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(The FASEB Journal. 2009;23:2831-2843.)
© 2009 FASEB

Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin

Jana Vojtova-Vodolanova*, Marek Basler*, Radim Osicka*, Oliver Knapp{dagger}, Elke Maier{dagger}, Jan Cerny{ddagger}, Oldrich Benada*, Roland Benz{dagger} and Peter Sebo*

* Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic;

{dagger} Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Würzburg, Federal Republic of Germany; and

{ddagger} Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic

1 Correspondence: Institute of Microbiology CAS, Vídenská 1083, CZ-142 20 Prague 4, Czech Republic. E-mail: sebo{at}biomed.cas.cz


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Bordetella adenylate cyclase-hemolysin (CyaA, ACT, or AC-Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation-selective pores. Indirect evidence suggested that these two activities were accomplished by different membrane-inserted CyaA conformers, one acting as an AC-delivering monomer and the other as an uncharacterized pore-forming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered pore-forming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membrane-inserted CyaA together with biotinylated CyaA-AC– toxoid. Membrane oligomers of CyaA could also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore-forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaA pores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomeric CyaA.—Vojtova-Vodolanova, J., Basler, M., Osicka, R., Knapp, O., Maier, E., Cerny, J., Benada, O., Benz, R., Sebo, P. Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin.


Key Words: blue native electrophoresis • planar lipid bilayer membranes • pore-forming activity • pulldown • repeats in toxin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE SECRETED REPEATS IN TOXIN (RTX) adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) is a key virulence factor of the human whooping cough agent Bordetella pertussis (1 2 3) . The toxin targets primarily myeloid phagocytic cells that possess the CD11b/CD18 integrin receptor ({alpha}Mβ2, CR3, or Mac-1) and induces phagocyte impotence (4 5 6 7) . This helps bacteria to escape surveillance of innate immunity effectors (2 , 3) . Recent work suggests that CyaA plays an even more versatile role in fooling host defense by inducing macrophage apoptosis (8) and driving dendritic cells into a semimature state, which potentially shapes the adaptive immune response and favors tolerance of the pathogen (9 , 10) .

CyaA is a 1706-residue protein consisting of a fusion of a cell-invasive N-terminal enzymatic adenylate cyclase (AC) domain (~400 residues) to a ~1300-residue C-terminal RTX hemolysin (Hly) moiety (11) . CyaA activity on cells requires binding of calcium ions into the numerous (~40) sites located in the Hly moiety that are formed by glycine and aspartate-rich nonapeptide RTX repeats harboring a conserved X-(L/I/V)-X-G-G-X-G-X-D nonapeptide motif (12 , 13) . Productive high-affinity binding to the CD11b/CD18 receptor and the capacity of CyaA to efficiently penetrate cells further depend on covalent posttranslational palmitoylation of the {epsilon}-aminogroup of Lys983 residue of proCyaA by a coexpressed protein toxin acyltransferase, CyaC (14 15 16 17) .

The toxin delivers its AC domain into cell cytosol, where AC is activated by binding of calmodulin and catalyzes uncontrolled, massive conversion of cellular ATP to the key signaling molecule, cAMP. This condition disrupts cellular signaling and bactericidal functions of CD11b+ phagocytes, such as opsonophagocytosis and oxidative burst (5 , 6 , 18 , 19) .

A unique feature of CyaA is the capacity of the toxin to bypass receptor-mediated endocytosis and to penetrate and deliver the AC domain into cytosol of target cells directly across their cytoplasmic membrane (20 21 22 23) . This allows CyaA to raise cAMP to readily detectable supraphysiological levels, also in a variety of cells lacking the CD11b/CD18 integrin receptor, such as epithelial cells, lymphocytes, or erythrocytes (7) . Besides that, CyaA can form small cation-selective pores in cell membranes that account for the moderate hemolytic activity of the toxin on erythrocytes (17 , 24 25 26 27) . Recent work suggests that this pore-forming activity is sufficiently potent to cause also lysis of CD11b+ cells and contributes to overall cytotoxicity of CyaA toward phagocytic cells (28) .

The recently discovered third activity of CyaA consists in its capacity to promote entry of calcium ions into CD11b+ cells by a mechanism that is not mediated by cAMP or CD11b/CD18 signaling, nor is it mediated by the CyaA pores. In turn, it is the translocating AC domain that appears to mediate calcium entry by participating in formation of a transiently opened Ca2+ path across the cell membrane (29) .

Translocation of the AC domain into cells and formation of toxin pores appear to be two parallel and divergent activities of CyaA, which can be dissociated by alterations of temperature and free calcium concentration or by specific mutations and different toxin acylation status (13 , 25 , 28 , 30 31 32 33 34 35) . While translocation of the catalytic domain into cell cytosol appears to be a linear function of toxin concentration—which suggests that monomers of CyaA may deliver the AC domain across membrane—the pore-forming (hemolytic) activity of CyaA is a higher-order function of toxin concentration, with a Hill cooperativity number ≥3. This finding suggests that oligomerization of several toxin molecules is involved in formation of CyaA pores (27 , 31 32 33) . Moreover, pores formed by CyaA in artificial membranes behave as frequently opening and closing membrane channels, which suggests an association-dissociation equilibrium between nonconducting CyaA monomers and conducting CyaA oligomers in the membrane (17) . Furthermore, the propensity of CyaA to form the presumably oligomeric membrane pores appears to be specifically modulated by acylation status of the toxin, since only the pore-forming (hemolytic) activity of CyaA, but not its membrane penetration and AC delivery capacities, are differentially affected by attachment of different fatty-acyl chains (17 , 31 , 34 , 35) . Unambiguous evidence that CyaA can form functional oligomers in erythrocyte membrane comes from in vitro complementation studies with individually inactive toxin mutants. When certain pairs of these mutant CyaA variants, bearing nonoverlapping deletions of various toxin segments, are mixed in the presence of target cells, fairly high levels of restoration of toxin capacity to penetrate and permeabilize erythrocyte membrane can be observed (36 , 37) .

Recently, we have proposed a model predicting that CyaA can insert into the membrane in two conformations, one being a precursor in the pathway of AC domain translocation into cell cytosol by toxin monomers, and the other being a conformer representing a pore precursor that oligomerizes within the membrane to form the hemolytic toxin pores in cell membrane (22 , 33) . To test this model, we examined here the formation of CyaA oligomers in sheep erythrocyte membranes by direct visualization and biochemical approaches, and the results provide the first physical evidence that CyaA forms membrane-inserted oligomers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids, antibodies, and reagents
Mouse monoclonal anti-CyaA antibodies 9D4 and 10A8 (38) were obtained from Erik L. Hewlett (University of Virginia School of Medicine, Charlottesville, VA, USA) and protein A-gold 5-nm particles from Jan Slot (University Medical Center, Utrecht, The Netherlands).

pCACT3 was used for coexpression of cyaC and cyaA genes that allowed production of recombinant CyaC-activated CyaA in Escherichia coli under control of the isopropyl-β-D-thiogalactopyranoside-inducible lacZ promoter (31 , 33) . The CyaA-derived proteins with site-directed substitutions of amino acid residues (CyaA-E581K and CyaA-E570K+E581P) were constructed by PCR mutagenesis, essentially as described earlier (22) .

Production and purification of the CyaA-derived proteins
Intact CyaA and its mutant derivatives were produced in the presence of the activating protein CyaC using the E. coli strain XL1-Blue (Stratagene, La Jolla, CA, USA), as described previously (22 , 33) .

Biotinylation of the CyaA-AC– protein
On-column biotinylation of CyaA toxoid was performed after the DEAE-sepharose purification step (22 , 33) . The toxoid sample was diluted 4x in ice-cold 50 mM Tris-HCl, pH 8.0, containing 1 M NaCl and loaded on a phenyl-sepharose column (1.5 ml/4 mg of protein). The resin was washed extensively with PBS (12 mM Na2HPO4; 2 mM KH2PO4, pH 7.4; 3 mM KCl; and 132 mM NaCl) and resuspended in 2 ml of PBS containing 40 nmol NHS-Sulfo-LC-Biotin (Pierce, Rockford, IL, USA) to reach a biotin:toxoid molar ratio of ~20:1. Biotin coupling was at room temperature and was stopped after 40 min by washing of the resin with 15 ml of 50 mM Tris-HCl, pH 8.0, and then extensively with PBS. Purified biotinylated toxoid was then eluted with 50 mM HEPES, 8 M urea, and 2 mM EDTA. Level of CyaA biotinylation was assessed by Western blotting, using detection with a streptavidin–peroxidase conjugate. The binding, cell-invasive, and hemolytic activities of the biotinylated CyaA were determined as described below and found to be similar (~80%) to those of untreated CyaA.

Assay of adenylate cyclase, binding, cell-invasive, and hemolytic activities of CyaA
Adenylate cyclase activity was determined in the presence of 1 µM calmodulin (39) . One unit of AC activity corresponds to formation of 1 µmol of cAMP formed per min at 30°C at pH 8.0. Specific binding activity of toxin was determined after incubation with washed sheep erythrocytes (5x108/ml) of 1 U/ml of purified toxin for 30 min at 37°C in 20 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 2 mM Ca2+ (TNC buffer). Under these conditions, no hemolysis of sheep erythrocytes was observed. The cell-associated AC activity was determined as described previously (40) . The cell-invasive activity was measured as the capacity of CyaA to raise intracellular cAMP level in sheep erythrocytes (5x108/ml) on incubation with 1 U/ml of purified toxin variants at 37°C for 30 min. The reaction was stopped by addition of 100 µl of 0.2% Tween-20 in 100 mM HCl, and the samples were boiled for 15 min at 100°C and neutralized by addition of 150 µl of 150 mM unbuffered imidazole. Concentration of cAMP was determined by an antibody competition immunoassay, as described previously (41) . The hemolytic activity was measured by photometric (A541) determination of the amount of hemoglobin released on incubation of washed sheep erythrocytes with the toxin in 20 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 2 mM Ca2+ at 37°C (36) .

Protein A-gold immunolabeling and pair correlation function (PCF) analysis
Sheep erythrocytes (5x108 cells/ml) were treated with 48 µg/ml of CyaA for 30 min at 37°C in the presence of 2 mM Ca2+, or 5 mM EDTA, respectively, in the incubation buffer containing 10 mM HEPES, pH 7.4; 140 mM NaCl; 1% BSA; and 75 mM sucrose (24) . On 30 min of incubation, erythrocytes were washed and the incubation was repeated with fresh input of CyaA. Erythrocytes were repeatedly washed (3x) in incubation buffer with 5 mM EDTA and loosely bound toxin molecules not integrated into the membrane, were stripped off with 150 mM NaCl and 100 mM Na2CO3, pH 11.5 (30 , 42) . The amount of bound toxin was determined as the amount of cell-associated adenylate cyclase enzyme (39) . Bound CyaA molecules were immunolabeled with the mouse monoclonal antibody 10A8 (an IgG2b subtype) diluted 1:80 (60 min at 4°C) and decorated by suspension of 5 nm protein A-gold (1:75, 60 min, 4°C) in incubation buffer. Labeled erythrocytes were hypotonically lyzed in 10 mM HEPES, pH 7.4, and erythrocyte ghosts were deposited onto glow-discharge activated grids coated with formvar-carbon film. Unstained samples of erythrocyte ghosts were examined using a Philips CM 100 electron microscope (FEI, Amsterdam, The Netherlands). It was verified prior to assay that the protein A-gold particle suspension as such was monodisperse and did not contain any particle aggregates.

Statistical evaluation of immunolabeling patterns was performed by PCF analysis on a set of randomly taken digitally recorded micrographs (n=35, 1267x1014 nm), as described by Philimonenko et al. (43) , using the Gold software available online (http://nucleus.biomed.cas.cz/gold). Only micrographs of smooth erythrocyte membrane were analyzed to avoid artificial clustering of gold particles due to membrane aggregation. Confidence intervals of the calculated function values were estimated by Monte Carlo simulations of the Poisson process for independent particles. Significance of clustering was tested by one-sided 1% test. The number of gold particles per cluster was determined around all detected particles using AnalySiS 3.1 software (Olympus Soft Imaging Solutions, Münster, Germany).

Coprecipitation of crosslinked CyaA with biotinylated CyaA-AC– from cell membranes
A mixture of unlabeled enzymatically active CyaA (10 µg) with biotinylated and catalytically inactive CyaA-AC– (10 µg) was added to sheep erythrocytes (5x108 cells/ml) resuspended in 1 ml of HNS (10 mM HEPES, pH 7.4; 140 mM NaCl; and 75 mM sucrose) supplemented with 2 mM CaCl2. Cells were incubated with the mixture for 30 min at 37°C, washed once in 1 ml of HNSE buffer (HNS supplemented with 2 mM EDTA) and 2 times in 1 ml of HNS, and then loosely bound toxin molecules were stripped off with 150 mM NaCl and 100 mM Na2CO3, pH 11.5. An aliquot (10 µl) was withdrawn for determination of total cell-associated CyaA, and the cells were resuspended in 40 µl of HNS. Cell-associated proteins were crosslinked for 1 h at 4°C by addition of 5 µl of 10 mM DSG (Pierce) in DMSO, before the reaction was stopped by addition of 5 µl of 1 M Tris-HCl, pH 8.0, and incubation for 10 min. Erythrocytes were collected by centrifugation, and cells were hypotonically lyzed in 500 µl of 50 mM Tris-HCl, pH 8.0, containing a protease inhibitor cocktail (Complete MiniTM, Roche, Nutley, NJ, USA). Erythrocyte membranes were collected at 13,000 RPM for 15 min at 4°C and solubilized in 300 µl of a NeutrAvidin agarose beads suspension (Pierce; 20 µl of packed beads/300 µl) in 0.1% SDS; 0.1% Tween-20; 50 mM Tris-HCl, pH 8.0; and 150 mM NaCl with Complete protease inhibitors. Prior to addition of cell membrane extract, the NeutrAvidin beads were preadsorbed with 1 µg of unlabeled enzymatically inactive CyaA-AC– for 40 min at 4°C, in order to reduce unspecific binding of CyaA. Specific binding of biotinylated CyaA-AC– extracted from cell membranes was allowed to proceed for 1 h at 4°C. Unbound proteins were removed by extensive washing from NeutrAvidin beads with 3 x 1 ml of IPW buffer (2% SDS; 2% Tween20; 50 mM Tris-HCl, pH 8.0; and 150 mM NaCl) at room temperature and 1 ml of IPW buffer at 37°C for 15 min with vigorous shaking. After another 2 washes with 1 ml TN (to remove detergents), the enzymatic AC activity of CyaA that associated with NeutrAvidin beads was determined as described above.

Blue native and 2-dimensional electrophoresis
Sheep erythrocytes were treated with 25 µg/ml of CyaA or its E581K and E570K+E581P variants for 30 min at 37°C. Washed erythrocytes were stripped with carbonate, pH 11.5, as described above and hypotonically lyzed with 10 mM HEPES, pH 7.4, containing 5 mM iodoacetamide and 1 mM Pefabloc as protease inhibitors. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed essentially as described by Schägger and von Jagow (44) . Briefly, a pellet of 5 x 108 toxin-treated cells was solubilized in 200 µl of the blue native lysis buffer (BNLB) consisting of 1% n-dodecyl β-D-maltoside, 5 mM iodoacetamide, 1 mM Pefabloc in 750 mM aminocaproic acid, and 50 mM Bis-Tris, pH 7.0; Coomassie Brilliant Blue G was added to the detergent extract to the final concentration of 0.25%; and aliquots of the samples were run on nondenaturing 3–15% polyacrylamide gradient gels. The separated proteins were electroblotted using native cathode buffer (15 mM Bis-Tris and 50 mM Tricine, pH 7.0) onto Immobilon-P membrane, detected by the indicated antibodies, and visualized by immunoperoxidase staining. Relative molecular mass was estimated using calibration curves prepared with the native protein standards (HMW Native Marker Kit, Amersham Biosciences, Piscataway, NJ, USA).

For protein separation by SDS-PAGE in the second dimension, separation lane strips were cut out from BN-PAGE gels, rinsed with 1% SDS, and mounted into the stacking gel for a second-dimension 5% SDS-PAGE. The stacking gel was overlaid with 5x concentrated SDS-PAGE loading buffer, and electrophoresis was performed at room temperature with the current limited to 0.25 mA/cm2.

For conventional SDS-PAGE and Western blot analysis, washed erythrocyte membranes were solubilized in SDS-PAGE sample buffer containing 2% SDS, 5 mM iodoacetamide, and 1 mM Pefabloc (SDS-lysis buffer). Membrane proteins were separated on 7.5% SDS-PAGE and electroblotted to a nitrocellulose membrane and detected with appropriate antibodies. Relative molecular masses were derived from a calibration curve obtained with a biotinylated protein ladder detection pack for SDS-PAGE (Cell Signaling Technology, Beverly, MA, USA) that consists of 10 engineered proteins ranging in apparent molecular weight from 10 to 200 kDa.

Densitometric analysis
Western blot signals were quantified using LAS-1000 (Luminiscence Analyzing System; Fuji, Tokyo, Japan) and AIDA 1000/1D Image Analyzer 3.28 (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany). The background was set up according to a minimum of signal intensity between third and fourth peak. The peak volumes over background were calculated by numeric integration of the gray values of each pixel within the peak area. Peak volumes of the individual forms of CyaA proteins were then normalized against the summation of all peak volumes for a given protein and expressed in percentages of total. For comparison between proteins, the relative values for the CyaA species were normalized to the relative values of the individual protein forms of intact CyaA taken as 100%.

Lipid bilayer experiments
The methods used for black lipid bilayer experiments were performed as described earlier (22) .

Statistical methods
To allow reliable quantification of differences between the relative amounts of individual oligomeric species formed by CyaA and its mutant variants, four independent BN-PAGE separations, followed by immunoblotting, were performed, each analyzing triplicate samples of intact CyaA and CyaA mutants loaded next to each other. The peak values of the triplicate samples were used to calculate mean values per blot. Means from four independent Western blots (n=4) were tested by the nonparametric Wilcoxon matched pairs test, using Statistica (StatSoft, Inc. Tulsa, OK, USA). Threshold for significance was set as P ≤ 0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CyaA molecules are localized in clusters in erythrocyte membrane
CyaA is a large polypeptide of 177 kDa (11) , and its oligomers in membranes could be expected to be visualized by negative staining transmission electron microscopy (NS-TEM), which previously allowed visualization of membrane-inserted oligomers of several other smaller toxins, including Staphylococcus aureus {alpha}-toxin oligomers of only 232 kDa (45) . However, despite extensive efforts, we failed to visualize any CyaA molecules or CyaA oligomers in negatively stained erythrocyte membranes containing inserted toxin (46) . This result suggested that if CyaA formed oligomers in erythrocyte membrane, these did not exhibit any compact regular structure that would protrude out of the plane of the membrane and could be directly observed by NS-TEM.

Therefore, we examined oligomerization of CyaA in erythrocyte membrane by immunoelectron microscopy, a method previously used to demonstrate formation of complexes of other proteins in cellular membranes (47 , 48) . Toxin-treated erythrocytes were vigorously washed, and loosely associated CyaA molecules were stripped off with 0.1 M carbonate, pH 11.5 (30 , 42) . Membrane-inserted CyaA molecules were then detected using a monoclonal antibody recognizing a C-terminal segment of CyaA (10A8), which was decorated using a monodisperse suspension of 5 nm protein A-gold particles reacting in a 1:1 ratio with the primary antibody (49 , 50) . Comparison of determined average numbers of CyaA molecules bound per square micrometer of erythrocyte membrane with the observed density of protein A-gold labeling showed that only ~25% labeling efficiency was achieved in these experiments. Nevertheless, as shown in Fig. 1 , in contrast to mock membranes that did not bind any protein A-gold particles (Fig. 1A ), the erythrocyte membranes that contained inserted toxin were labeled by isolated gold particles, pairs of particles, and particle clusters (Fig. 1B, C ). As further revealed by a pair correlation function analysis of labeling on ~45 µm2 of smooth membrane portions from 35 randomly picked erythrocyte ghosts, the observed particle clustering was highly significant up to the distance of 50 nm (P<0.01), and the highest degree of clustering was observed within the distance range of 10 to 20 nm, as shown in Fig. 1D . In fact, clusters of pairs of gold particles were the most frequently observed in erythrocyte membranes (Fig. 1E ). Given that the complex of a primary antibody and a protein A-gold (5 nm) particle decorating the CyaA molecule is 17 nm long (51) , the observed clustering of two gold particles within the distance of 10–20 nm would indicate at least two very tightly associated CyaA molecules. We propose that the pairs of gold particles represent CyaA oligomers, likely dimers or higher oligomers (trimers or tetramers), since the used antibody was bivalent for the toxin and monovalent for protein A-gold. These results do not exclude the presence of even higher CyaA oligomers in the erythrocyte membranes, since clusters of more than two tightly associated gold particles were detected.


Figure 1
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  		Figure 1. Immunodetection of CyaA by protein A-gold reveals oligomerization of CyaA in sheep erythrocyte membranes. A, B) Erythrocytes were repeatedly (2x) treated with 48 µg/ml of CyaA for 30 min at 37°C in the presence of 5 mM EDTA (A) or 2 mM Ca2+ (B). CyaA molecules were detected by indirect immunolabeling, using the 10A8 monoclonal antibody specific for C-terminus of CyaA (38) and 5-nm protein A-gold particles, visualized by transmission electron microscopy of unstained specimens. C) Selected examples of detected clusters of CyaA molecules. D) Statistical evaluation of protein A-gold distribution. A set of randomly taken micrographs (n=35, 1267x1014 nm) was tested by PCF analysis, where the PFC value (open bars) corresponds to a ratio of density of particles within a given distance around a gold particle to the average density of gold particles across the entire examined membrane surface (43) . The 1% confidence intervals (inserted solid bars) were obtained by Monte Carlo simulation of independent random processes. *P < 0.01. E) Number of gold particles per cluster was determined up to the cluster size of 50 nm. Results are representative of 2 independent experiments with 2 independent CyaA preparations.

CyaA homooligomers can be pulled down from erythrocyte membranes
To assess directly the existence of CyaA oligomers in the erythrocyte membranes, a pulldown assay was used to isolate CyaA oligomers. Unlabeled enzymatically active CyaA was mixed with biotinylated and catalytically inactive biotin-CyaA-AC– toxoid, and a mixture of the proteins was allowed to insert into erythrocyte membrane. After vigorous washing of cells, CyaA proteins inserted into the erythrocyte membrane were crosslinked with 1 mM DSG, a short-arm bifunctional reagent (7.72 Å) that can cross-link only tightly interacting proteins. When the biotin-CyaA-AC– toxoid was pulled down from detergent extracts of cells by NeutrAvidin beads, as documented in Fig. 2 , the enzymatically active CyaA copurified with biotin-CyaA-AC– toxoid and the isolated protein complex could catalyze the conversion of ATP to cAMP. However, when nonbiotinylated CyaA-AC– was used during pulldown assay, no catalytic activity could be determined (Fig. 2 ). The modest yield of only ~0.35% of membrane inserted AC activity recovered in the pulldowns from cellular extracts was found to be in line with the observation that overall yield of the pulldown procedure ranged between 10 and 15% and crosslinking efficiency was ~20% of membrane associated AC activity, respectively (data not shown). Hence, if 50% of membrane-inserted CyaA would occur in oligomers, maximally 25% of it could possibly be involved in oligomers containing both CyaA and biotin-CyaA-AC–, and the expected recovery of pulled down AC activity (e.g., 0.125x0.2x0.5x0.25=0.00313) would then range between 0.4 and 0.5% of total membrane inserted CyaA. In addition, 3.5x lower AC activity was determined when the CyaA and biotin-CyaA-AC– proteins were not crosslinked with DSG in the erythrocyte membrane before pulldown assay, suggesting lower stability of the formed CyaA oligomers (Fig. 2 ). All these results clearly demonstrate the capacity of CyaA to form oligomers in target cell membrane that can be efficiently isolated by pulldown assay.


Figure 2
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  		Figure 2. CyaA oligomers can be pulled down from erythrocyte membranes. Unlabeled catalytically active CyaA was premixed with biotinylated or nonbiotinylated (negative control) catalytically inactive CyaA-AC– (10 µg of each protein, in 1:1 ratio), and the mixture was incubated for 30 min at 37°C with sheep erythrocytes (5x108 cells). Cells were washed to remove loosely associated CyaA and exposed or not to 1 mM DSG for 1 h at 4°C. Cross-linker reagent was inactivated, and erythrocytes were then hypotonically lyzed. Cell membranes were collected, solubilized, and incubated with NeutrAvidin beads for 1 h at 4°C. Unbound proteins were removed by extensive washing of resin; AC activity associated with NeutrAvidin beads was determined and compared to AC activity associated with erythrocyte membranes.

CyaA oligomers can be separated from CyaA monomers by BN-PAGE
It was important to characterize the stoichiometry of CyaA oligomers in erythrocyte membranes. Since no CyaA oligomers are detected under the harsh conditions of SDS-PAGE (25 , 30) , BN-PAGE was used. This allows the estimation of size, relative abundance, and subunit composition of multiprotein complexes and has previously proven useful in analysis of the quaternary structure of several membrane proteins under nondenaturing conditions (52 53 54) .

Toward this aim, CyaA-treated erythrocytes were stripped with carbonate buffer, to remove loosely bound toxin, and integral membrane proteins were extracted with the mild detergent n-dodecyl β-D-maltoside (1%) and separated by BN-PAGE on 3–15% gradient gels (Fig. 3 ). As expected, no CyaA was detected by immunoblotting in control membranes exposed to toxin in the presence of 5 mM EDTA (Fig. 3A , lane 1). In contrast, when cells were treated with toxin in the presence of 2 mM calcium, to allow membrane insertion of CyaA, several CyaA species were resolved in membrane extracts. Compared to migration of soluble marker proteins, the relative mobility of these CyaA species corresponded to apparent molecular masses of ~200, 300, 410, and 470 kDa, respectively. Except for the ~200 kDa species, these values were notably higher than the deduced molecular mass of monomeric CyaA (177 kDa). Moreover, as further shown in Fig. 3A (lanes 3–6), on addition of increasing concentrations of SDS to extracts prior to separation by BN-PAGE, the slower-migrating CyaA species, with apparent masses of ~410 and 470 kDa, respectively, disassembled almost exclusively into the ~300-kDa CyaA species, with only a minor increase in the amount of the ~200-kDa form, as determined by densitometric analysis of Western blots (Fig. 3B ), indicating that the ~410- and 470-kDa species were oligomers of CyaA. Moreover, on treatment with SDS, the ~300-kDa species exhibited the same mobility in BN-PAGE (Fig. 3A , lanes 2–6), whereas the ~200-kDa CyaA species migrated faster, with an apparent mass of ~170 kDa on exposure to 1% SDS (Fig. 3A , lane 6).


Figure 3
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  		Figure 3. Oligomers, monomers, and processed CyaA molecules are present in sheep erythrocyte membranes. A) BN-PAGE of CyaA complexes extracted from 5 x 108 sheep erythrocytes that were treated with 25 µg/ml of CyaA for 30 min at 37 °C in the presence of 5 mM EDTA (lane 1) or 2 mM Ca2+ (lanes 2–6). Erythrocyte membranes were solubilized in 200 µl of BNLB containing 1% n-dodecyl β-D-maltoside, and extracts were treated with indicated concentrations of SDS (lanes 3–6). Aliquots (5 µl) of detergent extracts were subjected to blue native electrophoresis separation on linear gradient polyacrylamide gels (3–15%) and electroblotted onto Immobilon-P membrane, on which CyaA was detected by immunoperoxidase staining with anti-CyaA mAb 9D4. B) Relative abundance of various CyaA species in sample prior to and after dissociation of oligomers induced by addition of 1% SDS, as determined by densitometric analysis of individual Western blots; n = 3.

To address the identity of the 200-kDa CyaA species detected in BN-PAGE blots, the detergent extracts of toxin-treated membranes were subjected to SDS-PAGE separation and Western blotting under denaturing conditions. As documented in Fig. 4A , two CyaA species were detected. The top CyaA species corresponded well to the full-length CyaA form, which is known to migrate in SDS-PAGE slower than expected ~205 kDa, presumably because of lower relative binding of SDS to the acidic CyaA protein (55) . This ~205-kDa CyaA species was recognized in immunoblots by both the 9D4 mAb binding to C-terminal RTX repeats of CyaA, as well as by the 3D1 mAb that specifically recognizes the segment of the AC domain located between residues 373 and 400 (38) . In contrast, the 3D1 antibody failed to detect the bottom ~170-kDa CyaA species, while the same protein was detected unambiguously by the 9D4 antibody. These results show that the lower CyaA species was lacking the AC domain and most likely corresponds to the previously observed processed form of the toxin, having the AC domain cleaved off on translocation into cells by proteases inside the cytosol of erythrocytes (30) . Indeed, this processed CyaA form also accounted for the fastest-migrating ~200-kDa CyaA species in BN-PAGE, as it was again detected by the 9D4 antibody and was not recognized by the 3D1 antibody (Fig. 4B ).


Figure 4
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  		Figure 4. Monomeric processed CyaA form with the AC domain cleaved off is excluded from CyaA oligomers. Cells were treated with 25 µg/ml CyaA for 30 min at 37°C in the presence of 5 mM EDTA or 2 mM Ca2+. A) Extracts of toxin-treated cells were separated by SDS-PAGE and subjected to Western blotting using the 9D4 mAb and anti-mouse IgG-peroxidase conjugate to detect total CyaA or by the 3D1 antibody, recognizing a CyaA segment between residues 373 and 400 (38) . B) CyaA species were extracted from erythrocyte membranes and separated by BN-PAGE. CyaA was detected by immunoblotting with the 9D4 mAb or with the 3D1 antibody. C) Two-dimensional electrophoretic separation of a detergent extract (20 µl) of toxin-treated erythrocytes. After separation on a BN-PAGE 3–15% acrylamide gradient gel in the first dimension, gel slices of lanes containing resolved native CyaA species were excised and loaded on top of a 5% SDS-PAGE gel for further separation under denaturing conditions in the second dimension. Gels were blotted onto nitrocellulose membranes, CyaA species (processed plus full-length) were immunodetected with the 9D4 antibody to detect RTX repeats, and membrane was stripped and reprobed with 3D1 antibody to visualize only toxin molecules containing the AC domain. Results are representative of 1 of 3 independent experiments.

All these results suggest that CyaA can be extracted from erythrocyte membranes in several forms, representing a CyaA monomer lacking the AC domain (~200 kDa), an entire CyaA monomer (~300 kDa), and CyaA oligomers (~410 and 470 kDa) with higher apparent masses (deduced from BN-PAGE) than the species of CyaA lacking the AC domain (140 kDa), or monomeric (177 kDa) or dimeric CyaA (354 kDa). This deviation may be due to the fact that suitable calibration markers for BN-PAGE separations of membrane proteins are not available, and the apparent masses of CyaA species had to be estimated from biased migration of soluble marker proteins that bind lower relative amounts of detergent than do hydrophobic membrane proteins (53 , 56 , 57) .

To determine whether the CyaA oligomers contain both entire and processed CyaA forms, a 2-dimensional separation of membrane extracts was performed, with resolution of CyaA oligomers in the first dimension under native conditions by BN-PAGE and characterization of their CyaA subunit composition by denaturing SDS-PAGE in the second dimension. As shown in Fig. 4C , the BN-PAGE-resolved ≥300-kDa CyaA species contained exclusively the entire CyaA detectable by the 3D1 antibody. The processed CyaA was, in turn, detected exclusively as the monomeric ~200-kDa CyaA species in the 1D BN-PAGE and migrated as a ~170-kDa protein in the SDS-PAGE, respectively. Hence, these results suggest that the CyaA oligomers are exclusively formed by the entire CyaA molecules, with the AC domain not translocated into cell cytosol, where it could be cleaved off by intracellular proteases to yield the processed CyaA form.

Pore-forming activity mirrors the propensity of CyaA to form oligomers while the cell-invasive activity correlates with the presence of processed CyaA
Based on a large body of indirect evidence, it has repeatedly been proposed that oligomerization of CyaA molecules is required for formation of CyaA pores (17 , 22 , 27 , 31 32 33) . Without direct evidence to support this hypothesis, we examined the oligomerization propensity of two CyaA variants that were constructed in frame of a large screen for CyaA mutants and exhibit strikingly altered specific hemolytic (pore-forming) activities (Table 1 ). In the first, CyaA-E581K, the charge-reversing lysine substitution of glutamate 581 by a lysine residue was found previously to strongly enhance the specific hemolytic (pore-forming) activity of the protein (22) . In fact, despite a reduced cell-binding capacity, CyaA-E581K provoked much faster lysis of erythrocytes at equal protein concentrations than did intact CyaA (Fig. 5A ), and severalfold lower concentrations of CyaA-581K than that of CyaA were needed to produce comparable extent of cell lysis at a given time point (Fig. 5B ). On the contrary, the combination of a charge-reversing substitution of glutamate 570 by a lysine residue with that of a helix-breaking substitution of glutamate 581 by a proline residue (E570K+E581P) essentially ablated the hemolytic (pore-forming) activity of the protein, while still allowing binding of CyaA to cells (Fig. 5A, B ). Moreover, the highly hemolytic CyaA-E581K construct exhibited on average much longer pore lifetimes in black lipid bilayers (~10.5 s) than intact CyaA (~2.8 s), while the pore-forming activity of the CyaA-E570K+E581P mutant was extremely low under otherwise identical conditions (Table 1 ). Furthermore, these mutations also affected the structure of the formed pores and caused an ~2-fold reduction in their size (to ~20 pS), as compared to pores formed by intact CyaA (~50 pS) (Table 1 ).


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  		Table 1. Activities of different CyaA mutants on erythrocytes and black lipid bilayer membranes


Figure 5
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  		Figure 5. Specific oligomerization propensity correlates with specific hemolytic activity of CyaA variants. A) Time course of hemolysis induced by CyaA mutants. Sheep erythrocytes (5x108/ml) were incubated in TNC at 37°C with correspondingly adjusted protein concentrations (1 U/ml of CyaA-E581K and CyaA-E570K+E581P and 0.8 U/ml of intact CyaA, respectively), in order to achieve binding of equal amounts (8 mU) of both mutant and intact toxin per 5 x 108/ml cells in 30 min of incubation. This procedure allowed comparison of hemolytic activity of the three proteins at equal concentration of cell-associated toxin, despite the slightly reduced cell binding capacity of the mutants. Erythrocyte lysis was determined photometrically at 541 nm as the release of hemoglobin over time. B) Hemolytic activity of CyaA mutants as function of protein concentration. Sheep erythrocytes (5x108/ml) were incubated in TNC at 37°C with indicated toxin concentrations, and cell lysis was assessed as release of hemoglobin after 270 min. Results are presented as means ± SD (n=9) of 3 independent assays performed in triplicates. C) BN-PAGE of CyaA complexes isolated from sheep erythrocytes incubated with 25 µg/ml of intact CyaA, CyaA-E581K, and CyaA-E570K+E581P, respectively, for 30 min at 37°C in the presence of 2 mM Ca2+. Amounts of detergent extracts of CyaA mutants subjected to BN-PAGE were adjusted to compensate for the slightly reduced cell-binding activity of the mutant toxin variants. A representative immunoblot is shown. D) Proportion of oligomers and monomers detected in membrane extracts for intact CyaA, hyperhemolytic CyaA-E581K mutant, and nonhemolytic CyaA-E570K + 581P protein, respectively. Relative amounts of oligomers and monomers were determined by 1-D densitometric evaluation of 4 independent Western blots, with intact CyaA and CyaA mutants deposited in triplicate samples per blot. Results are expressed as percentage of relative amounts ± SD (n=4), taking intact CyaA values as 100%. *P < 0.01 vs. intact CyaA; nonparametric Wilcoxon matched pairs test.

We reasoned that if oligomerization were required for CyaA pore formation, then the CyaA-E581K mutant would form more stable CyaA oligomers occurring at higher relative amounts in erythrocyte membranes than the oligomers of intact CyaA. In turn, the low pore-forming activity of the CyaA-E570K+E581P mutant would then reflect a low capacity to form CyaA oligomers. As indeed documented in Fig. 5C, D , the relative amounts of CyaA oligomers detected in the extracts of erythrocytes treated with the CyaA-E581K construct were reproducibly found to be ~60% increased, as compared to the relative amounts of the CyaA oligomers detected for the intact toxin, following normalization to total CyaA protein amounts detected per given sample. In parallel, substantially lower relative amounts of the processed CyaA monomer (lacking AC) were detected in cells exposed to CyaA-E581K than for cells treated with intact CyaA (Fig. 5C, D ), in line with the reduced capacity of the mutant to deliver the AC domain into cytosol of erythrocytes (Table 1 ). In contrast, ~3x reduced relative amounts of CyaA oligomers were detected reproducibly in extracts of erythrocytes exposed to the nonhemolytic CyaA-E570K+E581P protein (Fig. 5C, D ). This was detected predominantly in form of a full-length monomer, with almost no processed monomeric form of CyaA-E570K+E581P detected (Fig. 5C, D ), again in agreement with its nearly nil capacity to deliver the AC domain into cells (Table 1 ). Taken together, these results indicate a direct relation between the specific hemolytic and pore-forming activity of a given CyaA protein and its specific capacity to occur in the form of toxin oligomer in erythrocyte membrane. Furthermore, a direct relation between the presence of a processed monomeric CyaA species and the capacity of the respective protein to deliver its AC domain into cells is evident. This finding suggests that CyaA molecules translocating the AC domain into cell cytosol, where it is cleaved off (30) , would be excluded from the CyaA oligomers and accumulate as processed monomers in cell membrane.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report here direct physical evidence showing that at least dimers and most likely also higher oligomers of CyaA are present in membranes of toxin-treated erythrocytes and account for the membrane pores formed by the toxin. The observed pattern of migration of CyaA complexes in BN-PAGE corresponded well to results of immunogold labeling of erythrocyte membrane, where monomers, dimers, and clusters of dimers or higher CyaA oligomers were specifically detected using an antibody against the C-terminal portion of CyaA.

Until this study, the existence and nature of CyaA oligomers remained elusive, despite an accumulated body of indirect evidence suggesting that pore-forming activity of CyaA involves association of at least two CyaA monomers. We and others have previously suggested that oligomerization of CyaA was a prerequisite of the pore-forming (hemolytic) activity of CyaA, since this was observed to be a nonlinear and highly cooperative (higher-order) function of toxin concentration (17 , 27 , 31 32 33) . A rather conclusive, though still indirect, piece of evidence that CyaA may form functional oligomers came from our early demonstration that, when coincubated with cells, individually inactive CyaA constructs (bearing nonoverlapping deletions) could complement each other in vitro by an acylation and calcium-dependent interaction, to yield a partially restored toxin activity (36) . Using different complementation partners, Bejerano et al. (37) could later obtain highly active CyaA complexes through association of an inactive CyaA{Delta}A protein, lacking a conserved sequence between residues 1636 and 1650 (block A), with a C-terminal CyaA fragment (residues 1490 to 1706) carrying the block A. More recently, Lee et al. (58) extended the observations of Iwaki et al. (36 , 59) that erythrocyte binding and hemolytic activities of intact toxin also can be enhanced in the presence of an inactive toxin variant forming functional oligomers with CyaA.

The question of whether functional CyaA oligomers form in solution, or only on contact with or insertion into target cell membrane, remains difficult to answer. This is due to the natural tendency of the toxin to form inactive aggregates following secretion or refolding from 8 M urea stocks, which notably hampered functional analysis of toxin oligomerization (14 , 25 , 60) . Recently, Lee et al. (58) have indeed documented an acylation-dependent interaction of intact CyaA monomers in the absence of membranes, using a pulldown assay for CyaA oligomers formed in solution from differently labeled toxin molecules. However, the CyaA oligomers had to be isolated in the presence of a high concentration of detergent (1% Nonidet P-40), suppressing the unspecific aggregation, and thus could not be tested for hemolytic or cell-invasive activity. Therefore, until a toxin oligomer formed in the absence of target membranes has been isolated and shown to retain the capacity to penetrate cells, it cannot be concluded formally whether formation of functional CyaA oligomers can occur already in solution and may precede toxin insertion into target membranes. Furthermore, Iwaki et al. (59) showed that sequential incubation of erythrocytes with CyaA at 4°C, followed by vigorous washing and incubation with a 100-fold excess of a CyaA variant lacking the AC domain (CyaA{Delta}AC), resulted in enhancement of translocation of the AC domain of the membrane-preassociated intact CyaA on reincubation of cells at 37°C. Vice versa, preincubation of cells with CyaA{Delta}AC also allowed increased binding and translocation of subsequently added CyaA. Thus, functional interaction between CyaA molecules, leading to AC translocation into cells, can definitely occur also following toxin interaction with cell membrane. Here, monomers of CyaA have also been detected in erythrocyte membranes, both by BN-PAGE and in the form of the isolated protein A-gold particles bound to erythrocyte membrane-inserted toxin molecules. This finding suggests that CyaA also can insert into and function within the membrane in the form of monomers. With the present data, it is therefore plausible to conclude that binding to and/or insertion of toxin monomers into membrane may facilitate and precede functional, productive oligomerization of the toxin, simply by concentrating it within a 2-dimensional space. It can, however, not be excluded at present that both pathways of CyaA oligomer formation, in solution and in the membrane, may be operating in parallel in a nonexclusive manner.

The low stability of short-lived oligomers within the membrane would allow establishment of a dynamic equilibrium of CyaA monomers and dimers/oligomers, thus allowing CyaA monomers to occur within the membrane and to deliver the AC domain into cells. Such a model would be compatible with the observation that CyaA pores are forming and disappearing frequently, exhibiting a rather short lifetime of ~2.8 s and behaving, thereby, as flipping, opening, and closing membrane channels, rather than typical membrane pores (17) . Formation of the CyaA pore would then reflect association of toxin monomers, with disappearance of the pore reflecting dissociation of toxin dimers and oligomers, respectively.

That CyaA oligomers (pores) are rather unstable structures is suggested by the results presented here, showing that 3x lower AC activity (amount of CyaA oligomers) was repeatedly isolated from membrane extracts in pulldown experiments when the CyaA and biotin-CyaA-AC– proteins were not crosslinked with DSG in the erythrocyte membrane before pulldown assay (Fig. 2 ). This also makes unlikely the possibility that rapid formation of high-affinity dimers of CyaA was missed in earlier experiments, addressing the linear toxin concentration dependency of AC delivery into erythrocytes, based on which it was concluded that CyaA inserts into membrane and delivers the AC domain into cells in form of a toxin monomer (31 , 32) . The data presented here would lend support to the hypothesis that predominantly monomers of CyaA may be delivering the AC domain into cells. The presence in cell membranes of the monomeric and processed form of CyaA correlated, indeed, with the capacity of the toxin variants to deliver AC into erythrocyte cytosol for processing by intracellular proteases (30) . Moreover, 2-D electrophoresis clearly showed that the processed CyaA monomers were excluded from the membrane oligomers of CyaA. These observations indicate that either the translocation competent conformation of CyaA, allowing AC domain delivery into cells, may be incompatible with CyaA association into an oligomer, or on delivery of the AC domain, the processed CyaA molecules dissociate from the oligomer and accumulate as monomers in the membrane. The latter possibility would be compatible with results of the complementation experiments discussed above, which clearly demonstrated that initial assembly of CyaA subunits into oligomer did not prevent subsequent AC delivery into cells. However, no definitive conclusion on this issue can be drawn at present, since it cannot be excluded that the oligomeric form may serve for membrane insertion only, and dissociation of the oligomer within the membrane may be a prerequisite for delivery of the AC domain to occur by thus generated CyaA monomers.

The results discussed above are schematically summarized in a model of interaction of CyaA with the target membrane that accounts for all available data (Fig. 6 ). It predicts that two conformational isomers of CyaA would exist in solution. One would yield membrane-inserted AC translocation precursors, and the other, CyaA pore precursors. These may enter several equilibria, yielding AC delivery into cells by a membrane-inserted CyaA monomer, or yielding formation of an oligomeric CyaA pore. Functional CyaA oligomers possibly may, however, also form in solution and insert into membrane to form the small cation selective pores. These would appear to be rather unstable within the membrane and subject to a dynamic association-dissociation equilibrium, which would thus allow CyaA monomers to occur within the membrane that would deliver the AC domain into cells to be cleaved-off by intracellular proteases. Once translocated, the processed CyaA monomers would become unable to form an oligomer, or to remain part of it.


Figure 6
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  		Figure 6. Schematic representation of proposed model of CyaA interaction with target membrane. In solution, translocation and pore precursors of CyaA exist in equilibrium; on membrane insertion, the translocation precursor delivers the AC domain into cell cytosol, while the pore precursor enters an association-dissociation reaction involved in formation of oligomeric CyaA pores (22 , 33) . Results of Iwaki et al. (59) further suggest a possible functional interaction between the translocation and pore precursors of CyaA, forming an intermediate mixed precursor oligomer. Complementation experiments with CyaA mutants (36 , 37 , 58) suggest that formation of functional CyaA oligomers may also occur in solution and may precede toxin insertion into target membranes. While the translocation and pore precursors of CyaA are in a dynamic equilibrium with their intermediate and oligomeric pore forms within the membrane, monomers of CyaA that translocate the AC domain into cell cytosol are excluded from this equilibrium, having their AC domain cleaved off by intracellular proteases. E581K substitution selectively ablates formation and membrane insertion of translocation precursors; however, its pore precursors associate with increased frequency, thereby giving rise to the highly increased specific pore-forming and hemolytic activity (22) . In turn, the E570K+E581P double substitution yields a low capacity to form oligomeric pores and, moreover, ablates CyaA capacity to translocate the AC domain into cell cytosol.

Due to low yield and stability of CyaA oligomers, making their isolation from membrane extracts difficult, it could not be analyzed whether the toxin oligomers consisted only of CyaA subunits, or whether these were complexes including also some erythrocyte membrane proteins. The latter possibility appears, however, quite unlikely, since CyaA does not seem to bind to any proteinaceous receptor in erythrocyte membrane, where binding of CyaA to erythrocytes is unsaturable and excessive protease digestion of erythrocytes with several different enzymes results, in fact, in enhancement of overall CyaA binding (40) .

It will be important to elucidate why CyaA oligomers formed by the intact CyaA migrated as two separate bands (~410 and 470 kDa) in BN-PAGE, while a single ~410-kDa oligomeric form was detected for the notably more stable oligomers of the hyperhemolytic CyaA-E581K mutant. A plausible explanation would be that the two different oligomeric forms of CyaA represent different conformations of the CyaA oligomer, with the Glu581 to Lys581 substitution possibly locking the oligomer in the conformation prone to form a pore (more hemolytic). This would be supported by the observation that while exhibiting identical selectivity for cations, the pores formed by CyaA and the CyaA-E581K differed markedly in size. Whereas the shorter-lived pores formed by intact CyaA exhibited about twice as high mean single-pore conductance (~40–50 pS) than the more stable ("locked") CyaA-581K pores, exhibiting a mean single-pore conductance of ~20 pS (Table 1 ). Indeed, short-lived pores exhibiting lower unit conductances of 20 to 30 pS were also observed rather frequently in the single-pore recordings obtained for intact CyaA (22) . These results, hence, show that the segment of CyaA, containing the Glu581 residue plays a crucial role in controlling oligomerization of the toxin.


   ACKNOWLEDGMENTS
 
The generous gift of monoclonal antibodies by Erik L. Hewlett (University of Virginia School of Medicine, Charlottesville, VA, USA) and protein A-gold by Jan Slot (University Medical Center, Utrecht, The Netherlands), as well as insightful advice of P. Hozak on pair correlation function analysis, are gratefully acknowledged. This work was supported by EU 6th FP contract LSHB-CT-2003-503582 THERAVAC; Deutsche Forschungsgemeinschaft SFB 487 project A5; Institutional Research Concept AVOZ50200510; Academy of Sciences grant IAA50020091h; and grant MSM0021620858 and Center of Molecular Immunology grant 1M0506 from the Ministry of Education, Youth and Sports of the Czech Republic.

Received for publication February 11, 2009. Accepted for publication March 26, 2009.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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