Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer Rémi Venezianoa, Claire Rossib, Alexandre Chenalc, Jean-Marie Devoissellea, Daniel Ladantc,1, and Joel Chopineaua,d,1 a Equipe Matériaux Avancés pour la Catalyse et la Santé, Unité Mixte de Recherche 5253, Centre National de la Recherche Scientifique-Ecole Nationale Supérieure de Chimie de Montpellier-Université Montpellier 2-Université Montpellier 1, Institut Charles Gerhardt Montpellier, 34296 Montpellier Cedex 5, France; bCentre National de la Recherche Scientifique Formation de Recherche en Evolution 3580, Université de Technologie de Compiègne, 60205 Compiègne Cedex, France; cCentre National de la Recherche Scientifique Unité Mixte de Recherche 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, 75724 Paris Cedex 15, France; and dUniversité de Nîmes, 30021 Nîmes Cedex, France

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study’s in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions. adenylate cyclase activity

| synthetic biomembrane | toxin internalization

ATP-cyclizing, CaM-activated catalytic domain (AC) is located in the 400 amino-proximal residues, whereas the carboxyl-terminal 1,306 residues are responsible for the hemolytic phenotype of B. pertussis (17–20). The C-terminal “hemolysin” moiety contains, between residues 500 and 750, several hydrophobic segments that are predicted to adopt alpha-helical structures and to insert into membranes to create the cation-selective pores responsible for the hemolytic activity (20, 21). The C-terminal part of the molecule (RD; residues 1,000–1,706) is involved in toxin binding to a specific cellular receptor (CD11b/CD18) (22, 23). This domain consists of approximately 40 copies of a calcium-binding, glycineand aspartate-rich nonapeptide repeat (residues 1,014–1,613) characteristic of a large family of bacterial cytolysins known as repeat-in-toxin (RTX) toxins (11, 13, 24, 25). The CyaA toxin is synthesized as an inactive precursor, proCyaA, which is converted into the active toxin form (CyaA) on specific acylation of two lysine residues (26, 27). Then CyaA is secreted across the bacterial envelope by a dedicated type I secretion machinery and binds to the CD11b/CD18 integrin expressed by a subset of leukocytes including neutrophils, macrophages, and dendritic cells (22, 28–30). However, CyaA can also invade a wide variety of cells that do not express this receptor, albeit with a lower efficiency (19, 31–35). The most unique property of CyaA is its capability to deliver its N-terminal catalytic domain directly across the plasma membrane of the eukaryotic target cells, a process that occurs independently of the CD11b/CD18 receptor (11–13). It is believed that CyaA first Significance

T

ransport of protein across the cell membrane is a complex process that usually involves multipart translocation machineries. Many protein toxins from poisonous plants or from pathogenic bacteria are able to penetrate into the cytosol of their target cells where they exert their toxic effects. Some of these toxins exploit the endogenous cellular machinery of endocytosis and intracellular sorting to gain access to the cell cytosol, but others carry their own translocation apparatus (1–4). These latter toxins provide a unique opportunity to analyze the molecular mechanisms and the physicochemical principles underlying polypeptide transport across biological membranes. Studies combining structural, biochemical, and electrophysiological approaches have begun to unravel the various strategies developed by these toxins to deliver their catalytic moieties across the cell membranes (5–10). The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the few known toxins able to invade eukaryotic cells through a mechanism of direct translocation across the plasma membrane of the target cells (11–13). CyaA is an essential virulence factor of B. pertussis that is secreted by virulent bacteria and able to enter into eukaryotic cells, where, on activation by endogenous calmodulin (CaM), it catalyzes high-level synthesis of cAMP, which in turn alters cellular physiology (14–16). CyaA is a 1,706-residue-long bifunctional protein organized in a modular fashion (Fig. 1A); the www.pnas.org/cgi/doi/10.1073/pnas.1312975110

Many bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, although how they pass through a lipid bilayer remains largely unknown. Bordetella pertussis adenylate cyclase (CyaA) toxin delivers its catalytic domain directly across the cell membrane. To characterize this unique translocation process, we designed an in vitro assay based on a tethered lipid bilayer assembled over a biosensor surface derivatized with calmodulin, a natural activator of the toxin. CyaA activation by calmodulin provided a highly sensitive readout for toxin translocation across the bilayer. CyaA translocation was calcium- and membrane potential-dependent but independent of any additional eukaryotic protein. This biomimetic membrane will permit in vitro studies of protein translocation in precisely defined conditions. Author contributions: D.L. and J.C. designed research; R.V., C.R., and A.C. performed research; J.-M.D. and D.L. contributed new reagents/analytic tools; R.V., D.L., and J.C. analyzed data; and R.V., D.L., and J.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence may be addressed. E-mail: [email protected] or joel. [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1312975110/-/DCSupplemental.

PNAS | December 17, 2013 | vol. 110 | no. 51 | 20473–20478

BIOCHEMISTRY

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved November 8, 2013 (received for review July 17, 2013)

immobilized CaM. With this highly sensitive assay, translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and application of a negative transmembrane potential, in agreement with previous studies on eukaryotic cells (36). Our results demonstrate that CyaA does not require any specific eukaryotic components apart from CaM to translocate across a membrane. They also suggest that the catalytic domain may be electrophoretically transported across the bilayer in a calcium-dependent manner. This study provides a direct in vitro demonstration of a toxin translocation across a tBLM (41) and suggests that the biomimetic tBLM/CaM structure may be a useful tool for characterizing the molecular mechanisms of protein translocation across biological membranes under precisely defined conditions. Results

Fig. 1. Principle of CyaA translocation assay on tBLM/CaM assembly. (A) Scheme of CyaA toxin structure showing the three major domains: the catalytic domain, AC; the hydrophobic region, H, responsible for insertion of CyaA into the membrane; and the Ca2+-binding, RTX-containing domain, RD. (B) Schematic illustration of the approach used to monitor CyaA translocation across the tBLM. (C) Schematic representation of the SPR sample cell cross-section and tBLM/CaM construction.

inserts its hydrophobic segments into the plasma membrane and then delivers its catalytic domain across the plasma membrane into the cell cytosol (19, 31, 32) (Fig. 1B). Previous studies have shown that the translocation process is dependent on the temperature (occurring only above 15 °C), the membrane potential of the target cells, and the presence of calcium ions in the mM range (32, 36). Inside the cell, on binding to CaM with a subnanomolar affinity, CyaA is stimulated by more than 1,000-fold and exhibits a high catalytic rate (kcat > 2,000 s−1) to produce supraphysiologic levels of cAMP (12, 19, 37). How the hydrophilic CyaA catalytic domain of approximately 400 residues is able to pass across the hydrophobic barrier of the plasma membrane remains largely unknown, and whether specific eukaryotic proteins and/or cell membrane components are involved in this process is also unclear (19, 32, 35, 38, 39). To characterize the molecular mechanisms of CyaA translocation across the membrane, we performed a functional in vitro assay that exploits a recently designed biomimetic membrane assembly composed of a bilayer membrane (tBLM) tethered over an amino-grafted gold surface derivatized with CaM (40). This multilayer biomimetic assembly exhibits the fundamental feature of an authentic biological membrane in creating a continuous, yet fluid phospholipidic barrier between two distinct compartments: a cis side, corresponding to the extracellular milieu, and a trans side, marked by the cytosolic protein CaM (Fig. 1C). We monitored the binding of CyaA to the tBLM by surface plasmon resonance (SPR) spectroscopy, and detected the translocation of the catalytic domain across the bilayer by CyaA activation by the 20474 | www.pnas.org/cgi/doi/10.1073/pnas.1312975110

Biomimetic Membrane Assembly. The tBLM/CaM structure was assembled in a homemade SPR cell comprising a gold- coated glass slide covered by a Teflon chamber (∼1 mL) with inlet and outlet tubes and mounted on an SPR optical bench instrument using the Kretschmann configuration (Fig. 1C and Fig. S1). The tBLM/CaM structure was obtained in two steps as described previously (40), using SPR spectroscopy to follow the grafting of the successive molecular layers (Fig. 2, Table S1, and Fig. S2). In the first step, the gold surface was amino-grafted by self-deposition of 2-aminoethanethiol on which CaM was covalently coupled through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) activation. The immobilized CaM was functional in binding CyaA, as demonstrated by SPR spectroscopy, and in stimulating its adenylate cyclase enzymatic activity (Fig. 2A; Table S1, experiment A; and Fig. S2A). In the second step, the tBLM was assembled over the CaM layer by incubating an L-α-phosphatidylcholine from egg yolk (egg-PC) vesicle suspension doped with 5% 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)3400]-succinimidyl propionate (DSPE-PEG3400-SPA) lipopolymer for 1 h at 25 °C to covalently anchor the vesicles to the aminecoated gold surface. The Teflon cell was then flushed extensively with HBS/EGTA buffer (20 mM Hepes-Na, 0.15 M NaCl, and 2 mM EGTA; pH 7.4), to remove unlinked vesicles and favor the formation of a continuous planar bilayer. The optical thickness of the tBLM assembled over the immobilized CaM was reproducibly ∼54 ± 2 Å (Fig. 2B; Table S1, experiment B; and Fig. S2B), a value consistent with the formation of a planar lipid bilayer, as reported previously (40, 42). The fluidity and continuity of the bilayer were confirmed by fluorescence recovery after photobleaching experiments, as described in our previous report (40). The tBLM could be efficiently removed by washing with HBS/EGTA buffer containing a nonionic detergent (0.5% Triton X-100) as indicated by a large decrease in the SPR signal (Fig. 2B; Table S1, experiment B; and Fig. S2B). After washing out the bilayer, we verified that the immobilized CaM was still functional in binding CyaA and activating its enzymatic activity (Fig. 2B; Table S1, experiment B; and Fig. S2B). This also indicated that nonionic detergent treatment did not affect CyaA binding and its activation by the tethered CaM. CyaA Binding to and Translocation Across the Biomimetic Membrane.

When CyaA was diluted (200 nM) in HBS/CaCl2 buffer (20 mM Hepes-Na, 0.15 M NaCl, and 2 mM CaCl2; pH 7.4) and injected on the top of the tBLM, it efficiently bound to the membrane, as demonstrated by the increased SPR signal (Fig. 2C; Table S1, experiment C; and Fig. S2C). After an extensive wash with HBS/ EGTA buffer, a large fraction of the bound CyaA remained attached to the tBLM (amounting to 110 ± 10 ng/cm2), likely as a result of insertion of its hydrophobic segments in the bilayer. After washing with HBS/EGTA buffer + 0.5% Triton X-100, the SPR signal again decreased significantly (Table S1, experiment C and Fig. S2C) to an optical thickness of ∼8 Å, close to the 6 Å signal found in the previous experiment with Veneziano et al.

tBLM only (Table S1, experiment B). This finding suggests that most of the membrane-bound CyaA had been washed away as well, and indeed, only background levels of enzymatic activity were detected on the surface of the SPR chip. Taken together, these results indicate that although CyaA had efficiently bound the tBLM, its catalytic domain could not interact with the immobilized CaM and thus was not retained on the chip on membrane removal. Thus, CyaA did not translocate under these experimental conditions. Based on the finding of Otero et al. (36) that a membrane potential is critical for CyaA intoxication of myocytes, we explored whether CyaA translocation could be achieved in our biomimetic device after application of an electrical potential across the tethered bilayer. In this set of experiments, CyaA was again allowed to bind to and insert into the tBLM as described above (Fig. 2D; Table S1, experiment D; and Fig. S2D). After the unbound CyaA was washed with HBS/CaCl2 buffer, an electrical potential of −80 mV was applied for 5 min between the gold surface and an Ag wire (serving as a reference electrode connected to ground) connected to the bulk medium above the tBLM (i.e., between the trans and cis sides of the membrane); following convention, the bulk medium potential was set to 0 mV. Current recordings of the tBLM with or without bound CyaA at different voltages (Fig. S3) indicate that the tBLM created a good ion-impermeable barrier between the trans and cis compartments. After the membrane was washed out with Triton X-100 (i.e., HBS/EGTA + 0.5% Triton X-100) the SPR signal decreased (Fig. 2D; Table S1, experiment D; and Fig. S2D) to a value (∼200 ng/cm2) significantly higher than that found without the application of electrical potential (80 ng/cm2; Table S1). This finding suggests that a fraction of the CyaA protein was retained on the SPR chip. This was confirmed by enzymatic assays, which detected a high level of adenylate cyclase activity on the chip (Fig. S4). We conclude that on application of a negative potential across the bilayer, the catalytic domain of CyaA was translocated across the tBLM and could interact with the immobilized CaM to acquire its enzymatically active form. Voltage and Calcium Dependence of CyaA Translocation Across the tBLM. We carried out additional experiments to measure the effi-

cacy of the translocation process as a function of membrane potential. CyaA was bound to the tBLM as described above, and after the unbound CyaA was washed out, various electrical potentials, ranging from −80 mV to +50 mV, were applied for 5 min. After Veneziano et al.

the tBLM was washed with Triton X-100, the enzymatic activity bound to the immobilized CaM was measured. Enzymatic activity bound to the tethered CaM was strictly dependent on the application of a negative potential across the membrane (Fig. 3). These data are in excellent agreement with the voltage dependence of the CyaA intoxication of myocytes reported by Otero et al. (36). Of note, if after CyaA binding to tBLM (obtained in the presence of calcium), HBS/EGTA buffer was added before application of the electric potential, then no enzymatic activity was detected on the chip. This indicates that translocation of the CyaA catalytic domain is strictly dependent on the presence of calcium in the mM range (Fig. 3, Inset), as previously observed on various eukaryotic cells (18–20, 30, 32, 35, 36, 38). Taken together, these data

Fig. 3. Voltage and calcium dependence of CyaA translocation across tBLM. CyaA (200 nM) was injected on top of the tBLM/CaM assembly in HBS/CaCl2 buffer. After extensive washing with HBS/CaCl2 (black triangle; 2 mM Ca2+ on the cis side) or HBS/EGTA (black square; 2 mM EGTA on the cis side), the indicated potentials were applied for 5 min. The potential in bathing medium (i.e., on the cis side) was set at 0. After extensive washing in HBS/EGTA, the tBLM was removed by washing with 0.5% Triton X-100 in HBS/EGTA, and AC enzymatic activity was measured. (Inset) CyaA binding to tBLM/CaM was carried out in HBS/CaCl2 buffer as above. The membrane was then washed with HBS buffer containing the indicated calcium concentrations, and a −80mV potential was applied for 5 min. The AC activity was measured after membrane removal as above, and expressed as a percentage of AC activity measured with 2 mM CaCl2. Values are mean ± SEM from at least three independent measurements for each condition.

PNAS | December 17, 2013 | vol. 110 | no. 51 | 20475

BIOCHEMISTRY

Fig. 2. SPR monitoring of tBLM/CaM construction and CyaA translocation. The optical thicknesses recorded at the different stages of the tBLM/CaM construction and subsequently, during CyaA binding and translocation events, were measured by SPR (SI Materials and Methods). The numbers above the bars indicate the AC activity (in nmol/min) measured on the chip after the final wash with Triton X-100 (0.5%). The data are extracted from the traces shown in Fig. S2. The experiments are described in detail in the legend of Fig. S2. (A) Optical thicknesses measured after, sequentially, (i) CaM coupling to the cysteaminecoated gold substrate (+ CaM), (ii) CyaA binding in HBS/CaCl2 buffer (+CyaA), and (iii) a final wash with 0.5% Triton X-100 (+ TX-100). (B) Optical thicknesses measured after (i) CaM coupling (+ CaM) as above, (ii) bilayer membrane tethering (+ tBLM), (iii) tBLM removal by Triton X-100 wash (+ TX-100), (iv) CyaA binding in HBS/CaCl2 (+ CyaA), and (v) a final wash with 0.5% Triton X-100 (+ TX-100). (C) Optical thicknesses measured after (i) CaM coupling (+ CaM), (ii) bilayer membrane tethering (+ tBLM), (iii) CyaA binding in HBS/CaCl2 (+ CyaA), and (iv) a final wash with 0.5% Triton X-100 (+ TX-100). (D) Optical thicknesses measured after (i) CaM coupling (+ CaM), (ii) bilayer membrane tethering (+ tBLM), (iii) CyaA binding in HBS/CaCl2 (+ CyaA), and (iv) application of voltage (−80 mV) for 5 min, followed by a final wash with 0.5% Triton X-100 (+ TX-100).

demonstrate that our in vitro translocation assay on tBLM is able to reproduce the fundamental properties of CyaA intoxication of target cells, that is, its calcium and membrane potential dependency. Probing tBLM Integrity with a Protease Protection Assay. To further validate the in vitro translocation assay, we performed control experiments to verify that application of a voltage across the membrane had not impaired the integrity of the tBLM by creating “holes” through which CyaA could have accessed the immobilized CaM. For this, we conducted a protease protection assay similar to that used to measure CyaA translocation into erythrocytes or other cells (18–20, 32). First, CyaA was bound to the tBLM in HBS/CaCl2 buffer, and membrane polarization was applied for 5 min to trigger translocation of the catalytic domain across the tBLM. Then a solution of trypsin (20 nM in HBS/ CaCl2 buffer) was injected, followed by incubation for 5 min at 25 °C, then extensive washing with a solution containing an excess of soybean trypsin inhibitor (40 nM in HBS/CaCl2) to stop proteolysis. After the tBLM was washed out with Triton X-100 (in HBS/EGTA buffer), AC enzymatic activity was measured as before. AC activities measured with or without trypsin treatment were very similar at all tested potentials from −80 mV to +50 mV (Fig. 4). However, when a higher positive potential (+80 mV) was applied, a significant fraction of AC activity was detected in the absence of trypsin treatment, but not after trypsin treatment (Fig. 4). We conclude that this high positive potential did not trigger CyaA translocation, but rather partially impaired the integrity of the bilayer, allowing access of CyaA to the immobilized CaM and, subsequently, access of trypsin as well. We also verified that when the trypsin treatment was applied after CyaA binding but before the application of voltage (i.e., before translocation), no AC activity was detected on the chip. This finding indicates that after binding and insertion of CyaA into the tBLM, the catalytic domain is readily accessible to trypsin degradation on the “external” side of the bilayer. Finally, we verified that the AC enzyme bound to the immobilized CaM after translocation was fully degraded by trypsin when the tBLM was previously removed by washing with Triton X-100. Based on these results, we conclude that the bilayer was not altered by application of electrical potentials ranging from

Fig. 4. Probing the tethered bilayer integrity by a protease protection assay. CyaA (200 nM) was injected onto tBLM/CaM in HBS/CaCl2 buffer. After extensive washing with HBS/CaCl2 buffer, the indicated potentials were applied for 5 min as described in Fig. 3 with 2 mM Ca2+ on the cis side. For the voltage + trypsin condition, 20 nM trypsin (in HBS/CaCl2) was injected in the device and incubated for 5 min, followed by the addition of 40 nM soybean trypsin inhibitor (STI). For the trypsin + voltage condition, CyaA (200 nM) was bound to tBLM as above, but the trypsin incubation (20 nM in HBS/ CaCl2 for 5 min, followed by the addition of STI) was carried out before application of the electrical potential. After extensive washing in HBS/CaCl2, the tBLM was removed in all samples with 0.5% Triton X-100 (in HBS/EGTA buffer), and AC enzymatic activity was measured. As a control (voltage + Triton X-100 + trypsin), CyaA was bound to tBLM, a −80-mV potential was applied as above, and trypsin proteolysis was carried out after removal of tBLM by washing with Triton X-100. Values are mean ± SEM from at least three independent measurements for each condition.

20476 | www.pnas.org/cgi/doi/10.1073/pnas.1312975110

−80 mV to +50 mV, and was still forming a continuous lipidic barrier impermeable to small proteins like trypsin. However, at a higher positive potential (+80 mV), the bilayer integrity apparently was partially impaired, allowing nonspecific access of proteins (CyaA or trypsin) to the underlying trans compartment. mAb 3D1 Blocks CyaA Translocation Across the tBLM. Gray et al. (33) previously reported that a monoclonal antibody, mAb 3D1, which recognizes an epitope located between residues 373 and 399 of CyaA at the C-terminal end of the catalytic domain, does not affect the adenylate cyclase activity of the toxin or its capacity to bind to target cells, but does inhibit delivery of the catalytic domain into the cytosol of target cells (erythrocytes or J774A.1 cells). Thus, we tested the effect of mAb 3D1 in our in vitro translocation assay. We found that the addition of a molar excess of mAb 3D1 to the CyaA solution before injection into the construction did not prevent binding to tBLM, but did significantly diminish translocation of the catalytic domain, as demonstrated by the AC activity bound to the immobilized CaM (Fig. 5). This finding confirms that our in vitro translocation assay closely mimics the biological process occurring on living cells. Nonacylated ProCyaA Cannot Deliver Its Catalytic Domain Across the Tethered Bilayer. It is well known that CyaA’s invasive capacity

depends on its acylation on the lysine residues K860 and K983 (26, 27). Thus, we tested nonacylated proCyaA in our in vitro tBLM/CaM translocation assay. ProCyaA and CyaA bound to the tBLM to a similar extent, as detected by SPR signals (Fig. 6); however, after application of a −80-mV potential in the presence of CaCl2, only background levels of enzymatic activity were detected on the chip with proCyaA, in contrast to the levels seen with CyaA. This result demonstrates that the acylation of CyaA is critical for conferring the ability to deliver its catalytic domain across the tBLM, similar to that observed on eukaryotic cells. Discussion CyaA is unique among the bacterial toxins for its capacity to deliver its catalytic domain (AC) into the cytosol of target cells directly across the plasma membrane. To characterize this original translocation process, we describe here an in vitro assay based on a biomimetic membrane model consisting of a tBLM assembled over a SPR biosensor surface derivatized with CaM. The tBLM forms a continuous, protein-impermeable boundary that fully insulates the immobilized CaM (trans side) from the medium above (cis side). The binding of CyaA to the tBLM can be monitored by SPR spectroscopy, whereas binding of CyaA to the shielded, immobilized CaM serves as a highly sensitive reporter of toxin translocation across the bilayer. We demonstrated with this in vitro tBLM/CaM system, that the translocation of the AC domain was strictly dependent on the presence of calcium and on application of a negative electrical potential across the membrane, in excellent agreement with previous studies of Otero et al. (36) performed on living eukaryotic target cells. The overall efficiency of translocation (approximately 80% of total tBLM-bound CyaA) observed in vitro compares favorably with that measured previously on sheep erythrocytes (32, 43). Furthermore, no translocation was observed with the nonacylated proCyaA protein or after preincubation of CyaA with mAb 3D1, which had been previously shown to block the entry of CyaA into eukaryotic cells (30, 33, 38). This finding further supports our contention that our in vitro assay captures the essential features of the authentic physiological processes occurring with living cells. Importantly, our data show that no additional eukaryotic components are needed for the transfer of the catalytic domain across the tBLM, indicating that the CyaA protein contains all of the elements required for this process. Thus, translocation of CyaA catalytic domain across the tBLM depends only on calcium and membrane potential, which could provide the driving force for polypeptide transport across the membrane via an electrophoretic process (36, 43). However, we cannot exclude the possibility that Veneziano et al.

the tight association of CyaA with the immobilized CaM may contribute in part to translocation of the AC across the bilayer. Indeed, it has been shown that for several toxins (e.g., diphtheria toxin, anthrax lethal factor, anthrax edema factor), the entry process is facilitated in vivo by specific interactions with dedicated cytosolic factors, such as the coatomer I complex, Hsp90, or thioredoxin reductase, which recognize specific amino acid sequences within the toxin polypeptide chains (44–47). These interactions likely contribute to pull the catalytic chains of these toxins through the membrane through a Brownian ratchet-like mechanism (48), and could aid toxin refolding within the cytosol. CaM likely can play such a chaperone-like role for CyaA (49). However, it has been shown that mutant CyaA toxins exhibiting low affinity for CaM are still able to efficiently deliver their AC domain into the cytoplasm of cells (50), suggesting that a high affinity of CyaA for CaM might not be critical for CyaA translocation. CyaA is known to invade a wide variety of cell types in vivo, albeit with varying efficacies and possibly through different pathways, and to target primarily innate immune system cells that express the CD11b/CD18 integrin receptor to which its binds with high affinity (22, 29, 30, 38, 39). CyaA binds with much lower affinity to cells that do not express this receptor (19, 26, 32–34, 36, 43, 51), but whether the translocation process per se is modulated by the CD11b/CD18 receptor remains unknown (30, 38, 39). A more sophisticated biomimetic tBLM/CaM construction harboring the CD11b/CD18 receptor inserted in the membrane might be feasible in the future.

Materials and Methods

Fig. 6. ProCyaA cannot deliver its catalytic domain across tBLM. CyaA or proCyaA (200 nM) was injected either onto the immobilized CaM without tBLM ( CaM) or onto the tBLM/CaM construction as in Fig. 2. After extensive washing with HBS/CaCl2, a –80-mV potential was applied for 5 min on the tBLM/CaM assembly or not applied, as indicated. For each condition, the quantity of protein bound to the tBLM or CaM was determined by SPR spectroscopy, after which the SPR surfaces were washed extensively with 0.5% Triton X-100 in HBS/EGTA buffer and AC enzymatic activity was measured. Protein amount (black bars) and AC activity (white bars) are expressed in percentages, taking the protein binding and AC activity measured on immobilized CaM (without tBLM) as 100%. Values are mean ± SEM from at least three independent measurements for each condition.

Veneziano et al.

Proteins were produced and purified in Escherichia coli and stored at −80 °C. For tBLM construction, CaM (140 nM in HBS/EGTA buffer: 20 mM Hepes-Na, 0.15 M NaCl, and 2 mM EGTA; pH 7.4) was covalently linked to the aminegrafted surface in the presence of EDC (350 nM). The CaM surface coverage thus obtained was approximately 35 ng/cm2 (40). tBLM egg-PC/DSPEPEG3400-SPA (95/5 wt/wt ratio) was achieved by injection of a small unilamellar vesicle suspension (1 mg/mL in HBS/EGTA buffer) on top of the CaM layer, followed by incubation at room temperature for 1 h. CyaA and proCyaA were incubated in the biomimetic system at 200 nM (with 2 mM CaCl2). For the mAb–CyaA complex, preincubation with CyaA and antibody (1:1 ratio) was performed for 20 min at 25 °C before injection into the SPR sample cell. SPR measurements were performed with a homemade optical setup in a Kretschmann configuration (Fig. S1). The goldcoated glass slide was vertically assembled with a Teflon sample cell (total volume, 925 μL; gold surface, 1.54 cm2). The optical thicknesses and amounts of lipids or proteins bound on the surfaces were determined as described in SI Materials and Methods.

PNAS | December 17, 2013 | vol. 110 | no. 51 | 20477

BIOCHEMISTRY

Fig. 5. mAb 3D1 blocks CyaA translocation across the tBLM. CyaA (200 nM) was injected above the tBLM/CaM in HBS/CaCl2 as in Fig. 2 (calcium or EGTA, or 3D1 + CyaA) after preincubation (20 min at 25 °C) with mAb 3D1 (200 nM). After extensive washing with HBS/CaCl2 (calcium or 3D1 + CyaA) or HBS/EGTA (EGTA), a potential of −80 mV was applied for 5 min. Then tBLM was removed with 0.5% Triton X-100 in HBS/EGTA buffer, and AC enzymatic activity was measured. For each condition, the quantity (ng/cm2) of protein bound to the tBLM was determined by SPR spectroscopy. Values are mean ± SEM from at least three independent measurements for each condition.

The precise molecular mechanisms by which bacterial toxins can cross biological membranes to reach the cytosolic compartment of mammalian cells remain largely unknown (1, 2). Reconstitution of toxin translocation across synthetic model membranes is crucial to better characterize the structural and biophysical principles at work in this process, but designing efficient in vitro systems for that purpose is challenging. The most effective approaches to date involve electrophysiological studies on planar lipid bilayers. Indeed, many toxins are known to form ion-conductive pores in model membranes (5, 6), and it has been shown that these pores can be transiently blocked by their catalytic subunits as they pass through the pore from the cis side to the trans side of the membrane (7–9). This highly sensitive method has been instrumental to the characterization of many structural and functional aspects of toxin translocation; however, direct demonstration of the passage of the catalytic subunit from the cis compartment to the trans compartment of the membrane is difficult, given the very low quantities of molecules delivered (7). More recently, Fisher et al. (10) developed a droplet-interface bilayer (DIB) approach to monitoring the transfer of anthrax toxin across a model membrane separating two submicroliter aqueous droplets. In this approach, a lipid bilayer membrane is formed at the interface between two droplets connected to electrodes. Because the droplets can be moved into contact or separated by a micromanipulator, it is possible to follow, with an exquisite sensitivity, the physical transit of material (e.g., proteins) from one droplet to the other through a protein pore. Such a system might be of general interest for many other toxins. We present here a novel approach to study protein translocation in tBLM. These biomimetic models have been widely used to characterize protein–membrane associations, binding of ligands to cellular receptors incorporated in the supported planar bilayers, and molecular interactions in cell adhesion processes (41, 42, 52, 53); however, up to now, few studies have been devoted to protein transport across reconstituted bilayers, owing to various technical difficulties. Our tBLM/CaM technique is particularly well adapted for characterizing protein translocation across membranes, allowing for direct detection of the passage of polypeptides from the cis side to the trans side of the membrane. This is clearly an advantage over the planar lipid bilayer and DIB approaches discussed above. However, if the tBLM is fully impermeable to protein, its electrical properties cannot be compared with those of planar lipid bilayers or DIB techniques, which remain unrivaled for studying channels or pore-forming proteins. The in vitro approach elaborated herein may be applicable to other toxins that associate with CaM, such as the anthrax edema factor, and also could be adapted for virtually any toxins that target specific cytosolic factors (1). These biomimetic constructions should provide opportunities to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined in vitro conditions.

The membrane translocation assay was performed in the SPR cell using two electrodes for application of a membrane potential. The gold functionalized glass slide was set as the trans electrode, and the cis Ag electrode was bathed in buffer (Fig. 1C and Fig. S1). Potentials were applied using a Bio-Logic BLM120 amplifier equipped with a 1-GΩ head, and the signal was attenuated by an 8-pole low-pass filter (AF-180; Bio-Logic). The potential was applied for 5 min, and the current was monitored with an oscilloscope (TDS3012; Tektronix). The trypsin proteolysis assay was initiated with trypsin (20 nM) added into the SPR sample cell for 5 min and stopped by the addition of trypsin inhibitor (40 nM).

The enzymatic activity assay was performed as described previously (40). In brief, tBLM was removed with Triton X-100, after which 0.5 mL of AC buffer containing ATP (2 mM) and pyrophosphatase (2 U/mL) was added and incubated for 10 min. Pi release was measured with a Pi ColorLock Kit (Innova Bioscience).

1. Alouf JE, Popoff MR (2006) The Comprehensive Sourcebook of Bacterial Protein Toxins (Elsevier, Amsterdam, The Netherlands), 3rd Ed. 2. Lemichez E, Barbieri JT (2013) General aspects and recent advances on bacterial protein toxins. Cold Spring Harb Perspect Med 3(2):a013573. 3. Lord JM, Smith DC, Roberts LM (1999) Toxin entry: How bacterial proteins get into mammalian cells. Cell Microbiol 1(2):85–91. 4. Sandvig K, et al. (2004) Pathways followed by protein toxins into cells. Int J Med Microbiol 293(7-8):483–490. 5. Blaustein RO, Koehler TM, Collier RJ, Finkelstein A (1989) Anthrax toxin: Channelforming activity of protective antigen in planar phospholipid bilayers. Proc Natl Acad Sci USA 86(7):2209–2213. 6. Oh KJ, Senzel L, Collier RJ, Finkelstein A (1999) Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain. Proc Natl Acad Sci USA 96(15):8467–8470. 7. Koriazova LK, Montal M (2003) Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nat Struct Biol 10(1):13–18. 8. Zhang S, Finkelstein A, Collier RJ (2004) Evidence that translocation of anthrax toxin’s lethal factor is initiated by entry of its N terminus into the protective antigen channel. Proc Natl Acad Sci USA 101(48):16756–16761. 9. Beitzinger C, et al. (2012) Role of N-terminal His6-Tags in binding and efficient translocation of polypeptides into cells using anthrax protective antigen (PA). PLoS ONE 7(10):e46964. 10. Fischer A, Holden MA, Pentelute BL, Collier RJ (2011) Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers. Proc Natl Acad Sci USA 108(40):16577–16581. 11. Carbonetti NH (2010) Pertussis toxin and adenylate cyclase toxin: Key virulence factors of Bordetella pertussis and cell biology tools. Future Microbiol 5(3):455–469. 12. Ladant D, Ullmann A (1999) Bordatella pertussis adenylate cyclase: A toxin with multiple talents. Trends Microbiol 7(4):172–176. 13. Vojtova J, Kamanova J, Sebo P (2006) Bordetella adenylate cyclase toxin: A swift saboteur of host defense. Curr Opin Microbiol 9(1):69–75. 14. Confer DL, Eaton JW (1982) Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science 217(4563):948–950. 15. Goodwin MS, Weiss AA (1990) Adenylate cyclase toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice. Infect Immun 58(10):3445–3447. 16. Harvill ET, Cotter PA, Yuk MH, Miller JF (1999) Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity. Infect Immun 67(3):1493–1500. 17. Glaser P, et al. (1988) The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: Cloning and expression in Escherichia coli. Mol Microbiol 2(1):19–30. 18. Hewlett EL, Gordon VM, McCaffery JD, Sutherland WM, Gray MC (1989) Adenylate cyclase toxin from Bordetella pertussis: Identification and purification of the holotoxin molecule. J Biol Chem 264(32):19379–19384. 19. Rogel A, et al. (1989) Bordetella pertussis adenylate cyclase: Purification and characterization of the toxic form of the enzyme. EMBO J 8(9):2755–2760. 20. Bellalou J, Sakamoto H, Ladant D, Geoffroy C, Ullmann A (1990) Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase. Infect Immun 58(10):3242–3247. 21. Benz R, Maier E, Ladant D, Ullmann A, Sebo P (1994) Adenylate cyclase toxin (CyaA) of Bordetella pertussis: Evidence for the formation of small ion-permeable channels and comparison with HlyA of Escherichia coli. J Biol Chem 269(44):27231–27239. 22. Guermonprez P, et al. (2001) The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18). J Exp Med 193(9): 1035–1044. 23. El-Azami-El-Idrissi M, et al. (2003) Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: Role of toxin acylation and identification of the main integrin interaction domain. J Biol Chem 278(40):38514–38521. 24. Rose T, Sebo P, Bellalou J, Ladant D (1995) Interaction of calcium with Bordetella pertussis adenylate cyclase toxin: Characterization of multiple calcium-binding sites and calcium-induced conformational changes. J Biol Chem 270(44):26370–26376. 25. Welch RA (2001) RTX toxin structure and function: A story of numerous anomalies and few analogies in toxin biology. Curr Top Microbiol Immunol 257:85–111. 26. Barry EM, et al. (1991) Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation. J Bacteriol 173(2):720–726. 27. Hackett M, Guo L, Shabanowitz J, Hunt DF, Hewlett EL (1994) Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science 266(5184): 433–435.

28. Cheung GY, et al. (2008) Transcriptional responses of murine macrophages to the adenylate cyclase toxin of Bordetella pertussis. Microb Pathog 44(1):61–70. 29. Kamanova J, et al. (2008) Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling. J Immunol 181(8):5587–5597. 30. Eby JC, Gray MC, Mangan AR, Donato GM, Hewlett EL (2012) Role of CD11b/CD18 in the process of intoxication by the adenylate cyclase toxin of Bordetella pertussis. Infect Immun 80(2):850–859. 31. Gordon VM, et al. (1989) Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis: Different processes for interaction with and entry into target cells. J Biol Chem 264(25):14792–14796. 32. Rogel A, Hanski E (1992) Distinct steps in the penetration of adenylate cyclase toxin of Bordetella pertussis into sheep erythrocytes: Translocation of the toxin across the membrane. J Biol Chem 267(31):22599–22605. 33. Gray MC, et al. (2001) Translocation-specific conformation of adenylate cyclase toxin from Bordetella pertussis inhibits toxin-mediated hemolysis. J Bacteriol 183(20): 5904–5910. 34. Dal Molin F, et al. (2006) Cell entry and cAMP imaging of anthrax edema toxin. EMBO J 25(22):5405–5413. 35. Eby JC, et al. (2010) Selective translocation of the Bordetella pertussis adenylate cyclase toxin across the basolateral membranes of polarized epithelial cells. J Biol Chem 285(14):10662–10670. 36. Otero AS, Yi XB, Gray MC, Szabo G, Hewlett EL (1995) Membrane depolarization prevents cell invasion by Bordetella pertussis adenylate cyclase toxin. J Biol Chem 270(17):9695–9697. 37. Wolff J, Cook GH, Goldhammer AR, Berkowitz SA (1980) Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci USA 77(7):3841–3844. 38. Bumba L, Masin J, Fiser R, Sebo P (2010) Bordetella adenylate cyclase toxin mobilizes its beta2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. PLoS Pathog 6(5):e1000901. 39. Martín C, Uribe KB, Gómez-Bilbao G, Ostolaza H (2011) Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity. PLoS ONE 6(2):e17383. 40. Rossi C, et al. (2011) A tethered bilayer assembled on top of immobilized calmodulin to mimic cellular compartmentalization. PLoS ONE 6(4):e19101. 41. Tanaka M, Sackmann E (2005) Polymer-supported membranes as models of the cell surface. Nature 437(7059):656–663. 42. Rossi C, Chopineau J (2007) Biomimetic tethered lipid membranes designed for membrane–protein interaction studies. Eur Biophys J 36(8):955–965. 43. Karimova G, et al. (1998) Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: Implication for the in vivo delivery of CD8(+) T cell epitopes into antigen-presenting cells. Proc Natl Acad Sci USA 95(21): 12532–12537. 44. Lemichez E, et al. (1997) Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery. Mol Microbiol 23(3):445–457. 45. Ratts R, et al. (2003) The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex. J Cell Biol 160(7):1139–1150. 46. Tamayo AG, Bharti A, Trujillo C, Harrison R, Murphy JR (2008) COPI coatomer complex proteins facilitate the translocation of anthrax lethal factor across vesicular membranes in vitro. Proc Natl Acad Sci USA 105(13):5254–5259. 47. Dmochewitz L, et al. (2011) Role of CypA and Hsp90 in membrane translocation mediated by anthrax protective antigen. Cell Microbiol 13(3):359–373. 48. Simon SM, Peskin CS, Oster GF (1992) What drives the translocation of proteins? Proc Natl Acad Sci USA 89(9):3770–3774. 49. Karst JC, et al. (2010) Calmodulin-induced conformational and hydrodynamic changes in the catalytic domain of Bordetella pertussis adenylate cyclase toxin. Biochemistry 49(2):318–328. 50. Heveker N, Ladant D (1997) Characterization of mutant Bordetella pertussis adenylate cyclase toxins with reduced affinity for calmodulin: Implications for the mechanism of toxin entry into target cells. Eur J Biochem 243(3):643–649. 51. Paccani SR, et al. (2011) The Bordetella pertussis adenylate cyclase toxin binds to T cells via LFA-1 and induces its disengagement from the immune synapse. J Exp Med 208(6):1317–1330. 52. Arslan Yildiz A, Yildiz UH, Liedberg B, Sinner EK (2013) Biomimetic membrane platform: fabrication, characterization and applications. Colloids Surf B Biointerfaces 103: 510–516. 53. Kaufmann S, et al. (2012) Supported lipopolysaccharide bilayers. Langmuir 28(33): 12199–12208.

20478 | www.pnas.org/cgi/doi/10.1073/pnas.1312975110

ACKNOWLEDGMENTS. We thank Agnes Ullmann for a critical reading of the manuscript and Jean-Yves Le Guennec for fruitful discussions. The project was supported by the Institut Pasteur and by the Centre National de la Recherche Scientifique (Unité Mixte de Recherche 5253, Institut Charles Gerhardt Montpellier and Unité Mixte de Recherche 3528, Biologie Structurale des Processus Cellulaires et Maladies Infectieuses).

Veneziano et al.

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer.

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model ...
715KB Sizes 0 Downloads 0 Views