Department of Biological Sciences1 and Department of Microbiology and Infectious Diseases2, University of Calgary, Calgary, Alberta, Canada T2N 1N4
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA3
Author for correspondence: Douglas G. Storey. Tel: +1 403 220 5274. Fax: +1 403 289 9311. e-mail: storey{at}ucalgary.ca
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ABSTRACT |
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Keywords: cystic fibrosis, Pseudomonas aeruginosa, exotoxin A, ADPRT, cytotoxicity
Abbreviations: ADPRT, ADP-ribosyltransferase; CF, cystic fibrosis; EF-2, elongation factor 2; ETA, exotoxin A; TSBDC, trypticase soy broth dialysate chelexed
The GenBank accession numbers for the toxA sequences are: strain 4384, AF227419; strain 5154, AF227420; strain 5166, AF227421; strain 5552, AF227422; strain 5585, AF227423; strain 5588, AF227424.
a Present address: Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
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INTRODUCTION |
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ETA production in the lungs of patients with CF has been demonstrated using a variety of methods. Storey et al. (1992) and Raivio et al. (1994)
demonstrated that toxA transcript accumulation was detectable in bacterial populations directly isolated from sputum of CF patients chronically infected with P. aeruginosa. Subsequently, Jaffar-Bandjee et al. (1995)
detected the presence of ETA in sputum samples from CF patients. High levels of circulating antibodies to ETA are also found in CF patients (Jaffar-Bandjee et al., 1995
; Klinger et al., 1978
; Moss et al., 1986
), suggesting repeated exposure to the ETA antigen. In addition, a strong correlation exists between high serum antibody levels to ETA and increased mortality in CF patients (Moss et al., 1986
). Preliminary evidence also indicates that patients with more severe pulmonary complications have P. aeruginosa populations that transcribe higher levels of the ETA-encoding toxA gene (T. L. Raivio & D. G. Storey, unpublished result). These data suggest that in vivo production of ETA is common and may be an important virulence determinant in patients with CF.
ETA possesses an ADP-ribosyltransferase (ADPRT) activity. In eukaryotic cells, ETA catalyses the transfer of the ADP-ribose moiety from NAD+ to elongation factor 2 (EF-2). As a result, protein synthesis ceases and intoxicated cells die since elongation of polypeptide chains no longer occurs (Iglewski et al., 1977 ). ETA causes cell intoxication via a three-step mechanism. The first step involves binding of ETA to a specific receptor, the
2-macroglobulin receptor/low-density lipoprotein (Kounnas et al., 1992
). The second step consists of the internalization of the toxin by target cells through endocytosis. In the third step, the toxin is cleaved by a cellular protease (Ogata et al., 1992
), reduced, and translocated to the cytosol, where it ADP-ribosylates EF-2. A better understanding of the mechanism of action of ETA was made possible by the characterization of its three-dimensional structure (Allured et al., 1986
). Several studies have been performed to characterize this protein and assign specific functions to its three domains (Allured et al., 1986
; Hwang et al., 1987
; Jinno et al., 1988
, 1989
). In addition, the importance of each domain in the cytotoxicity of ETA has been demonstrated (Jinno et al., 1989
; Ogata et al., 1992
; Seetharam et al., 1991
; Wick & Iglewski, 1988
).
Domain I, located at the N-terminal end of the protein, is required for binding of ETA to target cells. Hwang et al. (1987) showed that deletion of domain I abolished cell binding and decreased ETA cytotoxicity against Swiss 3T3 cells. Jinno et al. (1988)
demonstrated the importance of Lys-57 for cell binding by constructing a mutant that had decreased cytotoxicity and failed to compete for binding with native ETA.
Domain II has been implicated in the translocation of ETA into the cytosol. Hwang et al. (1987) determined that deleting portions of domain II reduces the toxicity of ETA without abolishing cell binding. Furthermore, mutation of all arginine residues in domain II resulted in a decrease in cytotoxicity, presumably due to reduced translocation efficiency (Jinno et al., 1989
). In addition, it was recently demonstrated that a deletion of the smallest helix in domain II, helix F, increased translocation and cytotoxicity of the mutated toxin (Taupiac et al., 1999
).
Domain III is responsible for the ADP-ribosylating activity of the protein (Hwang et al., 1987 ; Siegall et al., 1989
). This domain possesses an endoplasmic reticulum retention sequence which is critical for intracellular targeting of the toxin (Chaudhary et al., 1990
; Seetharam et al., 1991
). Using deletion analysis, Chow et al. (1989)
and Siegall et al. (1989)
demonstrated that amino acids 400608 were sufficient for full enzymic activity of ETA. In addition, several studies of domain III have indicated the importance of amino acids His-426, His-440, Tyr-470, Tyr-481 and Glu-553 for the enzymic activity and cytotoxicity of ETA (Brandhuber et al., 1988
; Carroll & Collier, 1987
; Han & Galloway, 1995
; Wick & Iglewski, 1988
; Wick et al., 1990
).
The role of ETA in chronic lung infections associated with CF has yet to be defined. In particular, it is curious that some bacterial populations in the lungs of the affected patients transcribe high levels of this toxin (Raivio et al., 1994 ; Storey et al., 1992
) but the infection rarely spreads beyond the lungs. In this study, to address the role of ETA in the pathogenesis of CF lung infections we analysed the ETA produced by a number of P. aeruginosa CF isolates. We found that P. aeruginosa strains from the lungs of patients with CF produced ETA with altered ADPRT activity and cytotoxicity, and identifed several mutations in the toxA gene that could account for the reduction in ADPRT activity.
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METHODS |
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Cloning of toxA from strain 4384.
Chromosomal DNA from P. aeruginosa strain 4384 was digested with EcoRI and PstI and ligated into the corresponding sites of pKK223-3. The ligation mixture was then transformed into Escherichia coli strain JM109. Colony hybridization using the internal 1·53 kb toxA fragment from pMS151-1 was performed to identify positive clones. Southern blot analysis was carried out as recommended by Schleicher & Schuell, using the internal 1·53 kb BamHI toxA probe. The toxA fragment was labelled with [-32P]dCTP. Hybridization products were visualized by autoradiography.
Sequencing of the toxA gene.
Various subclones were generated from pCG5 using specific restriction enzymes. All DNA fragments were subcloned into the pUC12 vector and sequenced using universal primers via the dideoxy chain-termination method (Sanger et al., 1977 ) by the University of Calgary Core DNA Services.
PCR amplification.
Amplification of domain III of ETA was performed using Ultma polymerase (Perkin-Elmer Cetus), which possesses 3' to 5' proof-reading ability, with primers complementary to the known DNA sequence. The amplified fragment was cloned into a pCR2.1 cloning vector (Invitrogen) and transformed into E. coli strain JM109. Mutations identified in domain III were confirmed by sequencing the cloned chromosomal DNA (Sanger et al., 1977 ).
ADP-ribosylation assays.
ADPRT activity was assayed as described by Chung & Collier (1977) . ADPRT assays were performed in triplicate on each supernatant sample and the results averaged.
SDS-PAGE and immunoblots.
Proteins were separated by the method of Laemmli (1970) using a 5% (w/v) stacking gel and a 10% (w/v) resolving acrylamide gel. To estimate the Mr of the proteins, a prestained standard of known Mr was run on each gel. Immunoblots were performed according to the method of Towbin et al. (1979)
. Primary antibodies to ETA were used at a 1:2000 dilution. Specific reactions were detected by incubation with the secondary antibody, anti-rabbit IgG coupled with horseradish peroxidase. Colour was developed with a solution of Tris/saline containing 3 mg horseradish peroxidase ml-1. Supernatants isolated from each CF strain were concentrated using a Microcon Centrifugal Filter Device. Briefly, the supernatant of each sample was collected at an OD540 of 4·0, and 500 µl of supernatant was applied to the filter possessing an Mr 10000 cut-off range and centrifuged at 8000 g for 30 min. The filter containing the concentrated samples was inverted and recentrifuged for another 15 min. The retentate was then applied to an SDS-PAGE gel.
Mixed toxin experiments.
Strains 4384, PA103 and PA103toxA:: were grown in low-iron conditions to an OD540 of 4, and 10 µl of supernatant was then collected and mixed with 10 µl of purified toxin from the Swiss Serum Institute (1/100 dilution). The samples were incubated for up to 6 h at 32 °C. The ADPRT activity was assayed as previously described (Chung & Collier, 1977
).
Cytotoxicity assays.
The cytotoxicity of ETA was measured using a colorimetric microtitre assay. Briefly, HeLa cells were grown to confluency (1x106 per well) and incubated overnight at 37 °C. Serial dilutions of ETA were added to the HeLa cells and incubated for 48 h at 37 °C. Twenty microlitres of tetrazolium dye (MTT; 5 mg ml-1) was added to each well and incubated for 4 h at 37 °C to allow the viable cells to reduce the tetrazolium dye to purple formazan. The dye was solubilized by the addition of 100 µl 2-propanol/HCl solution. The A570 of each well was measured using an EIA reader.
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RESULTS |
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Specific activity calculated from P. aeruginosa hypertoxigenic strain PA103 and CF isolate strain 4384
To confirm whether the activity of ETA from our six CF isolates was significantly different from that of PA103 we decided to further characterize the production and enzymic activity of ETA by one CF isolate, strain 4384. Of the six strains, 4384 was chosen because its level of transcription of toxA is similar to that of the hypertoxigenic strain PA103 (Storey et al., 1991 ). To analyse the ETA from 4384 it was necessary to concentrate the toxin using a microconcentrator filter. As a control, we concentrated ETA from strain PA103 using the same method. ADPRT assays revealed that concentrated ETA from strain 4384 still had reduced enzymic activity (approx. 800 c.p.m. per 10 µl supernatant) compared to ETA from strain PA103 (approx. 32000 c.p.m. per 10 µl supernatant). Subsequently, Western blot analysis using anti-ETA antibodies was performed on the concentrated supernatant from strains 4384 and PA103 grown in low-iron conditions. Fig. 1
shows that significantly less ETA was purified from strain 4384 (0·16 µg per 10 µl) as compared to strain PA103 (1·8 µg per 10 µl) even when the two strains were grown under comparable conditions. We used the results from these assays to calculate the specific activity of the two ETA preparations. The specific activity of ETA from PA103 was determined to be 58898 c.p.m. (µg protein)-1, whereas the specific activity of ETA from strain 4384 was only 5104 c.p.m. (µg protein)-1. Thus ETA produced by strain 4384 had approximately a 12-fold reduction in its enzymic activity when compared to ETA from strain PA103.
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Comparison of ADPRT activity from 4384 toxA to that from PA103 toxA in strain PA103toxA::
To obtain a more direct comparison of the 4384 and the PA103 ETAs we expressed each cloned toxA in the ETA-deficient strain PA103toxA::. The strains were grown to an OD540 of 4, in iron-depleted TSBDC. Culture supernatants from strain PA103toxA::
(pCG5), containing the 4384 toxA, had levels of activity (approx. 130 c.p.m. per 10 µl) that were only marginally higher than the negative control strain PA103toxA::
(approx. 68 c.p.m. per 10 µl) and strain 4384 itself (approx. 98 c.p.m. per 10 µl). In contrast, culture supernatants from PA103toxA::
(pMS151-1), containing a wild-type toxA, had ADPRT activity (approx. 890 c.p.m. per 10 µl) similar to that of the culture supernatants from the hypertoxigenic strain PA103 (approx. 700 c.p.m. per 10 µl). These results again suggest that 4384 ETA has a lower ADPRT activity than wild-type ETA even when produced in an isogenic strain.
Comparison of ETA production in strains 4384 and PA103
To compare the enzymic activity of ETA produced by each strain, ETA was purified from both 4384 and PA103. Western blot analysis, using anti-ETA antibodies, showed that ETA purified from strains 4384 and PA103 is a protein of Mr 66000 that co-migrates with purified ETA (Swiss Serum Institute) (Fig. 3). Fig. 3
also indicates that strain 4384 produces significantly less ETA than strain PA103. This may be due to the presence of high levels of proteases in strain 4384 (T. L. Raivio, D. E. Woods & D. G. Storey, unpublished result) which are absent in the protease-deficient strain PA103 (Liu, 1966
). The ADPRT activity of the two purified ETA preparations from strain 4384 (approx. 100 and 50 c.p.m. per 10 µl respectively) was lower than that of the two preparations from strain PA103 (approx. 1800 and 2500 c.p.m. per 10 µl). The highest level of ADPRT activity was obtained from the commercially available purified Swiss Serum ETA (approx. 2800 c.p.m. per 10 µl).
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Sequencing of the DNA encoding the enzymic moiety of toxA from five P. aeruginosa CF isolates
We cloned and sequenced the DNA encoding the enzymic moiety of toxA from the remaining CF strains in order to investigate the possibility of conserved mutations which could account for the reduced ADPRT activity (Fig. 6 ). All the CF strains tested had the same alteration of Ser-515 to Gly. The two other mutations identified in 4384, Ser-410 to Asn and Ala-476 to Glu, were also present in strain 5166. Strains 5552 and 5154 both had the mutation of Ala-476 to Glu. Two mutations in domain III of toxA from strain 4384 were absent from strains 5585 and 5588. In addition, strain 5154 contained a stop codon that would prematurely truncate the ETA protein in this strain, explaining the absence of an ETA band on the Western blots of strain 5154. Other point mutations were observed throughout the coding region for the enzymic moiety of toxA; however, these were unique to each strain and were not conserved among the six CF isolates examined.
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DISCUSSION |
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We selected one strain, 4384, to further characterize the ETA produced. Previously, CF isolate 4384 has been shown to transcribe toxA at a level similar to the hypertoxigenic strain PA103 (Raivio et al., 1994 ). The low ADPRT activity from this isolate could be due to a high level of protease production, a defect in secretion by strain 4384 or an inhibitory accessory factor, or it may be an inherent property of the toxin produced. Complementation studies, intracellular ADPRT activity and mixed toxin studies (Fig. 2
) showed that neither proteases, inhibitory accessory factors nor a secretory defect are likely to be responsible for the reduced toxin enzymic activity. An analysis of the specific activity of strain 4384 suggested that the reduced ADPRT activity of 4384 ETA may be due to an altered amino acid sequence rendering a functionally deficient protein.
It has previously been shown that amino acids 400608 in domain III were sufficient for full enzymic activity of ETA (Chow et al., 1989 ; Siegall et al., 1989
). In addition, several residues in domain III were shown to be critical for enzymic activity (Brandhuber et al., 1988
; Carroll & Collier, 1987
; Han & Galloway, 1995
; Wick & Iglewski, 1988
; Wick et al., 1990
). The observed reduction in the specific activity of strain 4384 ETA may be due to the presence of mutations within the domain III sequence which would render the toxin partially inactive. Sequence analysis revealed three mutations in domain III of toxA from strain 4384 (Fig. 6
). The position of mutation Ala-476 to Glu is within an
-helix which is believed to be an important part of the catalytic site (Domenighini et al., 1994
). The presence of a mutation within the helix may modify the dimensions of the active site, possibly interfering with substrate binding or transfer of the NAD+ moiety. A second mutation, which is at the protein surface, is a substitution of a Gly for Ser-515. This amino acid may be essential for the protein integrity or as a protein modification site. The third mutation, of Ser-410 to Asn, was outside the region normally considered to be involved in either substrate binding or the catalytic site. None of these mutations (positions 410, 476, 515) had previously been reported to be involved in ADPRT activity. These mutations alone may have an effect on the activity of the protein, or the mutations could act in concert to reduce the ADPRT activity of the ETA. The presence of these mutations may affect proper folding of the protein and render the catalytic site inaccessible to the substrate.
The ADPRT activity of ETA is directly responsible for the inhibition of protein synthesis in eukaryotic cells (Iglewski & Kabat, 1975 ; Iglewski et al., 1977
; Iglewski & Sadoff, 1979
). Our findings suggested that the specific activity of purified ETA from strain 4384 (approx. 11000 c.p.m. µg-1) was about 10-fold lower than that of the toxin from strain PA103 (approx. 110000 c.p.m. µg-1). As such, we expected that strain 4384 ETA would have a reduced cytotoxicity to coincide with its reduced ADPRT activity. However, Fig. 5
shows that despite the low ADPRT activity of this ETA, the cytotoxic activity of the toxin from strain 4384 was slightly higher than the toxin from strain PA103. An increased cytotoxic activity could be explained in a number of ways. Mutations in domains I and II have previously been shown to alter the cytotoxicity of ETA (Siegall et al., 1989
; Taupiac et al., 1999
). However, sequence analysis of the toxA from strain 4384 showed no mutations in either domains I or II. Alterations in the C-terminal end of the protein also seem to affect the cytotoxicity of the protein. Once again, sequence analysis of toxA from strain 4384 suggested no alterations in this portion of the gene. This leads us to hypothesize either that ETA may have a second cytotoxic activity in domain III or that the cytotoxicity of the toxin is not tightly linked to the ADPRT activity. The nature of the increased cytotoxicity remains to be elucidated.
A similar pattern of reduced ADPRT activity was also observed among five other CF isolates. To investigate this deficiency, we amplified and sequenced the ETA enzymic moiety of these five strains to see if the amino acid sequences differed from that of the wild-type. Surprisingly, we found a number of mutations which could potentially be responsible for the reduced ADPRT activity in the CF strains. A stop codon was identified in strain 5154, resulting in the production of a truncated protein. Thus, the lack of ADPRT activity from this strain results from lack of production of ETA. Interestingly, all five of the other strains had the conversion of Ser-515 to Gly. However, this mutation is predicted to be on the outside surface of the ETA molecule and therefore not directly involved in either substrate binding or catalysis. Four strains, including 4384, had the alteration of Ala-476 to Glu in a position predicted to be located in the active site of the enzyme. This mutation alone could potentially account for the alteration in ADPRT activity of the ETA in these strains. Finally, two strains (5166 and 5154) had the third mutation also present in strain 4384. This mutation, Ser-410 to Asn, is also outside the region believed to be directly involved in NAD+ binding and catalysis. Other point mutations were found in the five additional strains but none of those appear to be consistent among the strains. Except for the mutation of Ser-515, no clear pattern has emerged that could account for the reduction of ADPRT acitivity in all the strains. Further research will be necessary to clarify the role of these mutations.
Previous research has revealed that in strain PAO1, an insertion sequence in the region upstream of toxA altered the phenotype of the strain (Woods et al., 1991 ; Sokol et al., 1994
). In particular, when the insertion was present the strain became mucoid and the levels of ADPRT activity in the supernatant of the culture dropped by 20%. A possible explanation of our findings is that some or all of our CF isolates may have an insertion in the region upstream of toxA. However, both the purified 4384 ETA and the 4384 ETA produced in the transformed strain PA103toxA::
had reduced ADPRT activity. These results suggested that the ETA itself was modified. Additionally, we have shown that an inhibitory accessory factor is probably not involved in the lower ADPRT activity of ETA from strain 4384. While an insertion sequence may not play a role in the reduced ADPRT activity of strain 4384, it still could account for the reduced activity of the other strains used in this study.
Even though we have examined only a small number of isolates, our results suggest that mutations in toxA may be common among CF isolates. Our results also suggest that the proposed role of ETA in the pathogenesis of CF lung infection may have to be altered, especially during the chronic phase of the infection. It is likely that during the initial infections with P. aeruginosa, ETA plays a role in establishment and persistence of the infection. Once the infection becomes chronic, mutations may arise in the colonizing strains. Our research has indicated that these mutations occur in toxA, but they may arise in other genes as well (Taylor et al., 1992 ). As a result of these mutations, at least some of the bacterial population in the lungs may lose or have reduced ADPRT activity. Our results may explain why other researchers have found that CF isolates produce lower levels of ETA. The correlation between ETA production, the immune response to ETA and morbidity and mortality in CF suggests a role for ETA in the chronic phase of the disease (Moss et al., 1986
). Our data also suggest that the cytotoxicity of the toxin, rather than the ADPRT activity, may be the more important feature of the toxin in the chronic phase of CF lung disease. On the other hand, we cannot rule out the possibility that the mutations have left the organism less pathogenic, thereby allowing the host, and thus the bacteria, to survive for a longer period of time. The chronic production of an immunogenic protein such as ETA in this situation could lead to immunopathological effects in the lungs of patients with CF.
In summary, this work has shown that strains producing ETA with reduced ADPRT activity commonly occur among P. aeruginosa CF isolates. In at least one strain, the ETA produced had a slightly higher level of cytotoxicity despite a 10-fold reduction in ADPRT activity. Other clinical isolates are currently under investigation to determine the prevalence of these mutations and perhaps lead to a better understanding of the factors contributing to mutations among CF isolates.
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ACKNOWLEDGEMENTS |
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Received 20 September 1999;
revised 28 March 2000;
accepted 4 April 2000.