Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins

Res. Microbiol. 152 (2001) 889–900  2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923-2508(01)01272-4/FLA

Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins Sakina Gmira1 , Gouzel Karimova, Daniel Ladant∗ Unité de Biochimie Cellulaire, CNRS URA 2185, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France Received 11 May 2001; accepted 16 July 2001

Abstract – Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver CD8+ T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.  2001 Éditions scientifiques et médicales Elsevier SAS Bordetella pertussis / adenylate cyclase / recombinant toxin / cell delivery

1. Introduction Targeted delivery of proteins or polypeptides into the cytosol of eukaryotic cells represents a major advancement towards the development of novel therapeutic agents. Among the various strategies to achieve this goal, a particular class of bacterial toxins, the A-B toxins, appears to be a very promising vector system for transporting polypeptides into cells. These toxins are made up of two different protomers, A and B: the A protomer is an enzyme that acts on specific cytosolic targets and the B protomer is responsible for cell binding and membrane translocation of A [34]. In several instances, it has been shown that exogenous polypeptides can be genetically fused to the A protomer, without hampering its transport into the cell cytosol by the B moiety. Such recombinant toxins might represent new avenues for cell therapy either by delivering therapeutic polypeptides into cells or targeting antigens to the major histocompatibility

∗ Correspondence and reprints.

E-mail address: [email protected] (D. Ladant). 1 Present address: Science Faculty, Ismail Moulay University,

Meknes, Morocco.

complex (MHC) class I presentation pathway to induce specific immune responses [16, 31]. Diphtheria toxin (DT), Pseudomonas exotoxin A (ExoA) and anthrax toxins (lethal Factor, LF, and adenylate cyclase, EF) have already been used to deliver, into eukaryotic cells, either enzymes like barnase, dihydrofolate reductase (DHFR), and other catalytic subunits of different toxins, or proteins like acidic fibroblast growth factor or the HIV gp120 protein [2 – 4, 6, 15, 19, 29, 30, 33, 43, 44]. These toxins exploit the cellular uptake systems to reach the target cell cytosol [34]. After endocytic uptake, they gain access to the cytosol from early endosomes (like DT), late endosomes (LF/EF) or after migration through trans-Golgi and Golgi up to the endoplasmic reticulum (ExoA). We recently used one particular toxin, the adenylate cyclase toxin (CyaA) from Bordetella pertussis [27], the causative agent of whooping cough, to deliver small peptides, corresponding to CD8+ T-cell epitopes, into antigen presenting cells (APCs) in vivo [13, 38]. The CyaA toxin is able to enter into eukaryotic target cells [10] where it is activated by endogenous calmodulin (CaM) [45] to produce supraphysiological levels of intracellular cAMP that alter the cell physiology (for a review, see [27]). CyaA is synthesized as an inactive protoxin encoded by the cyaA

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gene [14] and post-translationally modified to the active toxin by the product of the B. pertussis cyaC gene [5, 20]. The CyaA toxin is a bifunctional protein of 1706 residues made up of an N-terminal catalytic domain of 400 amino acids and a C-terminal part of 1306 residues which is responsible for the binding of the toxin to target cell membrane and the subsequent delivery of the catalytic moiety into the cell cytosol [14, 36]. This part, which displays strong homology with the RTX (repeat in toxin) toxin family, also exhibits a weak hemolytic activity due to its ability to form cation-selective channels in biological membranes [7, 8]. The originality of the CyaA toxin stems from its unique mode of entry into eukaryotic target cells, that involves a direct translocation of the catalytic domain across the plasma membrane to the cytosol of the cells, where it associates with calmodulin [35]. The translocation of the catalytic domain into the cell cytosol is calcium- and temperature-dependent and depends upon the plasma membrane potential [32, 35]. The molecular mechanisms by which the toxin transports its N-terminus catalytic domain across the lipid bilayer remain largely unknown to date. We previously showed that peptides corresponding to CD8+ T-cell epitopes could be inserted into various permissive sites within the catalytic domain of CyaA without altering the biological activities of the molecule [25, 40] and that recombinant CyaA toxins could elicit specific MHC class-I-restricted cytotoxic T-cell (CTL) responses in mice [13, 38]. Recent studies indicated that these recombinant toxins are delivered to the cytosol of the APCs where they are proteolytically processed by the proteasome [18]. In order to examine the tolerance of CyaA to insertion of large polypeptides we constructed and characterized several recombinant CyaA toxins that harbor different passenger proteins inserted within the catalytic domain. Three of these recombinant toxins, carrying either HIV Tat (87 amino acids), Aspergillus fumigatus restrictocin (148 amino acids), or mouse dihydrofolate reductase (187 amino acids), were found to be invasive. Furthermore, unfolding of the passenger proteins appeared to be required to allow efficient translocation of the recombinant toxins across plasma membrane of target cells.

2. Materials and methods 2.1. Bacterial strains, plasmids and growth media

The Escherichia coli strain XL1-Blue (Stratagene) was used for DNA manipulation and the E. coli strain BLR (Novagen) was used for production of the recombinant toxins. The plasmids pACM224 and pCACT3 were already described [25, 40]. Transformants were selected on LB agar media containing 100 mg/L ampicillin. All bacteria were grown in liquid LB medium containing 100 mg/L ampicillin. 2.2. Constructions of recombinant CyaA toxins

All in vitro DNA manipulations were performed according to standard protocols [37]. All recombinant toxins were produced in E.coli by using a novel expression vector pTRCAG, described in figure 1. It is derived from plasmid pDL1312 [24] and harbors the two polypeptides’ coding regions of CyaA and CyaC. CyaC is involved in the conversion of proCyaA into the active toxin by post-translational palmitoylation of Lys983 of CyaA [20]. In pTRCAG, the cyaC and cyaA genes are placed in the same transcriptional unit under the control of the λ phage Pr promoter. The pTRCAG plasmid also encodes the thermosensitive λ repressor CI857 that strongly represses gene transcription at the λ Pr promoter at temperatures below 32 ◦ C [47]. The 3 end of the cyaC gene was modified to introduce before its stop codon, a ribosome binding site to enhance the translation initiation of the downstream cyaA gene (figure 1). The resulting modification of the CyaC polypeptide (the last 3 amino acid Gly-Thr-Ala at the C-terminus of CyaC were replaced by Asn-Arg-Glu-Glu) had no effect on its ability to acylate CyaA (data not shown). To facilitate construction of recombinant toxins with exogenous polypeptides inserted within their catalytic domains, a multicloning site sequence with new unique restriction sites was introduced in CyaA downstream from codon 224 (figure 1). In addition, codons 225 to 234 were deleted and codons 236, 238 and 239 were changed as shown in figure 1. These modifications were introduced to increase the local electrostatic charge, which was previously shown to be critical for the translocation [21]. The modified CyaA had similar invasive activity as the wild-type molecule (data not shown). The open reading frames of mature restrictocin, Rest [28], HIV Tat [42], HIV Nef [42], and mouse

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DHFR [9], were amplified by PCR using target DNA and appropriate synthetic oligonucleotide primers (sequences available upon request). The amplified DNA fragments were then gel-purified, cut by appropriate restriction enzymes, and subcloned into the corresponding sites of pTRCAG. Bovine neurocalcin δ was inserted in the CyaA catalytic domain using a different approach. Plasmid pDL1312 [24], encoding neurocalcin δ, was cut by NdeI and NaeI. The DNA fragment encoding codons 1 to 190 of neurocalcin was purified, blunted by incubation with T4 polymerase in the presence of the 4 dNTPs and subcloned into plasmid pACM224 cut by PstI, and blunted by T4 polymerase in the presence of the 4 dNTPs. Ligation in the correct orientation restored the reading frame at both ends of neurocalcin cDNA. The resulting plasmid, pTRAC-Neuro224, encoded a chimeric protein made up of residues 1 to 224 of CyaA fused to residues 1 to 190 of neurocalcin followed by residues 224 to 399 of CyaA. The neurocalcin insertion was reintroduced into pTRCAG by two successive subclonings. First, a 1.65 kb SacII/BamHI fragment of pTRAC-Neuro224 was subcloned into the corresponding sites of pTRCAG. This resulted in the deletion of codons 400 to 1706 of CyaA that were reinserted in a second step by subcloning a BstBI/BamHI fragment from pTRCAG. 2.3. Expression and purification of the wild-type and recombinant CyaA toxins

Figure 1. Schematic representation of the pTRCAG expression vector. (A) Schematic map of pTRCAG. CyaC, CyaA, CI857 and β-lactamase open reading frames are indicated as well as the ColE1 origin, the Pr promoter and some relevant restriction sites. (B) Intergenic region between CyaC and CyaA showing the new C-terminus of CyaC, the stop codon (underlined) and the ATG of CyaA. (C) Multicloning site sequence inserted within modified CyaA between codon Ala220 and Leu240 . The new unique restriction sites are indicated. The alignment of the modified polypeptide sequence with that of wild-type CyaA is displayed in the bottom part.

Expression of the recombinant toxins was carried out in the E.coli BLR strain. Cells harboring pTRCAG were grown in LB medium at 30 ◦ C until mid-log phase and then synthesis of CyaC and CyaA was induced by increasing the growth temperature to 42 o C. Bacteria were harvested after 3–4 h of induction, resuspended in 20 mM Hepes-Na, pH 7.5 and lysed by French press. After centrifugation, the inclusion bodies containing the CyaA proteins were solubilized by overnight agitation in 8 M urea, 20 mM Hepes-Na, pH 7.5. After centrifugation the supernatants were supplemented with 0.14 M NaCl, and applied to a DEAE-sepharose columns (1 volume of packed gel/volume of urea extract) equilibrated with 8 M urea, 0.14 M NaCl in 20 mM Hepes-Na, pH 7.5, and the columns were washed with 10-column volumes of the equilibration solution. The bound toxins were eluted with 8 M urea, 0.5 M NaCl in 20 mM Hepes-Na, pH 7.5. The eluted proteins were diluted 5 times with 20 mM Hepes-Na, 1 M NaCl, pH

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Table I. Characteristics of the passenger proteins inserted into CyaA.

Toxins

CyaA CyaA-Neuro

Protein name and origin

CyaA-Tat

– Neurocalcin (Ca2+ binding protein, cytosolic, bovine) Restrictocin (ribotoxin, cytosolic, Aspergillus restrictus) Dihydrofolate reductase (cytosolic enzyme, mouse) Tat (trans-activating factor, HIV)

CyaA-Nef

Nef (negative factor, HIV)

CyaA-Rest CyaA-DHFR

Inserted polypeptides Size Electrostatic (number of amino chargea acids) (R/K – D/E)

Kd CaM (nM)b

Translocation efficiencyc (% of CyaA)

192

−6

1−3 150−400

100 <5

148

+7

10−30

40−70

187

+5

900−2500

50−80

87

+14

200−400

20−40

206

−4

100−300

<5

a Calculated from the number of Lys and Arg residues minus the number of Asp and Glu residues. b Determined from dose-responses

curves of AC activity as a function of added CaM; the indicated values correspond to the concentrations of CaM required for halfmaximum stimulation of enzyme activity. c The given values correspond to the range observed in the different experiments performed using different batches of recombinant toxins and different lots of cells. The translocation efficiency of a given toxin was calculated from the ratio of internalized AC activity to bound AC activity and compared to the same ratio for the wild-type CyaA (taken as 100%) in the same set of experiments.

7.5 and applied to phenyl-sepharose columns equilibrated with the same buffer. After washing with 20 mM Hepes-Na, pH 7.5, the toxins were eluted with 8 M urea in 20 mM Hepes-Na. All toxins purified by this method, were more than 90% pure as judged by SDS-gel analysis (figure 2). The toxin concentrations were determined spectophotometrically from the absorption at 280 nm using a molecular extinction coefficient of 142,000 M−1 cm−1 (calculated from the content in amino acids Trp, Tyr and Phe) for wild-type CyaA. Alternatively, protein concentrations were measured with the Bio-Rad Bradford assay reagent using purified wild type CyaA as a standard. The isolated catalytic domain and the unmodified proCyaA toxin, which were used as controls in internalization assays, were purified as described previously [25, 41]. 2.4. Preparation of oxidized CyaA-Rest toxin

The purified CyaA-Rest protein, kept in 8 M urea +10 mM DTT, was renatured by 5× dilution into 20 mM Hepes-Na, pH 7.5, 0.5 mM CaCl2 (final urea concentration < 1.6 M) and incubated in batch with CaM-agarose for 2 h at 4 ◦ C. In these conditions, the catalytic domain of CyaA-Rest was able to form a tight 1:1 complex with the immobilized CaM. The

CaM-agarose resin was then washed extensively with 20 mM Hepes-Na, pH 7.5, 0.5 mM CaCl2 , to remove the unbound CyaA-Rest, urea and the reducing agent, DTT, and then equilibrated in 20 mM Hepes-Na, pH 7.5, containing a mixture of oxidized and reduced glutathione (2 mM/5 mM, respectively). This mixture was incubated overnight at room temperature to allow formation of disulfide bonds. After extensive washing of the CaM-agarose resin with 20 mM Hepes-Na, pH 7.5, the oxidized CyaA-Rest was eluted in 8 M urea, 0.5 M NaCl, 20 mM Hepes-Na, pH 7.5, and in the absence of reducing agents. 2.5. CyaA binding and translocation assays

Adenylate cyclase activity was measured as previously described [25] in a medium containing 50 mM Tris-HCl, pH 8.0, 6 mM MgCl2 , 0.1 mM CaCl2 , 0.1 mM cAMP, 5–6000 cpm of [3 H]cAMP, 1.25 µM CaM, and 2 mM [α-32 P]ATP (1–2 × 105 cpm/assay). One unit of adenylate cyclase activity corresponds to 1 nmol of cAMP formed in 1 min at 30 ◦ C and pH 8.0. Toxin binding and translocation into sheep erythrocytes were assayed essentially as previously described [21, 40]. Toxins in 8 M urea, 20 mM Hepes-Na were directly diluted (at least 200-fold) into suspensions of sheep erythrocytes (2% of dry

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100. The internalized adenylate cyclase activity protected from trypsin digestion was then measured and expressed as a percentage of total activity added. In control experiments, after exposure to the toxins, the washed erythrocytes were lysed with 0.1% Triton X100 prior to trypsin treatment. No adenylate cyclase activity could be detected in these conditions, indicating that the protection of AC activity required the cell membrane integrity (data not shown). 2.6. Cytotoxicity assays

Figure 2. SDS-Page analysis of the recombinant CyaA toxins carrying various passenger proteins. Five micrograms of the purified proteins were separated on a 5–15% polyacrylamide gel and stained by Coomassie blue. Note the reduced mobility of the toxins harboring passenger proteins compared to that of wild-type CyaA. Lane 1: molecular weight markers (from top to bottom: 98, 67, 45, 32, 22 kDa); lane 2: modified CyaA (encoded by pTRCAG); lane 3: CyaA-Rest; lane 4: CyaA-Tat; lane 5: CyaANef; lane 6: CyaA-DHFR; lane 7: CyaA-Neuro; lane 8: wild-type CyaA.

pellet) in buffer A (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM CaCl2 ) and incubated at 37 ◦ C for 15 to 30 min. An aliquot was removed to determine the total adenylate cyclase activity added to each sample (in the range of 0.8 to 1 unit per ml of cell suspension, corresponding to about 2 µg/mL of CyaA protein). The cell suspensions were chilled on ice, centrifuged at 4 ◦ C, and the pelleted cells were resuspended in buffer A and separated into two batches. One batch was centrifuged again and the pelleted cells were lysed with 0.1% Triton X-100. The enzymatic activity measured in this extract corresponds to that of the toxin bound to the membrane and was expressed as a percentage of total activity added to the cells. To the second batch, 20 mg of TPCK (L1-(tosylamino)-2-phenylethyl chloromethyl ketone)treated trypsin (Sigma) were added and the mixture was incubated for 10 min at room temperature to digest the adenylate cyclase that remained at the external surface of the erythrocytes. After addition of soybean trypsin inhibitor (5-fold excess), the erythrocytes were washed and lysed with 0.1% Triton X-

Intoxication of erythrocytes by CyaA toxins was also quantitated by measuring intracellular cAMP accumulation. Recombinant CyaA toxins were diluted into suspensions of sheep erythrocytes in buffer A (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM CaCl2 ) as above and incubated at 37 ◦ C for 30 min. The cells were then pelleted by centrifugation; the cell pellets were resuspended in 0.5 ml of 0.1 N HCl and boiled for 5–8 min. The suspension was then neutralized by addition of 0.1 ml of 0.5 N NaOH. Insoluble material was removed by centrifugation and the cAMP content was determined by an ELISA assay [22]. 2.7. Miscellaneous

Trypsin, soybean trypsin inhibitor and methotrexate were purchased from Sigma, DEAE-sepharose and phenyl-sepharose were from Pharmacia, Calmodulin-agarose, NADPH, ATP, and cAMP were from Sigma, 3 H-cAMP and ATP-α32P from New England Nuclear. Calmodulin was purified from an E. coli overproducing strain (D.L., unpublished results). Oligonucleotide synthesis and DNA sequencing were performed by Genaxis (France). Cultures in fermentors were performed by the “Service des Fermentations” facility of the Pasteur Institute. 3. Results and discussion 3.1. Construction of recombinant CyaA toxins carrying passenger proteins inserted within the catalytic domain

To examine the tolerance of CyaA to insertion of foreign polypeptides, we constructed five recombinant toxins harboring passenger proteins that were inserted at the same insertion position, between Arg224 and Arg235 of CyaA (table I). The inserted proteins included bovine neurocalcin δ, an intracellular

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Table II. Invasive activity of the recombinant CyaA toxins carrying passenger polypeptides.

Toxins CyaA CyaA-DHFR CyaA-Rest CyaA-Tat CyaA-Nef CyaA-Neuro

AC activitya Bound Internalized Bound Internalized Bound Internalized Bound Internalized Bound Internalized Bound Internalized

EGTA/37 ◦ C 0.96 0.001 0.40 0.001 0.87 0.001 0.40 0.001 0.56 0.001 0.77 0.001

Assay conditions CaCl2 /4 ◦ C 1.38 0.001 1.62 0.001 1.32 0.001 1.51 0.001 1.46 0.001 1.39 0.001

CaCl2 /37 ◦ C 1.15 0.62 0.57 0.30 0.91 0.51 0.88 0.51 1.05 0.001 0.97 0.001

cAMP accumulationb 3.6 2.4 2.8 1.4 < 0.1 < 0.1

a The indicated recombinant toxins were incubated with erythrocytes for 30 min at the indicated temperature, either in the presence of 2 mM EGTA or in the presence of 2 mM CaCl2 . The adenylate cyclase activities associated with (bound) or internalized into erythrocytes were determined and expressed as percentage of total input. The given values correspond to mean of three experiments (the standard error mean being less than 12% of the indicated values). b Erythrocytes were incubated with the indicated recombinant toxin for 30 min at 37 ◦ C, in the presence of 2 mM CaCl2 . Intracellular cAMP was determined as described in Materials and methods. Results are expressed as pmol cAMP/mL of erythrocyte suspensions/units of added enzymatic activity.

Ca2+ binding protein of 192 amino acids (aa), mouse DHFR, a cytosolic enzyme of 187 aa, the Tat and Nef proteins from HIV (87 and 206 aa, respectively) and restrictocin, a 148-aa-long ribonuclease from Aspergillus restrictus. The characteristics of these different protein inserts are given in table I. The recombinant toxins were expressed in E. coli and purified to homogeneity from inclusion bodies by a two-step procedure including DEAE-sepharose and phenyl-sepharose chromatographies (see figure 2). The purified proteins were stored in 8 M urea in the presence of reducing agents as in all cases, the inserted polypeptides contain two or more Cys residues (CyaA itself has no Cys residues). All five recombinant adenylate cyclase toxins were catalytically active although their affinity for the activator calmodulin was differently affected (table I). This was not unexpected, since the exogenous polypeptides were inserted in close proximity to the main calmodulin binding site (located within residues 235–254 of CyaA). 3.2. Recombinant CyaA toxins harboring passenger proteins can translocate into eukaryotic cells

The ability of these recombinant toxins to enter into eukaryotic cells was tested by using sheep ery-

throcytes as a model system. These cells are particularly appropriate for analyzing the translocation of CyaA across the plasma membrane as they are totally devoid of membrane traffic. Therefore, only the direct translocation of the catalytic domain across the plasma membrane is detected. The translocation of the catalytic domain into erythrocytes was determined by a trypsin protection assay. This assay allows precise quantification of the efficiency of translocation by measuring in parallel the fraction of CyaA toxin that associates with the cells and the fraction that reaches the cytosol of these cell (where it becomes resistant to the action of externally added trypsin; see Materials and methods). As shown in table II, CyaA-Tat, CyaA-Rest, and CyaA-DHFR were able to enter into eukaryotic cells as revealed by the presence of trypsin resistant adenylate cyclase activities within erythrocytes. The translocation efficiencies of these recombinant toxins, as estimated from the ratio of internalized versus bound activity, ranged from 40 to 80% of that of native CyaA (table II). In contrast, CyaA-Neuro and CyaA-Nef were unable to translocate across the plasma membrane of erythrocytes under the same conditions (table II). It is important to note that all of these recombinant toxins bound with similar efficiency to the tar-

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get cells, as did the native CyaA (table II). This indicates that the polypeptide sequences inserted in CyaA affected only translocation of the catalytic domain across the cell membrane, but not the binding of these modified toxins to the erythrocytes. Measurements of the intracellular cAMP levels in erythrocytes exposed to the different recombinant toxins (table II), confirmed that CyaA-Tat, CyaA-Rest, and CyaA-DHFR were delivered to the cytosol of erythrocytes in an enzymatically active form, whereas CyaA-Neuro and CyaA-Nef were unable to enter the erythrocytes. In all cases, the entry of recombinant adenylate cyclases into erythrocytes was abolished at 4 ◦ C or in the absence of extracellular calcium, as shown for the wild-type CyaA (table II). Furthermore, when cells exposed to the recombinant toxins were lysed by addition of nonionic detergents prior to exposure to trypsin, no adenylate cyclase activity could be detected (data not shown), indicating that, as expected, the protection of the AC enzymatic activities of recombinant toxins towards trypsin inactivation required erythrocyte membrane integrity. Altogether, these results show that CyaA can transport several passenger proteins inserted within its catalytic domain across the plasma membrane of eukaryotic cells. 3.3. Methotrexate and NADPH specifically block the entry of CyaA-DHFR into erythrocytes

Numerous studies have shown that, in general, polypeptides that are transported across a membrane need to be maintained in a loosely folded structure in order to be translocation-competent. To examine whether unfolding of the passenger protein inserted within CyaA is a prerequisite for the translocation of the recombinant toxin into erythrocytes, we analyzed the effect of the DHFR ligands, NADPH and methotrexate (MTX), on the internalization of CyaADHFR. The DHFR protein has been extensively used in membrane translocation studies following the pioneering work of Eilers and Schatz [11]. These authors demonstrated that DHFR, fused to a mitochondrial targeting sequence, could be efficiently imported into mitochondria. This import could be blocked by MTX, which binds with high affinity to DHFR and stabilizes its tertiary structure. DHFR has been used similarly to demonstrate that various translocation processes such as protein export in E. coli and in eukaryotic cells or toxin entry into target cells requires unfolding of the transported polypeptide [1, 6, 23, 39, 43].

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The wild-type CyaA and CyaA-DHFR toxins were first incubated with erythrocytes at 4 ◦ C in the presence of Ca2+ for 20 min. In these conditions, the toxins were bound to the cells but not internalized (figure 3). After washing unbound toxins, the cells were resuspended in the presence of Ca2+ and in the presence of either NADPH or MTX, or both ligands or with no ligand. The different samples were then incubated at 37 ◦ C for 30 min to allow translocation of the cell-bound toxins into the cytosol. As shown in figure 3, the internalization of wild-type CyaA into erythrocytes was not significantly affected by the presence of MTX (1–50 µM) or NADPH (1 mM) or the presence of both ligands. In contrast, the translocation of CyaA-DHFR was inhibited by these ligands. Addition of either 50 µM MTX or 1 mM NADPH reduced by 40–60% the translocation of CyaA-DHFR (figure 3). When both ligands were added, less than 15% of CyaA-DHFR was translocated into erythrocytes. These results indicate that stabilization of the 3dimensional structure of the passenger protein DHFR as a result of ligand binding impedes the translocation of CyaA-DHFR across target cell membranes. It is interesting to note that the presence of both DHFR ligands was required to significantly inhibit entry of CyaA-DHFR, as previously observed by Arkowitz et al. [1] for the arrest of translocation of a proOmpADHFR polypeptide by the sec machinery of E. coli. This suggests that the translocation process might drive unfolding of DHFR, when only one of the two ligands is added. 3.4. Internal disulfide bridges within the restrictocin polypeptide block the entry of CyaA-Rest into erythrocytes

To further demonstrate the influence of the threedimensional structure of the passenger protein on the translocation of recombinant CyaA, we analyzed the effects of disulfide bridge formation on the internalization of CyaA-Rest. We reasoned that if unfolding of the passenger protein is a prerequisite for internalization, formation of intramolecular disulfide bridges would stabilize its structure and prevent the translocation of the recombinant toxins, as was shown previously for the diphtheria toxin [12]. CyaA itself has no Cys residues, whereas restrictocin has 4 Cys that are engaged, in the native state of the protein, into two disulfide bonds [46], which link Cys 6 to Cys 148, and Cys 76 to Cys 132 (numbering corresponds to the mature restrictocin). We therefore examined the effects

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Figure 3. Methotrexate and NADPH block the translocation of CyaA-DHFR into erythrocytes. Erythrocytes were incubated with wild type CyaA or CyaA-DHFR toxins at 4 ◦ C in the presence of 2 mM CaCl2 for 20 min. After eliminating unbound toxins by washing, the cells were resuspended in buffer A containing 2 mM CaCl2 and separated into five batches. One batch was kept at 4 ◦ C, whereas the others were incubated at 37 ◦ C for 30 min, with 1 mM NADPH and 50 µM MTX where indicated. After a second washing, the adenylate cyclase activities associated with or internalized into erythrocytes were determined in each batch. Results are expressed as a percentage of the initial total activity added to the erythrocyte suspension. Int/bound: ratio of the internalized activity versus bound activity under the different experimental conditions. The graph is representative of eight separate experiments performed on three independent CyaADHFR preparations.

of internal disulfide bond formation on the translocation capability of CyaA-Rest. In order to favor the formation of correct internal disulfide bonds, the oxidation of cysteine residues of CyaA-Rest was performed while the toxin was bound to CaM immobilized on agarose beads. As the restrictocin polypeptide is inserted in the middle of the catalytic domain between two subdomains (called T25 and T18) that both interact with CaM [25, 26], we expected that upon binding of CyaARest to CaM, the two extremities of the restrictocin polypeptide, including Cys6 and Cys148, would be brought in close proximity so that the formation of a disulfide bond between these two residues would be strongly favored. Therefore, the CyaA-Rest toxin was first bound to a CaM-agarose resin and after extensive washing (to remove the unbound CyaARest and the reducing agent DTT), was incubated with a mixture of oxidized and reduced glutathione to allow formation of disulfide bonds (see Materials and methods). After washing, the CyaA-Rest was eluted from the CaM-agarose resin under denaturing conditions and in the absence of reducing agents (figure 4A). The translocation capabilities of oxidized CyaARest were then analyzed either in the presence or in the absence of DTT. As shown in figure 4B, the oxidized CyaA-Rest fraction was able to translocate into cells only upon preincubation with DTT. We conclude from these experiments that, after oxidation, intramolecular disulfide bridges within the restrictocin moiety of CyaA-Rest prevented the translocation of the catalytic domain across the plasma membrane of target cells. Disruption of these disulfide bridges by addition of DTT restored the translocation capacity. Therefore these results support the hypothesis that the passenger proteins inserted in the catalytic domain of CyaA must unfold in order to be translocated. 3.5. Concluding remarks

In this work we have constructed and characterized five chimeric CyaA toxins that harbor different passenger proteins inserted within the catalytic domain of CyaA. Three main conclusions can be drawn from our study: (1) All five recombinant toxins were catalytically active, indicating that the catalytic domain is able to accommodate insertions of up to 206 amino acid residues without major effects on the structure and

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A

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B

Figure 4. Translocation of the reduced and oxidized CyaA-Rest toxins. (A) SDS-PAGE analysis of the oxidized CyaA-Rest. Equivalent volumes of the different fractions of CyaA-Rest from the CaM-agarose chromatography were run on a nonreducing 5% polyacrylamide SDS gel and stained with Coomassie blue. Lane 1: starting material (i.e. reduced CyaA-Rest); lane 2: unbound CyaA-Rest; lane 3: CyaA-Rest eluted in 8 M urea + 0.5 M NaCl (i.e. oxidized CyaA-Rest); lane 4: same as lane 3 but heated in the presence of DTT; lane 5: wild-type CyaA. (B) Invasive activity of oxidized CyaA-Rest. Wild-type CyaA or oxidized CyaA-Rest toxins were preincubated for 30 min with 20 mM DTT where indicated, then diluted into erythrocyte suspensions in buffer A containing 2 mM CaCl2 , and further incubated for 30 min at the indicated temperatures. After washing, the adenylate cyclase activities associated with or internalized into erythrocytes were determined as described in Materials and Methods, and expressed as percentage of total input. The graph is representative of five separate experiments.

stability of the enzyme. Previous studies had shown that the catalytic domain is constituted of two subdomains, T25 and T18, that are both required for enzymatic activity. These two fragments could be separated by proteolytic cleavage and could reassociate with calmodulin in a functional complex [26]. In the present study, the exogenous polypeptides were inserted within the region connecting these two subdomains that therefore appears to be a highly permissive site [25]. Furthermore, all the chimeric toxins were able to bind efficiently to target cells. This indicates that the modifications of the N-terminal catalytic domain did not affect the C-terminal part of the toxin responsible for target cell membrane binding. These findings confirm the structural independence of the 2 functional regions – catalytic and cell-binding do-

mains – of the CyaA toxin that was suggested in previous studies. (2) Several passenger proteins could be genetically inserted within the catalytic domain of CyaA without preventing its entry into erythrocytes. The translocation of these recombinant toxins was calcium-and temperature-dependent and followed the same internalization kinetics as the native CyaA. Therefore, most likely, these recombinant toxins follow the same pathway for penetration into eukaryotic cells as the wild-type CyaA, that is, a direct translocation across the plasma membrane of target cells. These results extend our previous studies, which demonstrated that CyaA could deliver small peptides into eukaryotic cells [13, 18, 40]. (3) Unfolding of the heterologous protein inserted within CyaA is required to allow internalization

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of the recombinant toxin into target cells. Indeed, stabilization of the passenger protein fold, either by addition of ligands, in the case of DHFR, or by formation of internal disulfide bridges, in the case of restrictocin, blocks the translocation of the corresponding recombinant CyaA into erythrocytes. It has been repeatedly observed that translocation of polypeptides across biological membranes requires their unfolding. This was extensively documented for protein import into the ER or the mitochondria of eukaryotic cells, as well as for sec-dependent protein secretion in E. coli [1, 11, 39]. Similarly, unfolding has been shown to be a prerequisite for the entry of different toxins into eukaryotic target cells [6, 12, 23, 43, 44]. The failure of the recombinant CyaAs carrying neurocalcin or HIV Nef to internalize into erythrocytes might be explained by the inability of the inserted proteins to unfold and adopt a translocation competent form in the experimental conditions of the intoxication assay. However, it should be noted that both neurocalcin and Nef have in common a net negative charge, in contrast to the three translocationcompetent proteins that have a net positive charge (table II). As we recently showed that the electrostatic charge of the central region of the catalytic domain of CyaA is critical for its translocation [21], it could also be possible that the negative charges of the grafted neurocalcin and Nef protein block the translocation process. Our present results illustrate the remarkable tolerance of CyaA to insertion of large passenger polypeptides and suggest that CyaA could be an attractive vehicle to transport proteins into eukaryotic cells. In particular, these findings could have direct applications for the design of recombinant toxins to stimulate cellular immunity [16]. The present demonstration that not only small peptides (corresponding to given epitopes) but full-length proteins could be inserted within the catalytic domain of CyaA without altering the biological activities of the toxin clearly expands the range of application of CyaA as a vaccine vehicle. The tolerance of CyaA to insertions of large polypeptide fragments is eminently attractive when considering the recent demonstration that CyaA uses the αM β2 integrin (CD11b/CD18) as a cell receptor [17]. As this integrin is expressed by a restricted subset of leukocytes including dendritic cells, CyaA appears as a very promising vector to target antigens to these professional APCs.

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