Genetically Engineered Clostridial C2 Toxin as a Novel Delivery

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Bioconjugate Chem. 2010, 21, 130–139

Genetically Engineered Clostridial C2 Toxin as a Novel Delivery System for Living Mammalian Cells Jo¨rg Fahrer,† Rainer Plunien,† Ulrike Binder,† Torben Langer,† Hartmut Seliger,‡ and Holger Barth*,† Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, and Research Group on Chemical Functions in Biosystems, University of Ulm, Albert-Einstein-Allee 47, D-89081 Ulm, Germany. Received August 17, 2009; Revised Manuscript Received November 30, 2009

The C2 toxin of Clostridium botulinum is a binary bacterial protein toxin, comprising the enzyme component C2I and the separate binding/translocation component C2IIa. C2IIa mediates the transport of C2I into the host cell cytosol. The N-terminal domain of C2I (C2IN) is enzymatically inactive but essential for C2IIa-mediated internalization of C2I. Here, we exploit the C2IIa/C2IN system to generate a recombinant C2IN-streptavidin fusion protein allowing for the delivery of biotinylated molecules into the cytosol of mammalian cells. C2IN-streptavidin overproduced in E. coli was affinity-purified and capable of binding biotinylated proteins in a concentration-dependent manner. Real-time surface plasmon resonance confirmed the biotin-mediated interaction yielding a KD-value of ∼0.75 µM. Internalization of C2IN-streptavidin into the cytosol of epithelial cells and macrophages was demonstrated by immunoblot analysis and confirmed by confocal microscopy. Cell viability studies showed no cytotoxic effects of the novel transporter. Furthermore, Vero cells treated with biotin-fluorescein or biocytin-Alexa488 as model cargo displayed a specific C2IN-streptavidin/C2IIa-dependent uptake, providing proof-of-principle for the functionality of this novel delivery system.

INTRODUCTION C2 toxin produced by C. botulinum is the archetype of binary actin-ADP-ribosylating toxins, comprising the two nonlinked proteins C2I and C2II (1). The enzyme component C2I harbors ADP-ribosyltransferase activity, whereas C2II represents the binding/translocation moiety. C2II is converted by proteolytic cleavage into its biologically active form C2IIa (2), which directs the uptake of C2I into the host cell cytosol. C2IIa forms ringshaped heptamers, also referred to as prepores, and binds to complex carbohydrate receptors on the cell surface of eukaryotic cells (3, 4). Subsequently, cell-surface bound C2IIa serves as a docking platform for C2I, allowing toxin assembly on the cell surface, which in turn triggers receptor-mediated endocytosis (5). Acidification of early endosomes results in a structural change of C2IIa heptamers to the so-called pore conformation, triggering their insertion into the endosomal membrane and subsequent pore formation (6). Translocation of C2I through this pore is driven by a pH gradient between the cytosol and the endosomal lumen. This process is facilitated by the host cell factors Hsp90 and cyclophilin A, as recently demonstrated (7, 8). In the cytosol, C2I catalyzes the covalent modification of actin with ADP-ribose, inducing the depolymerization of the actin cytoskeleton (9). The N-terminal domain of C2I (C2IN) is enzymatically inactive but essential for C2IIa-mediated internalization of C2I (10). The adaptor domain C2IN has been successfully used for the cellular import of various proteins, such as the virulence factor SpvB from S. enterica (11) or dihydrofolate reductase (12). Furthermore, C2IN has been genetically fused to bacterial C3 ADP-ribosyltransferases, which are selective inhibitors of * Corresponding author. Dr. Holger Barth, Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-EinsteinAllee 11, D-89081 Ulm, Germany, Tel.: +49-731-50065503; Fax: +49731-50065502; E-mail: [email protected]. † University of Ulm Medical Center. ‡ University of Ulm.

Rho GTPases. This allowed the delivery of C3 proteins into numerous eukaryotic cell lines to study Rho-mediated signaling and turned out to be a powerful pharmacological tool (10, 13). Streptavidin, produced by Streptomyces aVidinii, is a tetrameric protein which exhibits an extraordinary affinity for biotin (KD ) 10-15 M) (14). In addition, streptavidin is an exceptionally stable protein, showing a high resistance against heat, denaturants, or extreme pH values. Owing to these properties, it has been used in a wide range of applications including life sciences, (nano-) medicine, and drug delivery (15-17). Streptavidin and its chicken homologue avidin have recently been used as cellular delivery systems in conjunction with molecular Trojan horses. These systems comprise monoclonal antibodies directed against cell-surface receptors, such as the transferrin receptor, that induce receptor-mediated endocytosis, thus enabling the passage through biological membranes (18, 19). In this approach, avidin or streptavidin are either genetically fused to the respective monoclonal antibody or covalently linked by chemical means. Previously, a bioconjugate consisting of streptavidin and an antibody to the human insulin receptor was applied for the intracellular delivery of radiopharmaceuticals in vivo (20). Streptavidin was also fused to the cell-penetrating peptide TAT and demonstrated to promote the internalization of a biotinylated cargo into mammalian cells (21, 22). Moreover, an antitransferrin receptor-avidin fusion protein was used to deliver biotinylated molecules in myeloma and T cell lymphoma cells (23). Recently, streptavidin mutants have been generated allowing for a reversible biotin binding due to a decreased affinity. Sano and co-workers described the construction of a dimeric streptavidin, which is capable of biotin binding, however, with reduced affinity (KD ) 1.5 × 10-7 M) (24). In the present study, we exploit the transport mechanism of the clostridial C2 toxin in conjunction with dimeric streptavidin allowing for the delivery of exogenous biotinylated molecules into mammalian cells. We report the generation, functional characterization, and application of a chimeric C2IN-streptavidin fusion protein. Notably, C2IN-streptavidin applied in combina-

10.1021/bc900365b  2010 American Chemical Society Published on Web 12/23/2009

C2 Toxin as Delivery System

tion with the binding/translocation component C2IIa was detected in the cytosol of various tested mammalian cell types, emphasizing its versatility as a delivery system. We provide proof-of-principle for this novel transport system using biotin-fluorescein or biocytin-Alexa488 as cargo.

EXPERIMENTAL PROCEDURES Cloning of C2IN-Streptavidin. Plasmid pGEX2T-C2I (10), harboring a GST-tagged version of C2I, was digested with BamHI to remove the C-terminal part of C2I and religated yielding pGEX2T-C2IN. Streptavidin cDNA was amplified by PCR from plasmid TSA43 (24) using the primers StrepI (5′GAATTCAGATCTGGCATCACCGGCACC-3′) and StrepII (5′-GAATTCGGATCC CTACACCTTGGTGAA-3′) to introduce a BglII and a BamHI site, respectively. The PCR product was ligated in the pCR-Blunt vector using the Zero Blunt PCR cloning kit (Invitrogen, Karlsruhe, Germany) and transformed in competent E. coli DH5R cells. Isolated pCR-streptavidin was then digested with BglII and BamHI, ligated with BamHIdigested pGEX2T-C2IN, and transformed in competent E. coli cells. Subsequently, the C2IN-streptavidin construct was sequenced with the pGEX2T 5′- and 3′-sequencing primers. In addition, the C2IN-streptavidin boundary was analyzed with the sequencing primers 5′-SeqI (5′-TTAGATAGGGATGCTATAGGC-3′) corresponding to position 280-300 and 3′-SeqII (5′-GTCACGATGAAGGTCGAGCC-3′) mapping to position 709-728 of the C2IN-streptavidin construct. Expression and Purification of Recombinant Fusion Protein. C2IN was overproduced in E. coli BL21 and purified as described previously (10). C2IN-streptavidin was overexpressed as GST-tagged fusion protein in E. coli BL21 and isolated by affinity chromatography. Briefly, E. coli BL21 transformed with pGEX2T-C2IN-streptavidin was grown at 37 °C in Luria-Bertani (LB) medium supplemented with 100 µg/ mL ampicillin to an optical density of 0.6-0.8. To induce protein expression, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and cultures were incubated at 29 °C for 6 h. Bacteria were harvested by centrifugation for 10 min at 4 °C at 4400 g, resuspended in buffer containing 0.1% Triton X-100, and disrupted by sonication. Cellular debris was sedimented for 15 min at 4 °C at 20 000 g, and the clear supernatant was incubated with glutathione-agarose beads (Macherey-Nagel, Du¨ren, Germany) for 2 h at room temperature. After centrifugation for 5 min at 1000 g, the beads were washed 2 times with lysis buffer and 2 times with PBS pH 7.4. To remove the GST-tag, the immobilized protein was incubated with thrombin (15 NIH units/ L culture) for 1 h at room temperature, and supernatant containing C2IN-streptavidin was obtained by centrifugation at 10 000 g for 1 min. To eliminate thrombin, the supernatant was further incubated with benzamidine beads (GE Healthcare, Mu¨nchen, Germany) for 10 min and collected. Isolated C2IN-streptavidin was analyzed for homogeneity by SDSPAGE followed by Coomassie staining. Protein identity was confirmed by Western blot analysis with an anti-streptavidin antibody (Abcam, Cambridge, UK) and an anti-C2IN antibody raised in rabbits (25), respectively. Tomonitorthetime-dependentexpressionofC2IN-streptavidin, 300 µL aliquots of the suspension culture were collected and analyzed by SDS-PAGE and Coomassie staining. In addition, the GST-tagged protein was detected on a nitrocellulose membrane using an anti-GST antibody (BD Biosciences, Heidelberg, Germany). SDS-PAGE and Immunoblot Analysis. Proteins were separated by SDS-PAGE on 12.5% gels according to the method of Laemmli (26). For immunoblot analysis, the proteins were transferred onto a nitrocellulose membrane (Whatman, Dassel,

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Germany) by electroblotting using a semidry system (PeqLab, Erlangen, Germany) or alternatively with a wet-blot chamber (GE Healthcare, Mu¨nchen, Germany). The membranes were blocked for 1 h at room temperature with 5% nonfat dry milk in PBS containing 0.1% Tween 20 (PBS-T) followed by 1 h incubation with the respective antibodies in blocking solution. After washing the membranes with PBS-T, the blots were probed with the appropriate secondary antibodies coupled to horseradish peroxidase (1:2000), and proteins were detected by enhanced chemiluminescence. Overlay Blot Assay. Biotinylation of proteins was carried out using the EZ-Link Micro Sulfo-NHS-Biotinylation Kit (Pierce, Bonn, Germany). To assess the biotin-binding properties of the generated fusion protein, increasing amounts of C2IN-streptavidin as well as the negative control C2IN (each protein 1-50 pmol) were subjected to SDS-PAGE. After separation, the proteins were transferred on a nitrocellulose membrane by wet blotting and the membrane was blocked. Subsequently, the membrane was incubated with biotinylated BSA in PBS-T (5 µg/mL) or biotinylated C3bot (1 µg/mL). To disrupt unspecific binding, the membrane was washed with PBS-T containing 500 mM NaCl followed by three washing steps in PBS-T. Bound biotin-labeled BSA or C3bot were detected with streptavidin-POD (Roche, Mannheim, Germany) diluted 1:2000 in PBS-T. Surface Plasmon Resonance. All SPR experiments were performed with a Biacore 2000 system equipped with a CM5 sensor chip (GE Healthcare). C2IN-streptavidin in sodium acetate buffer (pH 4.5) was immobilized onto a CM5 sensor chip using the amine coupling procedure to obtain a final response level about 2500 RU. An activated reference cell without immobilized ligand was used to subtract buffer refractive effects and as a control for unspecific binding. The following concentrations of biotin-labeled C3bot protein from Clostridium botulinum were injected at room temperature: 750 nM, 1 µM, 2.5 µM, 5 µM, 7.5 µM, 10 µM. Flow rate was set to 30 µL/ min with the following parameters: 130 s contact time, 1800 s dissociation time. Dissociation was carried out in HBS buffer pH 7.4 without a regeneration step. Baseline levels were stable throughout all cycles. Data evaluation was carried out using BIAeValuation software v 3.2. Cell Culture and Cytotoxicity Assays. HeLa and Vero cells were cultured at 37 °C and 5% CO2 in Minimum essential medium (MEM), and CHO cells were maintained in a 1:1 mixture of Ham’s F12/ Dulbecco’s modified Eagle medium (DMEM), containing 10% heat-inactivated fetal calf serum (FCS), L-glutamate (2 mM), 100 U/mL penicillin, and 100 µg/ mL streptomycin. Cells were routinely trypsinized and reseeded three times per week. J774A.1 cells (a kind gift of Dr. Singh Chhatwal, Braunschweig, Germany) were cultured at 37 °C and 5% CO2 in DMEM, containing 10% heat-inactivated FCS, L-glutamate (4 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL). Cells were routinely scraped off and reseeded three times per week. Cell viability was determined by trypan blue exclusion. Briefly, Vero cells were seeded in a 12 well plate (4 × 104 cells/well) and grown overnight. Subsequently, varying concentrations of C2IN-streptavidin and C2IIa were added in serum-free medium. As a positive control, cells were exposed to 1 µM staurosporine. After 2 h, the medium was removed and the cells were incubated for another 3 h with MEM supplemented with FCS. Thereafter, cells were collected by trypsination and mixed 1:1 with 0.4% trypan blue solution. Viable and nonviable (i.e., blue) cells were counted with a hemocytometer permitting the calculation of the cell viability. In addition, cell proliferation was assessed over a period of 48 h using the Cell Titer 96 Aqueous nonradioactive cell proliferation

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assay (Promega, Mannheim, Germany). Briefly, Vero cells were seeded in 96 well plates at a density of 5 × 103 cells per well. The next day, cells were incubated with different combinations of C2IN-streptavidin and C2IIa, as well as 0.5 µM staurosporine as a positive control. Cell viability was determined after 5, 24, and 48 h, respectively, by measuring absorbance at 490 nm in an ELISA reader. Cell Fractionation by Digitonin Treatment. To obtain the cytosolic fraction of cultured Vero cells, digitonin-based cell fractionation was performed according to the method described by Sørensen and co-workers (27).Vero cells grown in 12 well plates were preincubated at 37 °C in serum-free medium for 30 min. Subsequently, a combination of C2IN-streptavidin (2 µg/mL) and C2IIa (4 µg/mL) was added to the medium and cells were incubated for up to 4 h with the complex. As a control, cells were left untreated or incubated without the binding/translocation component C2IIa. Cells were then washed twice with PBS to remove unbound toxin from the surface. Thereafter, cells were incubated in the presence of digitonin (20 µg/mL in PBS) for 5 min at 25 °C to permeabilize the cell membrane and for additional 25 min at 4 °C to allow the cytosol to diffuse into the buffer. The supernatant was carefully collected and adherent extracted cells were scraped off in Laemmli loading buffer. The samples were then subjected to SDS-PAGE and transferred to a nitrocellulose membrane by wet blotting, and C2IN-streptavidin was detected with a polyclonal antiC2IN antibody as described before. Thereafter, the blot membrane was stripped, and the identity of the cytosolic fraction was confirmed by immunoblot analysis of cytosolic marker protein Hsp90, using a monoclonal anti-Hsp90 antibody (Santa Cruz Biotechnology, Heidelberg, Germany). Importantly, the absence of early endosomes in this fraction was verified in immunoblot with an antibody directed against rab5 (BD Biosciences, Heidelberg, Germany), a marker protein for early endosomes. Immunofluorescence Microscopy of C2IN-StreptavidinTreated Cells. For immunocytochemical analysis of C2INstreptavidin uptake, cells were seeded on sterile coverslips at a density of 3.5 × 104 per well and incubated in 12-well culture dishes overnight. Cells were then treated with a combination of 1 µg C2IN-streptavidin and 2 µg C2IIa in serum-free medium for 2 h. Medium was exchanged and cells were incubated for another 2 h. As controls, cells were left untreated or incubated with the single components, i.e., C2IN-strepavidin or C2IIa. Subsequently, cells were fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, and blocked with 5% dry milk powder in PBS-T. C2IN-streptavidin was then visualized with an R-streptavidin antibody (1:1000 in PBS) in conjunction with a secondary antibody-Alexa488 conjugate (1:400 in PBS). Finally, nuclei were counterstained with Hoechst 33342 (1:20 000 in PBS), and coverslips were mounted with ProLong Gold antifade reagent (Invitrogen, Karlsruhe, Germany). Confocal microscopy was performed with a Zeiss Axiovert 200 M microscope equipped with a LSM510 Meta laser scanning device (Zeiss, Oberkochen, Germany). 1-µmthick sections were recorded with LSM Image Examiner and processed with ImageJ software (NIH, USA). Confocal Microscopy to Monitor Biotin-Fluorophor Uptake into Vero Cells. To analyze the uptake of biotin-fluorescein (Sigma, Deisenhofen, Germany) or biocytin-Alexa488 (Invitrogen, Karlsruhe, Germany), Vero cells were grown overnight on sterile coverslips. After preincubation of 2 µg C2INstreptavidin and an excess of fluorescein-biotin or biocytinAlexa488 for 30 min, the mixture was supplemented with 4 µg C2IIa, and complex formation was allowed to occur for 30 min at 4 °C. Subsequently, the complex was added to the cells and incubated for 2 h. Untreated cells, cells incubated with bioti-

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nylated fluorophor alone or with the complex omitting C2IIa, served as controls. After 2 h, the medium was replaced by serum-containing MEM, and cells were further incubated for 2 h. Cells were then processed for immunofluorescence as described above. After fixation and permeabilization, cells were blocked and early endosomes were stained with a primary antibody directed against EEA1 (Novus Biologicals, Littleton, USA) in conjunction with an Alexa 647-labeled secondary antibody. Cells embedded in ProLong Gold antifade reagent were visualized using a LSM510 Meta confocal system connected to a Zeiss Axiovert 200 M microscope (Zeiss, Oberkochen, Germany). z-Stack images were recorded in 1 µm confocal sections using LSM Image Examiner and processed with ImageJ software. Reproducibility of the Experiments and Statistics. All experiments were performed independently at least twice. Results from representative experiments are shown in the figures. Values (n g 3) are calculated as mean ( standard deviation (S.D.) using the GraphPad Prism4 software.

RESULTS Cloning, Expression and Purification of C2IN-Streptavidin. To generate a recombinant C2IN-streptavidin fusion protein, dimeric streptavidin was cloned in frame to C2IN, yielding the expression vector pGEX-2TGL-C2IN-streptavidin. The fusion construct features the N-terminal domain of C2I, which mediates the interaction with C2IIa to trigger cellular uptake, and a dimeric streptavidin, thus providing a versatile adaptor for biotinylated molecules (Figure 1A). The GST-tagged fusion protein was overproduced in E. coli BL21, and time-dependent expression was monitored. Western blot analysis with a monoclonal R-GST antibody revealed maximum expression 6 h post induction (p.i.), whereas longer expression times resulted in degradation of the fusion protein (Figure 1B). Thus, bacterial cultures were harvested after an expression period of 6 h. Then, C2IN-streptavidin was purified using glutathione-agarose affinity chromatography and analyzed by an immunoblot with a polyclonal R-streptavidin antibody (Figure 1C). After immobilization on the beads, the GST-tag was removed by thrombin cleavage releasing C2IN-streptavidin into the supernatant (Figure 1C, elution). C2IN-streptavidin was not completely soluble, as it could still be detected in the bacterial pellet. However, genetic fusion to C2IN markedly enhanced the solubility compared to the expression of dimeric streptavidin, which has to be purified under denaturing conditions (24). The purified protein was then checked for homogeneity by SDSPAGE and Coomassie staining and revealed about 85% purity as calculated by ImageJ (Figure 1D, left panel). In addition, protein identity was confirmed by Western blot analysis with a polyclonal R-C2IN antibody (Figure 1D, right panel). Biotin Binding Properties of the Novel C2IN-Streptavidin Fusion Protein. In order to characterize the binding of biotinlabeled macromolecules to C2IN-streptavidin, an overlay blot technique was employed. To this end, we have selected three biotinylated proteins differing in size and origin: bovine serum albumine (BSA), the ADP-ribosyltransferase C3bot from Clostridium botulinum, and calf alkaline phosphatase, Increasing amounts of the fusion protein and C2IN as negative control were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. Following incubation with 5 µg/mL biotinylated BSA, unspecifically interacting biotinylated protein was removed by high-salt washes, and bound BSA was detected using streptavidin-POD. Biotinylated BSA specifically interacted with C2IN-streptavidin in a concentration-dependent manner, whereas the negative control C2IN did not show any signal (Figure 2A). In addition, C2IN-streptavidin was incubated with streptavidin-POD omitting biotinylated BSA. Here, no signal was



Figure 1. Cloning, expression, and purification of C2IN-streptavidin. (A) Cloning strategy to generate the novel fusion protein C2IN-streptavdin. (B) Time-course of C2IN-streptavidin expression in E. coli BL21. Aliquots of the suspension culture were removed after the time points post induction (p.i.) as indicated and analyzed by Western blotting with a monoclonal R-GST antibody. The arrow highlights the GST-C2IN-streptavidin fusion protein (C) Purification of GST-C2IN-streptavidin by affinity chromatography. Fractions obtained during the purification process were collected and C2IN-streptavidin was detected using a polyclonal R-streptavidin antibody. The upper arrow indicates the GST-tagged C2IN-streptavidin. The GST tag was removed by specific thrombin cleavage, yielding the 37 kDa fusion protein (designated by the lower arrow). (D) Characterization of isolated C2IN-streptavidin. 2.5 µg of the fusion protein was subjected to SDS-PAGE and visualized by Coomassie staining (left panel). Moreover, protein identity was confirmed by Western blot analysis with a polyclonal R-C2IN antibody (right panel).

observed, thus excluding a direct interaction of both streptavidin conjugates (data not shown). Next, biotinylated C3bot was shown to bind to immobilized C2IN-streptavidin with an affinity comparable to biotinylated BSA (Figure 2B), which was also demonstrated for biotin-coupled alkaline phosphatase (data not shown). To obtain more quantitative information on the biotin-streptavidin mediated interaction, binding kinetics were assessed by real-time surface plasmon resonance (28, 29). C2IN-streptavidin was covalently immobilized on a Biacore CM5 sensor chip, and biotinylated C3 was injected in HBS buffer pH 7.4 at various concentrations ranging from 750 nM to 10 µM. Concentration-dependent binding was monitored and allowed the determination of binding kinetics resulting in a KD value of 7.57 × 10-7 M (Figure 2C and Table 1). Signal was corrected for unspecific binding and buffer changes using an activated reference cell. Taken together, the fusion protein C2IN-streptavidin is functionally active with regard to biotin binding. As expected, the binding affinity was clearly reduced in comparison to wild-type streptavidin, but is in the range reported for dimeric streptavidin (24). Specific Internalization of C2IN-Streptavidin into the Cytosol of Epithelial Cells and Macrophages. As a next step, the time-dependent uptake of C2IN-streptavidin in living Vero cells was monitored after cell fractionation by digitonin. C2IN-streptavidin and the translocation/binding component C2IIa were preincubated on ice and then added to the culture medium. At the time points indicated, cells were permeabilized with digitonin to extract the cytosolic proteins. The cytosolic fraction as well as the remaining extracted cells were then subjected to SDS-PAGE and analyzed by Western blotting using

a polyclonal R-C2IN-antibody (Figure 3A). Increasing amounts of C2IN-streptavidin were observed in the cytosolic fraction in a time-dependent manner, reaching a maximum after 4 h, which was confirmed by time course experiments over a period of 8 h (data not shown). However, the majority of the fusion protein was detected in the extracted cells representing cellbound or vesicular-internalized C2IN-streptavidin. Treatment of cells with C2IN-streptavidin in the absence of C2IIa resulted in weak nonspecific binding or uptake, which could only be observed after long exposure times. The identity of the cytosolic fraction was attested by immunoblot analysis of cytosolic marker protein Hsp90 (Figure 3B), showing approximately 50% of Hsp90 in the extracted cytosolic fraction, which is in line with previous studies applying digitonin extraction (30). Importantly, Rab5 as endosomal marker protein was found exclusively in extracted cells, ruling out a contamination of the cytosolic fraction by early endosomes (Figure 3C). Consistent with this observation, EEA1 as another marker for endosomes was only detected in extracted cells (data not shown). Furthermore, the specific internalization of C2IN-streptavidin into the cytosol of other mammalian cells was demonstrated for human epithelial HeLa cells, hamster epithelial CHO cells, and murine macrophage-like J774A.1 cells (Figure 3D). The uptake kinetic of C2IN-streptavidin into the cytosol of HeLa cells was similar to Vero cells, culminating 4 h after treatment (Figure 3D, upper panel). CHO cells exhibited the most efficient internalization of C2IN-streptavidin into the cytosol, whereas incubation of cells without the transport component C2IIa resulted in a weak background signal of C2IN-streptavidin (Figure 3, middle panel). Compared to the epithelial cell lines,


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Figure 2. Characterization of C2IN-streptavidin with regard to biotin binding. (A) Overlay blot using biotinylated BSA. Increasing amounts of C2IN-streptavidin and C2IN as negative control were separated by SDS-PAGE and transferred onto a nitrocellulose membrane stained with Ponceau S. Following incubation with 5 µg/mL biotinylated BSA, unspecifically interacting biotinylated protein was removed by highsalt washes and bound BSA was detected using streptavidin-POD. (B) Overlay blot using biotinylated C3bot protein. This experiment was performed as describe above, except that 1 µg/mL C3bot protein was applied. (C) Real-time SPR analysis. C2IN-streptavidin was immobilized on a Biacore CM5 sensor chip and increasing concentrations of biotinylated C3bot protein were injected (750 nM up to 10 µM). Black lines show experimentally obtained data, whereas red lines indicate the calculated curve fit according to a Langmuir 1:1 model. Table 1. Binding Kinetics of C2IN-Streptavidin and Biotinylated C3bot Obtained from Real-Time SPR Analysis ka (M-1 s-1)

kd (s-1)

KD (M)

χ2

6.75 × 103

5.11 × 10-3

7.57 × 10-7

0.174

J774A.1 macrophages displayed a pronounced nonspecific binding/internalization of the fusion protein in the absence

Figure 3. Uptake of C2IN-streptavidin into the cytosol of Vero cells. (A) Vero cells were treated with a combination of C2IN-streptavidin and C2IIa. As control, cells were left untreated or exposed to C2IN-streptavdin alone. After 0.5, 2, or 4 h, respectively, the cells were permeabilized by digitonin treatment to extract cytosolic proteins. The cytosolic supernatant was collected, and the remaining extracted cells were lysed. Both fractions were subjected to SDS-PAGE and analyzed by immunoblotting with a polyclonal R-C2IN-antibody. Lanes 1 and 6: control cells, 4 h. Lanes 2 and 7: cells treated with C2IN-streptavidin alone, 4 h. Lanes 3-5 and 8-10: cells treated with C2IN-streptavidin and C2IIa, 0.5, 2, and 4 h. Equal protein loading was confirmed by Ponceau S staining (data not shown). (B) Immunodetection of Hsp90, a cytosolic marker protein, on the same blot. (C) Visualization of Rab5, a marker protein for early endosomes, by Western blotting to exclude a possible contamination of the cytosolic fraction with early endosomes during digitonin treatment. (D) Treatment of HeLa, CHO, and J774 cells with a combination of C2IN-streptavidin and C2IIa as described under (A).

of C2IIa, most likely due to pinocytosis (Figure 3D, lower panel). Unlike the other cell types tested, lower amounts of C2IN-streptavidin were detected in the cytosol of J774A.1 cells.



Figure 4. Confocal microscopy of internalized C2IN-streptavidin. Vero cells grown on coverslips were incubated with C2IN-streptavidin and C2IIa (1 + 2 µg). After 4 h, cells were fixed and C2IN-streptavidin was visualized by an R-streptavidin antibody and a secondary antibody coupled to Alexa488. Cells embedded in ProLong Gold antifade reagent were then analyzed by confocal microscopy and optical thin layer sections obtained were processed with ImageJ. Representative pictures thereof are shown. Scale bars correspond to 10 µm.

In addition, confocal laser-scanning microscopy was performed to analyze the intracellular localization of C2INstreptavidin in more detail. Vero cells grown on coverslips were incubated with C2IN-streptavidin and C2IIa. After 4 h, cells were fixed and C2IN-streptavidin was visualized by an R-streptavidin antibody together with a secondary antibody coupled with Alexa488. Strikingly, confocal microscopy showed the uptake of the novel fusion protein as revealed by bright intracellular foci and diffuse staining (Figure 4). In contrast, untreated cells and cells treated with C2IN-streptavidin alone displayed weak diffuse background staining. In conclusion, we demonstrated the specific, i.e., C2IIa-dependent, uptake of C2IN-streptavidin into the cytosol of different mammalian cell types, which underscores the versatility of this novel delivery system. Absence of Cytotoxic Effects of the Novel Transporter C2IN-Streptavidin/C2IIa. To reveal any possible cytotoxic effect of the fusion protein generated, Vero cells grown overnight were incubated with different combinations of C2IN-streptavidin and C2IIa for 5 h. Staurosporine, a wellknown inducer of apoptosis, was used as positive control. Cell viability was determined by trypan blue assay, which is based on the selective uptake of trypan blue dye in dead cells, whereas living cells exclude the dye (Figure 5A). No effect was observed even at high concentrations (1 µg/mL C2IN-streptavidin + 2 µg/mL C2IIa), whereas the positive control staurosporine markedly decreased the cell viability to 60%. To monitor longterm effects on cell proliferation, cells were seeded in 96 well plates and treated with increasing concentrations of C2INstreptavidin and C2IIa ranging from 100 ng/mL + 200 ng/mL up to 2.5 µg/mL + 5 µg/mL. After 5, 24, and 48 h, respectively, cell viability was assessed using the MTS proliferation assay. No cytotoxicity of the novel transporter could be recorded

(Figure 5B). It should be noted that full-length C2I induces delayed caspase-dependent apoptosis after 24 h due to its intrinsic and persistent ADP-ribosyl-transferase activity (31), which has been removed in the novel fusion protein. In contrast to C2IN-streptavidin, staurosporine dramatically reduced the cell viability to about 25% after 48 h of incubation. Moreover, putative adverse effects of C2IN-streptavidin on the actin cytoskeleton were analyzed by immunofluorescence microscopy using the actin-specific probe phalloidin-Alexa 594, but no alterations were observed compared to control cells (data not shown). C2IN-Streptavidin/C2IIa-Mediated Uptake of BiotinFluorescein in Vero Cells. Finally, biotin-fluorescein was used as model cargo to test the functionality of the novel transport system. Vero cells grown on coverslips were treated with a complex of biotin-fluorescein and the transporter C2INstreptavidin/C2IIa. After 4 h, cells were fixed and permeabilized with Triton X-100. To visualize the endosomal marker protein EEA1, cells were stained with a polyclonal R-EEA1 antibody in conjunction with an Alexa647-coupled secondary antibody. Confocal laser scanning microscopy showed several bright foci in cells treated with the transporter together with fluoresceinlabeled biotin (Figure 6A). These spots were obviously localized in the cytosol and did not overlap with early endosomes as evidenced by the vesicular EEA1 staining. In contrast, cells exposed to biotin-fluorescein alone or with the transporter omitting C2IIa revealed no signal, demonstrating the specific uptake mediated by the transport system. Since the acidification of early endosomes may diminish the fluorescence signal of fluorescein, we used the polar tracer biocytin-Alexa488, which is cell-impermeant and shows enhanced pH stability. As observed for biotin-fluorescein, the C2IN-streptavidin/C2IIa delivery system mediated the uptake of biocytin-Alexa488 in


Figure 5. Determination of putative cytotoxic effects caused by C2IN-streptavidin. (A) Trypan blue exclusion. To reveal putative adverse effects on cell viability, Vero cells grown overnight were incubated with different combinations of C2IN-streptavidin and C2IIa for 5 h at 37 °C. Staurosporine, a well-known inhibitor of protein kinases and inducer of apoptosis, was used as a positive control. Subsequently, cell viability was determined by trypan blue assay using phase contrast microscopy. (B) To monitor long-term effects, cells were seeded in 96 well plates and treated with increasing concentrations of C2IN-streptavidin and C2IIa. After 5, 24, and 48 h, respectively, cell viability was assessed using the MTS proliferation assay. As above, staurosporine treatment was included as positive control. Data in (A) and (B) are expressed as mean ( SD, n ) 2 (each experiment performed as quadruplicate).

Vero cells (Figure 6B). Confocal microscopy showed bright green foci and partial colocalization with early endosomes (Figure 6B), which is attested by the yellow staining in the merged channel. Control experiments were performed as described for biotin-labeled fluorescein and displayed no signal in the green channel (data not shown). In summary, our data clearly demonstrate the successful application of the novel transporter as delivery system for biotinylated molecules.

DISCUSSION In the present study, we describe the successful generation of a novel molecular Trojan horse based on genetic engineering of the clostridial C2 toxin and a dimeric streptavidin, which is superior to biochemical conjugation with regard to homogeneity

Fahrer et al.

and its site-directed character. The novel fusion protein C2IN-streptavidin was overexpressed in E. coli and purified to homogeneity followed by removal of the GST tag. This native isolation procedure resulted in moderate yields, but led to soluble and functionally active protein. Alternative purification under denaturing conditions provides higher yields, but the protein precipitated on a biotin-agarose column, most likely due to improper refolding (data not shown). The purified fusion protein displayed a moderate affinity for biotinylated proteins as shown by an overlay blot technique and real-time SPR measurements. Compared to the dimeric streptavidin mutant (KD ) 1.5 × 10-7 M) (24), the affinity is slightly reduced probably due to steric constraints of the fusion protein affecting the biotin binding. This decreased affinity should allow for the reversible binding of internalized biotinylated molecules. In particular, endogenous free biotin generated by biotinidase (32) could displace biotinylated cargo by competition on the binding sites after internalization into the cytosol. On the other hand, this feature may be unfavorable with regard to the stability of the C2IN-streptavidin-biotin complex in vivo, which may be disrupted by serum proteins. Molecular Trojan horses employing wild-type avidin or streptavidin exhibit a very tight (strept) avidin-biotin binding, which is stable in the bloodstream in vivo (33, 34). Moreover, we demonstrated the successful delivery of C2IN-streptavidin into the cytosol of diverse eukaryotic cell lines of human, rodent, and monkey origin. The observed slight variations in the internalization efficiency of the protein among the different cell types might be due to different expression levels of the C2 toxin receptor and/or for different velocities of endocytosis and intracellular protein translocation processes. Importantly, all hitherto tested vertebrate cell lines and primary cultured cells express the C2 toxin receptor on their cell surface, thus rendering these cells sensitive for C2 intoxication (13). Therefore, our novel delivery system comprising C2INstreptavidin and C2IIa is suitable for a plethora of eukaryotic cell types. In contrast, conventional molecular Trojan horses encompassing receptor-specific antibodies are species-specific and limited to the expression pattern of the receptor. In addition, the novel C2-derived transporter did not affect cell viability over a time period of 48 h, nor did it induce any morphological changes, thus permitting its unrestricted application in cell cultures studies. Finally, we provided proof-of-principle for our novel delivery system using biotin-fluorescein as cargo, which was specifically internalized into Vero cells. Interestingly, biotin-fluorescein was detected in the cytosol of Vero cells and showed no colocalization with early endosomes. However, fluorescein exhibits no fluorogenic properties below pH 6, which may result in loss of signal during the acidification of endosomes. Therefore, biotin-fluorescein localized to early/late endosomes may not be detectable by confocal microscopy. To overcome this limitation, the fluorescent tracer biocytin-Alexa488 was applied. This conjugate is cell-impermeable due to its polarity and offers superior pH stability, allowing for the fluorescence detection even in acidic cellular compartments. Intriguingly, biocytin-Alexa488 was specifically delivered into Vero cells in a C2IN-streptavidin/C2IIa-dependent manner and showed partial colocalization with early endosomes. The uptake mechanism of the binary C2 toxin of C. botulinum has been extensively characterized and is well-understood in most aspects, which also holds true for the novel fusion protein C2IN-streptavidin. Unlike the C2 toxin, the entry pathways of other delivery vectors such as cell penetrating peptides are still unclear and seem to involve different mechanisms (35, 36). With regard to the binary C2 system, C2IIa has two major functions. First, C2IIa mediates the binding of C2I via its N-terminal domain to the cell surface, and second, it facilitates



Figure 6. C2IN-streptavidin/C2IIa-mediated uptake of biotin-labeled fluorescent tracers into Vero cells. (A) Vero cells grown on coverslips were treated with a complex consisting of biotin-fluorescein in combination with the transporter C2IN-streptavidin/C2IIa. After 4 h, cells were fixed and permeabilized with Triton X-100. To visualize the endosomal marker protein EEA1, cells were stained with a polyclonal R-EEA1 antibody in conjunction with an Alexa647-coupled secondary antibody. Cells embedded in ProLong Gold antifade reagent were then analyzed by confocal microscopy and recorded thin layer z-stack images were processed and merged with ImageJ. Representative images thereof are displayed. Scale bars correspond to 10 µm. (B) Vero cells were treated and analyzed by confocal microscopy as describe above, except that biocytin-Alexa488 was used instead of biotin-fluorescein.

the membrane translocation of C2I from endosomes into the host cell cytosol by pore formation. The translocation channel formed by the C2IIa pore was shown to be 25-32 Å in width (37), which allows the passage of small molecules as attested by the transport of biotin-fluorescein into the cytosol of Vero cells in the present study. However, the narrow inner diameter of the translocation pore may limit the transport of larger molecules such as biotinylated proteins into the cytosol, thus trapping them in endosomes. To solve this problem, pHresponsive polymers such as poly(propylacrylic acid) (PPAA) could be used, which trigger endosomal membrane disruption upon luminal acidification (38). Biotinylated PPAA was reported to promote the internalization and endosomal release of an antibody complex containing streptavidin (39). It has also been successfully applied together with TAT-streptavidin to direct

the uptake of a biotinylated protein to the cytosol, representing a useful tool for delivery systems (21). With regard to immunogenicity, humans are only slightly responsive to the chicken homologue avidin, which may result from consumption of egg white causing oral tolerance (40). In contrast, streptavidin was shown to be immunogenic in humans, which might be a drawback for long-term or repetitive treatments, e.g., during cancer radioimmunotherapy (RIT) (41). Nevertheless, the use of streptavidin in cancer RIT led to promising clinical results, increasing the tumor/background ratio and the delivery of higher therapeutic doses (15). Lack of glycosylation is another advantage of streptavidin compared to chicken avidin, which shows unspecific binding due its carbohydrate modification, impairing its use in several applications (42).


The use of bacterial toxins as delivery systems is well-known and protein toxins such as diphtheria toxin (DT) have been employed as so-called immunotoxins to selectively kill cancer cells (43). For that purpose, the binding domain of DT has to be replaced by ligands that interact with surface antigens abundant on cancer cells, e.g., epidermal growth factor, allowing for a concentration of the toxin on the target cell. After translocation into the cytosol, DT catalyzes the ADP-ribosylation of elongation factor 2, thereby blocking protein synthesis, which results in cell death (44). As pointed out above, the binary C2 toxin represents a universal transport system due to the ubiquitous expression of its corresponding receptor on many eukaryotic cells. Previously, several chimeric fusion proteins based on the adaptor domain C2IN were created by genetic engineering (10, 11), allowing the cellular uptake and cellbiological characterization of proteins, which do not enter cells. However, the construction and expression of recombinant fusion proteins is time-consuming and cumbersome, whereas the use of the chimeric C2IN-streptavidin as versatile binding platform for a variety of biotinylated molecules is convenient and applicable to nearly all systems. Therefore, the novel cellular transporter C2IN-streptavidin/C2IIa should be a valuable tool for the uptake of biotinylated siRNA, peptides, and even proteins. A promising approach would be the targeted silencing of genes, e.g., involved in inflammation or carcinogenesis by siRNA. Very recently, Xia and co-workers reported the successful delivery of siRNA into the cytosol of human endothelial cells mediated by a molecular Trojan horse, showing efficient RNA interference similar to cationic polyplexes (45). In summary, we have successfully established C2INstreptavidin as a novel delivery system, which can be used as molecular Trojan horse in a variety of eukaryotic cells. Furthermore, we demonstrated C2IN-streptavidin/C2IIa-mediated internalization of fluorescent biotinylated tracers as cargo in Vero cells, providing proof-of-principle for this universal transporter.

ACKNOWLEDGMENT We are grateful to Dr. Takeshi Sano, Boston, USA, for the generous gift of dimeric streptavidin cDNA and Bernd Betzler, University of Ulm, Germany, for the expression vector harboring streptavidin cDNA. We would like to thank Dr. Angelika Ru¨ck, ILM, University of Ulm, Germany, for kindly providing the confocal microscope facility and Dr. Alexander Bu¨rkle, University of Konstanz, Germany, for critically reading the manuscript.

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Genetically Engineered Clostridial C2 Toxin as a Novel Delivery

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