INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

The Bordetella pertussis RTX (for repeat in toxin family protein) adenylate cyclase toxin (ACT, adenylate cyclase-hemolysin [AC-Hly], or CyaA) can penetrate into a variety of immune effector cells, where upon activation by calmodulin, it catalyzes conversion of cellular ATP to cyclic AMP (cAMP) and interferes with cellular signaling and microbicidal functions (9, 26). AC toxoids with disrupted catalytic activity are, however, still cell invasive and are becoming a potent new tool for delivery of vaccinal CD8⁺ T-cell epitopes (recognized by CD8⁺ cytotoxic T lymphocytes) into cytosol of major histocompatibility complex (MHC) class I antigen-presenting cells. Indeed, recombinant AC toxoids have been successfully used for antigen delivery and induction of protective antiviral, as well as therapeutic antitumor CD8⁺ cytotoxic T-cell responses in mice (11, 12, 16, 32, 33).

Translocation of the AC domain into cells is poorly understood, and whether some other portions of the ACT molecule also penetrate into cell cytosol was unknown. ACT is a large 1,706-residue-long toxin consisting of an amino-terminal AC domain of 400 residues and an RTX moiety of 1,306 residues, that can form small cation-selective membrane channels and exhibits some hemolytic activity (4, 6, 13, 28, 36). This Hly moiety itself consists of an hydrophobic channel-forming domain (residues 500 to 700) (6), of the characteristic calcium-binding RTX nonapeptide repeats, rich in glycine and aspartate residues (last 700 residues) (10, 29, 38) and of a fatty acylation domain (residues 700 to 1000), where the essential posttranslational activation by amide-linked palmitoylation of lysine 983 is taking place in the presence of the accessory acyltransferase, CyaC (1, 17). The entire and acylated AC-Hly fusion is needed for AC delivery into target cells (20, 30), while the membrane insertion and channel-forming (hemolytic) activities of the toxin do not require the AC domain and are entirely located at the Hly moiety (20, 30). ACT can penetrate directly across the cytoplasmic membrane of cells and/or erythrocytes without the need for endocytosis (5, 14). The translocation path of the AC domain across target membrane, however, does not appear to involve the hemolytic channel (6), and translocation of the AC into cells and formation of the membrane channels appear to be two independent membrane activities of ACT (7, 15, 18, 27, 29). Indirect evidence suggests that the AC domain might be delivered into cells by ACT monomers, while formation of ACT channels might require oligomerization (3, 7, 15, 18, 20, 27, 28). Structural data on ACT are, however, missing because of the size of ACT (177.6 kDa) and due to the tendency of ACT to rapidly aggregate and lose the cell-invasive activity in solutions that do not contain high concentrations of chaotropic agents.

Further progress in understanding the topology and mechanisms of membrane insertion and translocation of ACT will largely depend on the use of site-specifically-tagged toxin molecules. Identification of permissive sites for insertion into ACT of epitope tags recognized by monoclonal antibodies and insertion of unique cysteine residues along the ACT molecule, allowing its site-specific labeling with thiol-reactive probes are, therefore, of importance for generation of suitable tools for structure-function studies on ACT. In parallel, identification of such permissive sites is important for further development of the ACT as a new carrier molecule for delivery of vaccinal epitopes. In this study, we have used random linker insertion mutagenesis to map sites along the ACT molecule, where the above-mentioned structural alterations can be performed and foreign peptides can be placed without interfering with toxin function. As a first approach to the elucidation of ACT membrane topology, the obtained mutant ACTs were used to delineate portions of ACT that are capable of delivering epitopes into the cytosolic MHC class I presentation pathway.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains, growth conditions, and plasmids. The Escherichia coli strain XL1-Blue (Stratagene) was used throughout this work for DNA manipulation and for expression of ACT. Bacteria were grown at 37°C in Luria-Bertani medium supplemented with 150 µg of ampicillin, 30 µg of chloramphenicol, and/or 200 µg of kanamycin per ml when appropriate.

View larger version (322317K): [in this window] [in a new window]   FIG. 1.   Insertional mutagenesis of cyaA. (A) Schematic description of the employed linker insertion mutagenesis procedure. The gene denominations are as follows: lacZp, lacUV5 promoter operator sequence derived from pTZ19R (Pharmacia); blaM, -lactamase gene conferring ampicillin resistance; cyaC, gene for the accessory protein CyaC required for posttranslational activation of ACT by covalent palmitoylation at lysine 983; cyaA, gene for the ACT protoxin; KmR, cartridge containing the kanamycin resistance gene from pUC4K (Pharmacia). (B) Schematic depiction of the location of the characterized insertions within the ACT polypeptide, with respect to its functional domains. The numbers indicate the residues at which the various peptides described in the text were placed. (C) Representation of the outcome of insertion of the TGTACA oligonucleotide in the three possible reading frames into cyaA. Unique cysteine codons were introduced at the level of the unique BsrGI sites with TGTACA linkers in frames 2 and 3, by insertion of a palindromic adapter encoding a hexapeptide or a nonapeptide comprising a cysteine codon.

Insertional mutagenesis of pT7CACT1. The insertional mutagenesis with the Km^r-BsrG cassette is explained in detail in Fig. 1A and was done by standard procedures for DNA manipulation in vitro (31). The plasmid for expression of CyaC-activated ACT (pT7CACT1) was randomly linearized by limited digestions with the HaeIII and BstUI enzymes, cutting the G:C-rich cyaA DNA at 69 and 68 sites, respectively. Linearized pT7CACT1 was purified by preparative agarose gel electrophoresis, and the plasmid was recircularized with the purpose-designed mutagenic cassette Km^r-BsrG, containing a kanamycin resistance gene flanked by BsrGI restriction site. About 5,000 kanamycin-resistant transformants were selected, and their plasmid DNA was pooled. The kanamycin resistance gene was then excised with BsrGI, leaving in the mutagenized plasmids a randomly inserted hexanucleotide linker, TGTACA, and introducing a unique BsrGI site. The insertion points were mapped by restriction analysis and determined by DNA sequencing. For this purpose, the Km^r-BsrG cassette was reintroduced into the unique BsrGI sites of the plasmids from chosen mutants by using selection for gain of kanamycin resistance. Sequencing was performed with double-stranded miniprep DNA by using the Thermosequenase cycle sequencing kit (Amersham) and ³²P-terminally-labeled primers 5'-CAATGTAACATCAGAGATTT and 5'-ATGAGTCAGCAACACCTTCTT, respectively, which are complementary to 3' and 5' ends of the Km^r-BsrG cassette.

Insertion of hexa- or nonapeptides with unique cysteine residues. Unique cysteine codons were introduced at the BsrGI sites of the mutant pT7CACT1 plasmids by replacing the Km^r-BsrG cassette with purpose-designed and cysteine-encoding palindromic adapters (Fig. 1C), obtained by self-annealing of synthetic oligonucleotides (5'-GTACGATGCATC and 5'-GTACTGCAGG ATCCTGCA, respectively). Plasmids with the inserted adapters were readily identified by screening for sensitivity to kanamycin and gain of one of the new adapter-born restriction sites for Mph1103I and BamHI, respectively. Restoration of the reading frame upon adapter insertion was verified by assay for AC secretion (see below) and verification of expression of the full-length toxin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The exact sequence of all inserts was verified by DNA sequencing of the respective constructs.

Insertion of OVA epitope sequences into ACT. Three double-stranded synthetic oligonucleotides encoding the ovalbumin (OVA) epitope in the required reading frames were used for insertion into the unique BsrGI sites generated within the cyaA gene by linker mutagenesis (see above). The oligonucleotide pairs 5'-GTACTTCAATAATTAATTTCGAAAAGCTTC and 5'-GTACGAAGCTTTTCGAAATTAATTATTGAA, 5'-GTACTCAATAATTAATTTCGAAAAGCTTCA and 5'-GTACTGAAGCTTTTCGAAATTAATTATTGA, and 5'-GTACTCTCAATAATTAATTTCGAAAAGCTTCA and 5'-GTACAAGCTTTTCGAAATTAATTATTGAGA, respectively, were designed (i) to introduce a unique HindIII restriction site for rapid identification of insertion clones, (ii) to stop ACT synthesis when inserted in the inverted orientation, and (iii) to destroy the original BsrGI insertion site upon ligation. The oligonucleotide sequences encoded the K^b-restricted CD8⁺ T-cell epitope SIINFEKL corresponding to residues 257 to 264 of OVA plus flanking residues, as indicated in Table 2. The orientation and exact sequence of all inserted oligonucleotides were verified by DNA sequencing. After the cell-invasive activity of the generated ACT-OVA fusions was characterized, according to their AC activity, the constructs were detoxified by ablation of their catalytic activity. For this purpose, a synthetic BamHI linker, 5'GGATCC, encoding a dipeptide, GlyPhe, was inserted into the EcoRV site between codons 188 and 189 of cyaA, and this resulted in destruction of the ATP binding site of the resulting ACT and loss of all AC activity, as described previously (12, 16).

AC secretion assay. Because the cyaBDE gene products which account for ACT secretion in Bordetella do not, for some unknown reason, constitute a functional apparatus for secretion of ACT in the heterologous E. coli host, the hlyBD genes were used for secretion of ACT. pT7CACT1 derivatives were transformed into XL1-Blue bearing the compatible plasmid pLG575 with genes encoding the HlyBD proteins of the HlyA translocator (25). Secreted AC activity was determined in cell supernatants of 2-ml overnight cultures of the individual transformants as previously described (35).

Screening for production of active ACT. We have previously shown that preparations which are suitable for reliable determination of ACT activities are obtained by 8 M urea extraction of cell debris after sonication of IPTG-induced cells (20, 34). Therefore, 5-ml cultures of the individual E. coli transformants were grown to mid-exponential phase and induced for ACT production by addition of 1 mM IPTG for 4 h. Accumulated ACT was extracted from cell pellets with 8 M urea-50 mM Tris-HCl (pH 8.0)-0.2 mM CaCl₂ upon ultrasonic disruption in an icewater bath. The crude extracts were cleared by centrifugation and used for determination of cell-invasive AC activity.

Standard techniques. SDS-PAGE analysis, determination of protein concentration, and in vitro DNA manipulations were performed according to standard protocols (31).

Characterization, production, and purification of the CyaA-derived proteins. For characterization and purification, the wild-type ACT and the different mutant derivatives were produced upon IPTG induction (1 mM) in liquid cultures in the presence of the activating protein CyaC by using E. coli XL1-Blue (Stratagene) and the respective constructs derived from pT7CACT1. For activity determinations, urea extracts were prepared from individual 50-ml cultures, and their ACT concentrations were equalized on the basis of the AC enzyme content, as previously described (20). For protein purification, 500-ml cultures were processed. The urea extracts of insoluble cell debris after sonication were prepared in 8 M urea-50 mM Tris-HCl (pH 8.0)-0.2 mM CaCl₂, as described previously (34). When appropriate, 10 mM -mercaptoethanol was added to extraction buffer in order to prevent formation of disulfide bonds of the cysteine-containing ACT variants. Some of the proteins were further purified by ion-exchange chromatography on DEAE-Sepharose (Pharmacia) as described previously (30). For the purpose of epitope delivery tests, an additional hydrophobic chromatography purification step on phenyl-Sepharose was added (2). In the final step, the proteins were eluted with 8 M urea-50 mM Tris-HCl (pH 8.0)-10 mM -mercaptoethanol (when appropriate) and stored at 20°C. The activities of the purified proteins were compared with toxin activities determined with the initial urea extracts. As expected, when expressed as milliunits of internalized AC enzyme per unit of AC added to cells, both types of toxin preparations exhibited very similar toxin activities, for all mutant proteins tested (Table 1). These results confirm that, as previously observed with intact ACT (20, 34), the urea extracts can be used for activity screening and characterization of the mutant ACTs without the need for purification.

Assay of AC, cell binding, and invasive and hemolytic activities. AC activities were measured as previously described in the presence of 1 µM calmodulin (22). One unit of AC activity corresponds to 1 µmol of cAMP formed per min at 30°C (pH 8.0). Because alterations outside the AC domain do not affect the specific AC activity (35), concentrations of mutant ACTs in the extracts were equalized on the basis of their AC content prior to activity testing. For constructs bearing inserts in the catalytic domain at positions 107 and 335, which had markedly reduced or nil AC-specific activity, the concentration of the ACT in extracts was adjusted by comparison of the band intensities on SDS-PAGE in the extracts. Purified proteins were tested at equal AC and/or total protein concentrations. Cell-invasive AC was determined as previously described (5), by determining the AC enzyme activity that reached the intracellular location within erythrocytes after 30 min of incubation, and was, hence, protected against externally added trypsin. To circumvent any potential differential effect on the specific AC activity of intact AC and of the AC variants altered by peptide insertion by the presence of membranes, the translocation of the AC enzyme into cells was not determined by measuring the amount of cAMP that had accumulated inside toxin-treated cells. Instead, the cell-associated (bound) AC and/or AC internalized into cells was determined upon solubilization of cell membranes in buffer containing 0.1% Triton X-100 and subsequent dilution of the extracted enzyme in a reaction mixture containing 0.1% Triton X-100 and saturating concentrations of ATP and the activator calmodulin under first-order kinetics conditions in the absence of membranes (5). The hemolytic activity was determined as hemoglobin release upon incubation of the toxins for 270 min with washed sheep erythrocytes at 5 × 10⁸ cells per ml (5). Erythrocyte binding of the toxins was determined as described in detail previously (20).

In vitro assay for MHC class I presentation of the ACT-OVA constructs. The stimulation of B3Z cells, a CD8⁺ T-cell hybridoma specific for the SIINFEKL-H-2K^b complex, was monitored by interleukin 2 (IL-2) release in the supernatants of cultures in the presence of antigen-presenting cells (APCs) (21). B3Z cells (10⁵ cells/well) were cocultured in flat-bottom 96-well culture plates for 18 h in the presence of various ACT concentrations and splenocytes (which originated from C57BL/6 mice, 6 to 8 weeks old; Janvier, France) as APCs (10⁵/well) in a 200-µl final volume of complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10⁵ M 2-mercaptoethanol, 100 U of penicillin per ml, and 100 µg of streptomycin per ml). After 18 h, the supernatants were harvested and frozen for at least 2 h at 70°C. Then, 10⁴ cells of the cytotoxic T-lymphocyte CTLL cell line per well, a line which proliferates specifically in response to IL-2, were cultured with 100 µl of supernatant in a 0.2-ml final volume (8). Two days later, [³H]thymidine (NEN Life Science, Boston, Mass.) was added, and the cells were harvested 18 h later with an automated cell harvester (Skatron, Lier, Norway). Incorporated thymidine was detected by scintillation counting. In all experiments, each point was done at least in duplicate and more often in triplicate. Results are expressed in cpm (cpm in the presence of ACT  cpm in the absence of ACT). It has been verified that treatment of splenocytes with detoxified ACT alone did not induce any production of IL-2 (data not shown).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Insertional mutagenesis of ACT. The ACT molecule was scanned for permissive peptide insertion sites by linker insertion mutagenesis of its structural gene, cyaA (Fig. 1), and functional characterization of the obtained mutant ACT. In order to allow straightforward mutagenesis of the large, 5.1-kb-long cyaA gene, a purpose-designed and excisable mutagenic cassette carrying the gene for resistance to kanamycin (Km^r) was employed (23). The cassette was modified so that, upon excision, it left at the insertion site a hexanucleotide, TGTACA (Fig. 1A), corresponding to a unique BsrGI restriction site and encoding a di- or tripeptide insert, depending on the respective reading frame at the insertion point. This procedure enabled antibiotic resistance selection of insertion mutants, rapid sequencing of all insertion points with a single pair of primers matching the ends of the mutagenic cassette, and a high-throughput antibiotic sensitivity screening for replacement of the mutagenic cassette with an oligonucleotide of choice.

Permissive peptide insertion sites and mutations highlighting important functional structures of ACT. It was important to test how insertion of longer peptides at defined positions will affect biological activities of ACT. Moreover, active ACT constructs with unique cysteine residue inserts would be amenable to site-specific labeling with thiol-reactive probes and subsequently serve in ACT-membrane interaction studies. Therefore, palindromic oligonucleotide adapters, encoding either a hexapeptide, VRCIVH (E2-Cys), or a nonapeptide, VYCRILQYT (E3-Cys), were inserted into the BsrGI sites (Fig. 1C). Each of these longer inserts also introduced a unique cysteine residue and allowed generation of a second set of mutant toxins with cysteine residues placed at 25 different positions along the toxin (Table 1).

View larger version (66K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE analysis of the purified ACT preparations. Seventeen of 25 cysteine-containing ACT proteins were purified from urea extracts by DEAE-Sepharose chromatography as previously described (30), and 2 to 4 U of each enzyme was analyzed together with the wild-type ACT on a 7.5% acrylamide gel. It can be seen that due to reduced specific AC activity of the ACT108/E2-Cys protein, a several-fold-larger amount of this protein was loaded, compared to the amounts of the other constructs.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Sequence of the repeat region of ACT organized in repeat blocks. The sequence representing the repeats of ACT was aligned according to the consensus repeat nonapeptide motif. The points of linker insertions within the repeat blocks are indicated by arrowheads or by an encircled residue which was substituted for by the insert. The dispensable repeat sequences, which were deleted, are boxed.

Delineation of cell-invasive portions of ACT suitable for delivery of foreign CD8⁺ T-cell epitopes. Recently, the cell-invasive ACT with various peptides inserted at residue 224 of the AC domain has been successfully used for CD8⁺ T-cell epitope delivery into the MHC class I presentation pathway and the activation of specific CD8⁺ cytotoxic T-cell responses against tumors and viruses (11, 12, 32). The insertional mutagenesis and screening procedure described here allowed definition of a set of an additional 10 permissive sites along the entire ACT molecule, at which insertion of up to nine-residue-long peptides does not substantially alter the capacity of the toxin to penetrate into cells. It was, therefore, important to test which of the newly identified permissive sites allow CD8⁺ T-cell epitope delivery to the MHC class I molecules. For this purpose, a model K^b-restricted CD8⁺ T-cell epitope, SIINFEKL, corresponding to residues 257 to 264 of OVA, was inserted at 12 positions along the toxin molecule. As summarized in Table 2, eight of the constructs retained a cell-invasive capacity identical or close to that of the intact ACT, and for one construct, ACT336/OVA, the invasive activity could not be assessed because of the loss of AC activity (see below). This confirms that at least eight and potentially nine of the new sites were permissive as well to insertion of other peptides than those used for initial screening. Two additional constructs, ACT594/OVA and ACT926/OVA, respectively, exhibited a reduced or nil capacity to associate with target cells and were therefore used as convenient negative controls in the epitope delivery experiments.

View larger version (71K): [in this window] [in a new window]   FIG. 4.   SDS-PAGE analysis of the detoxified ACT-OVA preparations. The detoxified OVA-containing ACT constructs were purified from urea extracts by a combination of DEAE-Sepharose and phenyl-Sepharose chromatographies as previously described (2). One to 3 µg of each protein was analyzed on a 7.5% acrylamide gel and stained with Coomassie blue. wt, wild type.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Location of the CD8+ T-cell epitope within ACT determines its presentation to CD8+ T cells. Splenocytes used as APCs were incubated in the presence of various concentrations of the ACT proteins harboring the OVA epitope at different sites and cocultured with B3Z, an anti-OVA CD8+ T-hybridoma selectively recognizing cell-surface-presented complexes of the H-2Kb MHC class I molecules with bound OVA epitope (SIINFEKL). IL-2 secretion by the stimulated B3Z CD8+ T cells was determined by the CTLL proliferation method. Results are expressed in cpm of incorporated [3H]thymidine (cpm in the presence of ACT  cpm in the absence of ACT). wt, wild type.

Concluding remarks. We report here the identification and use of a set of new permissive peptide insertion sites along the ACT molecule. This enabled insertion of peptides containing unique cysteine residues at 20 different positions of ACT, where these peptides mostly had a minor or undetectable effect on ACT activities and are amenable to site-specific tagging at the cysteine residues with thiol-reactive probes for toxin-membrane interaction studies. Furthermore, the identified permissive sites within all functional domains have now allowed construction of ACT molecules tagged with epitopes recognized by commercially available monoclonal antibodies and their use for analysis of ACT topology within target membranes (J. Loucká et al., unpublished data).