but it had no significant effect on either the growth of P. aeruginosa or its total protein production. Moreover, at a concentration of 4 micrograms/ml, AZM suppressed exoenzyme production by other strains of P. aeruginosa more than did EM. These findings indicate that AZM, EM, RXM, and RKM each has an inhibitory effect on exoenzyme production separate from the antimicrobial effect and that, of these macrolides, AZM has the strongest virulence-suppressing effect.

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.

Pseudomonas aeruginosa exotoxin A is synthesized with a secretion signal peptide typical of proteins whose final destination is the periplasm. However, exotoxin A is released from the cell without a detectable periplasmic pool, suggesting that additional determinants in this protein are important for recognition by a specialized machinery of extracellular secretion. The role of the N terminus of the mature exotoxin A in this recognition was investigated. A series of exotoxin A proteins with amino acid substitutions for the glutamic acid pair at the +2 and +3 positions were constructed by mutagenesis of the exotoxin A gene. These N-terminal acidic residues of the mature exotoxin A protein were found to be important not only for efficient processing of the precursor protein but also for extracellular localization of the toxin. The mutated exotoxin A proteins, in which a glutamic acid at the +2 position was replaced by a lysine or a double substitution of lysine and glutamine for the pair of adjacent glutamic acids, accumulated in precursor forms in the mixed cytoplasmic and membrane fractions, which was not seen with the wild-type exotoxin A. The processing of the precursor form of one exotoxin A mutant, in which the glutamic acid at the +2 position was replaced with a glutamine, was not affected. Moreover, a substantial fraction of the mature forms of all three mutants of exotoxin A accumulated in the periplasm, while wild-type exotoxin A could be detected only extracellularly. The periplasmic pools of these variants of exotoxin A could therefore represent the intermediate state during extracellular secretion. The signal for extracellular localization may be located in a small region near the amino terminus of the mature protein or could consist of several regions that are brought together after the polypeptide has folded. Alternatively, the acidic residues may be important for ensuring a conformation essential for exotoxin A to traverse the outer membrane.