Journal of Bacteriology, June 2004, p . 3760-3765, Vol . 186, No . 12

Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

César Martín,^1, M.-Asunción Requero,^1, Jiri Masin,^2,3 Ivo Konopasek,^2,3 Félix M . Goñi,¹ Peter Sebo,² and Helena Ostolaza^1*

Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), Departamento de Bioquímica, Universidad del País Vasco, 48080 Bilbao, Spain,¹ Institute of Microbiology, Czech Academy of Sciences,² Faculty of Sciences, Charles University, Prague, Czech Republic³

Received 30 December 2003/ Accepted 2 March 2004

 
Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough . ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents . The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents . ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types . In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT . A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents . Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux . Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE . These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures . Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux . Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux . Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers .

 
Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium responsible for whooping cough . The 1,706-residue protein can enter eukaryotic cells, where, upon activation by endogenous calmodulin, it increases the intracellular levels of cyclic AMP, leading to severe alterations in cellular physiology, often referred to as intoxication (see reference 28 for a review) . ACT belongs to the so-called RTX (repeats in toxin) family of proteins, characterized by a Ca²⁺-binding nonapeptide repeated in tandem several times, up to 30 to 38 repeats in the case of ACT, depending on the stringency of repeat definition . This toxin represents the most evolutionarily divergent example of the family (for reviews of RTX proteins, see references 40 and 41) . Unlike most other members of the family, ACT remains associated with the bacterial surface after secretion, apparently associated with filamentous hemagglutinin (42) .

In common with other members of the RTX family, and apart from its unique adenylate cyclase activity, ACT has a capacity to induce cell lysis, usually demonstrated as hemolysis . ACT-induced hemolysis requires higher toxin concentrations (by more than 1 order of magnitude) and occurs more slowly than intoxication (17) . Active ACT is acylated at two positions inside the chain, and the acylation pattern appears to affect hemolysis, rather than intoxication (19) . Moreover, dose-response experiments suggest that intoxication can be triggered by ACT monomers, while hemolysis is a more cooperative event, mediated by at least trimers (5, 17, 32) . These and other observations have led to the conclusion that hemolysis and intoxication occur through separate mechanisms (17, 28, 32, 34) .

Unlike intoxication, ACT-induced cell lysis has received relatively little attention . Benz et al . (4) and Szabo et al . (39), using planar lipid bilayers, demonstrated that ACT increased membrane conductance, giving rise to small, transient, cation-selective channels . These authors also found that ACT was less active in this respect than -hemolysin (HlyA), another member of the RTX family, secreted by Escherichia coli (4) . In general, the mechanism of HlyA-induced hemolysis has been studied in more detail (see references 16 and 40 for reviews) . In particular, studies in one of our laboratories have examined the capacity of HlyA to destroy the permeability barrier of pure-lipid vesicles (liposomes) . HlyA was found to cause efflux of high-molecular-weight dextrans from liposomes under isotonic conditions (33) . A mechanism of action was proposed according to which the toxin would be inserted into the membrane outer monolayer, thereby increasing lateral tension as more monomers were cooperatively incorporated, until membrane collapse and reorganization ensued (37, 40) .

Here we describe our analysis of ACT-induced cell lysis, which combined studies with red blood cells and liposomes (large unilamellar vesicles [LUV]) . Recombinant ACT, expressed in E . coli, was used . There are some indications that the palmitoylation pattern is not the same in the native and recombinant proteins (19, 36), although the practical consequences of such a difference are a matter of debate (3) but are unlikely to influence the main conclusions of our work . Our results suggest that red blood cells do not contain specific receptors for ACT . Moreover, a detailed analysis of the effect of the lipid composition on ACT-induced efflux of liposomal contents demonstrates that bilayer disruption is facilitated by the presence of lipids that favor inverted nonlamellar phase formation . Finally, we show that ACT-induced membrane lysis is accompanied by trans-bilayer lipid motion ("lipid scrambling") .

 
Materials. Egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE) were from Lipid Products (South Nutfield, United Kingdom) . Dielaidoyl PE, dioleoyl PE, lyso PC, cholesterol (Ch), and PE transphosphatidylated from egg PC were supplied by Avanti Polar Lipids (Alabaster, Ala.) . ANTS (8-aminonaphthalene-1,2,3-trisulfonic acid) and DPX [p-xylene-bis(pyridinium bromine)] were from Molecular Probes (Eugene, Oreg.) . Horse red blood cells were supplied by Biomedics (Alcobendas, Spain) . Human red blood cells came from a local blood bank . Tetramethyl rhodamine-conjugated goat anti-mouse immunoglobulin G (IgG) was from Molecular Probes .

ACT expression and purification. E . coli K-12 strain XL1-blue (Stratagene) transformed with the pT7CACT1 plasmid (31) was used for expression of ACT . Exponential 500-ml cultures were induced with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 3 h, during which time the culture optical density (A₅₀₀) increased from ca . 0.5 to 1.5 . The extracts of insoluble cell debris after sonication were prepared in 8 M urea-50 mM Tris-HCl (pH 8.0)-0.2 mM CaCl₂ as described previously (36) The protein was further purified by ion-exchange chromatography on DEAE-Sepharose and phenyl-Sepharose (Amersham Pharmacia Biotech) as previously described by Karimova et al . (27) . In the final step, the protein was eluted with 8 M urea-50 mM Tris-HCl (pH 8.0) and stored at –20°C .

Hemolysis assays. A red blood cell suspension was used that was obtained by diluting the erythrocytes with saline so that 37.5 µl of the mixture in 3 ml of distilled water gave an A₄₁₂ of 0.6 . The hemolysis assay was performed in 1-ml test tubes by mixing the erythrocyte suspension with the desired amounts of ACT in buffer (150 mM NaCl, 10 mM CaCl₂, 20 mM Tris-HCl, pH 8.0) . The mixtures were incubated at 37°C for 2 h with gentle shaking and then centrifuged in an Eppendorf centrifuge for 1 min . The A₄₁₂ of the supernatants, appropriately diluted with distilled water, was read . The blank (zero hemolysis) consisted of a mixture of appropriate volumes of buffer and erythrocytes .

LUV. Lipids in organic solution were mixed in the appropriate proportions, and the solvent was thoroughly evaporated . The resulting dry lipid film was hydrated in buffer, with gentle shaking, to form multilamellar vesicles . These were treated with 10 cycles of freezing and thawing, followed by 10 cycles of extrusion through polycarbonate filters (pore size, 0.1 µm; Nuclepore, Pleasanton, Calif.) . The buffer used contained 150 mM NaCl, 10 mM CaCl₂, and 20 mM Tris-HCl (pH 8.0), unless otherwise stated . The diameter of the resulting LUV was ca . 100 nm, according to quasielastic light scattering measurements . More details about the preparation of these vesicles can be found in reference 30 .

Efflux of liposomal contents. Leakage of vesicular aqueous contents was assayed with ANTS and DPX trapped in the liposomes . ANTS is a water-soluble fluorophore . DPX is also water soluble; it forms complexes with ANTS and quenches the fluorescence of the latter . When both ANTS and DPX are trapped in a vesicle, they exist in the form of a nonfluorescent complex . When vesicle efflux occurs, ANTS and DPX become highly diluted, the complex dissociates, and free ANTS emits fluorescence . LUV were prepared in 70 mM NaCl-10 mM CaCl₂-12.5 mM ANTS-45 mM DPX-20 mM Tris-HCl (pH 8.0) . This buffer is isotonic with 150 mM NaCl-20 mM Tris-HCl (pH 8.0), which was used as an external elution buffer . Nontrapped probes were removed by passing the LUV through a Sephadex G-75 column eluted with 150 mM NaCl-10 mM CaCl₂-20 mM Tris-HCl (pH 8.0) . Assays were performed with 100 µM lipid in a total volume of 1 ml . Lipid phosphorus was assayed as described by Bartlett (2) . The assay was started by adding the required amount of ACT . Measurements were monitored in a Perkin-Elmer LS50 spectrofluorimeter at 37°C with a continuously stirred cuvette . ANTS fluorescence was recorded continuously (excitation wavelength, 355 nm; emission wavelength, 520 nm; slits, 5 and 5 nm) . Triton X-100 was added (final concentration, 0.1% [wt/vol]) to induce 100% release . Percent release was computed as follows: % release = [(F_f – F₀)/(F₁₀₀ – F₀)] x 100, where F_f, F₁₀₀, and F₀ were the respective fluorescence intensities observed after addition of ACT, after addition of Triton X-100, and before any addition .

Trans-bilayer lipid movement. The trans-bilayer lipid movement assay is based on the phenomenon of fluorescence resonance energy transfer (FRET) . When two different fluorophores are in very close proximity, such that the emission spectrum of the first one overlaps the excitation spectrum of the second one, it is possible to excite the fluorescence of the first one (donor) and record the fluorescence emitted by the second one (acceptor) . In the present case, the donor and acceptor are, respectively, 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) and rhodamine . NBD is bound to a phospholipid initially located only in the inner monolayer of the membrane, and rhodamine is bound to a large protein (antibody) located outside the vesicle, so that at time zero no energy transfer can occur . However, when trans-bilayer lipid motion occurs, some of the NBD-lipid is transferred to the outer monolayer and can interact with the external, protein-bound rhodamine . To prepare LUV labeled in the inside bilayer with NBD-PE, PC-PE-Ch (2:1:1) LUV containing 0.6 mol% NBD-PE (about 1 NBD-PE molecule in 170 nonfluorescent lipid molecules) were treated with membrane impermeant sodium dithionite (10 mM) . The excess dithionite was removed by gel filtration through a Sephadex G-75 column . These NBD-PE-containing liposomes (0.1 mM final concentration) were incubated with the toxin (10 µg) under constant stirring . At different times, small aliquots of the suspension were removed and incubated with rhodamine conjugated to an antibody that was also membrane impermeant . Measurements were monitored in a Perkin-Elmer LS50 spectrofluorimeter at room temperature with a continuously stirred cuvette . Excitation was set at 460 nm, and emission was recorded between 510 and 640 nm, with slits of 5 nm for both monochromators . A cutoff filter (515 nm) was used to prevent contribution from scattered light . For more details, see reference 7 . In control experiments, heat-denatured ACT was used, which was obtained by heating the toxin in a boiling water bath for 5 min .

 
Membrane lysis and solute efflux. RTX toxins usually possess the capacity to break down the permeability barrier of cell membranes . The membrane-lytic ability of ACT was tested on red blood cells and on liposomes (LUV) containing entrapped fluorescent probes (ANTS and DPX) . ACT caused lysis of erythrocytes and release of entrapped dyes from liposomes . A representative experiment of ACT-induced ANTS and DPX release from liposomes is shown in Fig. 1A . The detergent Triton X-100 was added to mark 100% release . Dose-response curves of both hemolysis and liposome efflux are shown in Fig . 1B . Under the experimental conditions used, data for hemolysis and liposomal efflux above 30 nM ACT were virtually superimposable; 150 nM ACT caused the maximum effect in both cases . At lower ACT concentrations, LUV appear to be more sensitive than red blood cells . Experiments carried out with human erythrocytes revealed an ACT dose-response curve similar to that of horse cells (data not shown) . In similar studies with another member of the RTX family, -hemolysin from E . coli, Cortajarena et al . (9) found that red blood cells were always much more sensitive than liposomes to the toxin, and this observation led to the discovery that the erythrocyte membrane integral protein glycophorin acts as a receptor for -hemolysin . The data in Fig . 1B suggest that red blood cells contain no specific receptor for ACT . This notion was supported by an experiment in which red blood cells were treated with trypsin under conditions in which this protease cleaves the extramembranous part of glycophorin and of other membrane proteins . Hemolysis caused by ACT was the same in control and trypsin-treated erythrocytes (Fig. 1B, inset) .

 
Role of nonlamellar lipids. Liposomal assay of ACT activity is a convenient way of studying the influence of membrane lipid composition . Previous experiments with the related toxin -hemolysin from E . coli (33) had suggested that the lytic effect of that protein was favored by those lipids that tend to facilitate the formation of nonlamellar, inverted phases (lipids favoring negative curvature or type II lipids) (12, 20, 22) . ACT-induced liposomal efflux was studied in this context . Figure 2 shows some representative results of experiments in which release of contents from liposomes with various lipid compositions was induced by the same amounts of ACT . Pure-PC liposomes released 40% of their aqueous contents in 30 min, but when PC-PE (2:1 molar ratio) liposomes were used, 80% efflux was observed . PE is well known for its ability to induce negative curvature and eventually form non-bilayer (mostly hexagonal II) phases (13, 25) . The effect of PE was compensated by lyso PC, a lipid whose geometry is the opposite of that of PE (22) . Lyso PC induces a positive curvature in the monolayers where it is located and counters the lamellar-to-hexagonal II transition . As shown in Fig . 2, lyso PC neutralized the effect of PE when both lipids were present in the bilayer, so that ACT-induced efflux from liposomes containing PC-PE-lyso PC (2:1:1 molar ratio) was the same as with pure-PC vesicles . Efflux in the absence of ACT remained at the noise level, i.e., below 3%, after 30 min irrespective of the bilayer composition (data not shown) .

 
The correlation between the lipid propensity to form a non-bilayer phase and the ability of ACT to break the membrane permeability barrier is also supported by the experiment described in Fig . 3, in which different molecular species of PE were added to a bilayer originally composed of pure egg PC, at a final PC-PE molar ratio of 2:1 . Three different PEs were used, namely, dioleoyl PE, whose T_h (lamellar-to-hexagonal phase transition temperature) is 8°C; egg PE (19% C_16:0, 24% C_18:0, 21% C_18:1, 14% C_18:2, 14% C_20:4, 8% other), whose T_h is 25 to 35°C; and a transphosphatidylated form of egg PE (33% C_16:0, 11% C_18:0, 31% C_18:1, 16% C_18:2, 9% other), whose T_h is 40 to 55°C . As shown in Fig . 3, ACT-induced efflux is larger the lower the T_h of PE, i.e., the greater the tendency of PE to form an inverted hexagonal phase . In all of the experiments in Fig . 2 and 3, the total lipid and ACT concentrations were kept constant at 0.1 and 50 nM, respectively .

 
The inhibitory effect of lyso PC on ACT-induced lysis was further explored by using 100% PC liposomes that contained increasing proportions of lyso PC . As shown in Fig . 4, lyso PC inhibition was a dose-dependent phenomenon that became significant beyond 10 mol% in the PC bilayer . This confirms the observations described in Fig . 2 .

 
Ch displays a variety of effects when added to phospholipid bilayers . Ch facilitates the lamellar-to-nonlamellar transition (10, 25) and increases the molecular order of the phospholipid acyl chains (23), among other properties . In our system, Ch greatly enhanced the rate of ACT-induced liposomal efflux in a dose-dependent manner, and the effect became significant above 20 mol% in PC bilayers (Fig . 5) .

 
Trans-bilayer lipid movement. The above results appear to indicate that ACT insertion and/or membrane permeabilization may be accompanied by transient formation of nonlamellar structures in the membrane . In turn, such nonlamellar intermediates would explain the observed solute efflux . If they are indeed formed, those transient nonlamellar structures would lead to some degree of trans-bilayer lipid movement (flip-flop or lipid scrambling [6]) . In order to test this possibility, we applied an assay recently developed in our laboratory (7) . In this assay, LUV initially containing 0.6 mol% NBD-PE homogeneously distributed throughout the bilayer are treated with membrane-impermeant sodium dithionite . This reagent reduces the number of NBD molecules located in the outer monolayer and irreversibly quenches their fluorescence so that fluorescent NBD-PE is now located only in the inner monolayer . These liposomes are incubated with the toxin, and at different time intervals aliquots of the suspension are incubated with rhodamine conjugated to an antibody that is also membrane impermeant . NBD and rhodamine can undergo FRET when in close contact . Under our conditions, FRET will only occur if NBD-PE is transferred from the inner to the outer monolayer, so that it can contact the IgG-rhodamine in the outer solution . In principle, the system is stable for hours, with no FRET being detected . However, in the presence of ACT, FRET occurs (Fig . 6), the intensity of NBD fluorescence decreases and that of rhodamine increases simultaneously, and the phenomenon occurs in the time scale of liposomal efflux (Fig . 1A and 4) . The effect is specific for active ACT . Heat-denatured toxin does not induce FRET (Fig . 6B, open symbols) . The effect could not be explained by IgG-rhodamine gaining access to the interior of the vesicles, because ACT did not allow passage of fluorescein-derivatized dextrans with molecular masses of >4 kDa, while IgG has a molecular mass of 150 kDa (M.-A . Requero, unpublished data) .

 
ACT has in common with the other members of the RTX family a capacity to disrupt the permeability barrier of model and cell membranes, leading to efflux of vesicle or cell contents . This process is often referred to as cell lysis . It appears to be the endpoint of a complex series of events . In order to gain information on ACT-induced membrane lysis, the above results will be examined in the light of data obtained with other RTX toxins, or eventually other proteins .

Several RTX toxins bind specific receptors in the target cells: E . coli HlyA, A . actinomycetemcomitans LtxA, and P . haemolytica LktA bind a ß2 integrin in leukocytes (26, 29), and HlyA also binds glycophorin in red blood cells (8, 9) . ACT binds the human myeloid phagocytic cells, which are its natural targets, via the CD11b/CD18 receptor (18) .

However, ACT is rather promiscuous and can penetrate, intoxicate, and eventually lyse, with some efficiency, a variety of cell types from different hosts (40) . This speaks against an absolute requirement for specific cellular receptors, and our results in Fig . 1 support the lack of a receptor in erythrocytes . Thus, ACT interaction with the membrane lipid bilayer appears to be the first step in ACT-induced cell intoxication and lysis . Note that the latter are two very different effects and that lysis requires higher toxin concentrations than intoxication (17) . Lysis may constitute a further, more severe form of cell damage occurring in B . pertussis infection . In the present work, the mechanism of cell lysis is specifically addressed .

Once the toxin has reached the lipid bilayer, interaction with the hydrophobic matrix would occur . Toxin insertion into the membrane, which would be accompanied by a change in protein conformation and by changes in bilayer architecture leading to release of cell contents, is certainly the least understood process in RTX-dependent cell lysis . Our results shed light on two little-known aspects of this obscure stage of ACT-membrane interaction, namely, the involvement of non-bilayer lipid intermediates (Fig . 2 to 5) and the toxin-induced flip-flop lipid movement (Fig . 6) . The facts that (i) lipids inducing negative curvature (PE and Ch), i.e., favoring inverted nonlamellar phases (15, 20, 22), increase the ACT lytic effect (Fig . 2 to 4) and (ii) lyso PC, a lipid inducing positive curvature, counteracts the effect of PE (Fig . 2) and reduces ACT-caused efflux (Fig. 5) point together to the transient formation of nonlamellar intermediates during the irreversible, Ca²⁺-dependent step of ACT insertion into membranes . A similar suggestion had been made in relation to the lytic effect of E . coli HlyA (33) . The phenomenon is not limited to RTX proteins and may be more general in membrane protein insertion . Alonso et al . (1) reported that lipids favoring inverted-phase formation enhance the ability of aerolysin, a non-RTX toxin from Aeromonas hydrophila, to permeabilize liposome bilayers . These authors suggest that lipids inducing negative curvature of bilayer leaflets lower the surface pressure at the interface and that this in turn may favor access of the protein to the hydrophobic matrix . Outside the field of toxins, Dan and Safran (11) explained the requirement of non-bilayer lipids for the activity of some integral proteins in terms of the relationship between the lipid properties and the tension exerted on membrane proteins . A number of "nonpermanent" (14) membrane proteins are known to bind more easily when the bilayer contains negative-curvature lipids (13, 24, 38) . In general, the presence of non-bilayer lipids appears to increase the structural plasticity and functional versatility of bilayers .

Equimolar proportions of Ch in PC bilayers increase the rate of ACT-induced efflux about threefold compared to the pure-PC vesicles (Fig . 5) . This is not limited to ACT insertion . Scott and Zakim (35) observed that Ch lowered the energy barrier for insertion of integral membrane proteins into bilayers . Ch certainly favors the formation of nonlamellar structures (10, 25); it also has a number of other effects on the physical properties of the bilayer . The ability of Ch to increase the static order of phospholipid hydrocarbon chains may be relevant in this context . Ch ordering effects lead to the formation of "liquid-ordered phases." In PC-Ch mixtures, a liquid-ordered phase predominates above 20% Ch (21) . It is interesting that it is precisely in this range of concentrations that Ch clearly stimulates liposomal efflux (Fig . 5) .

Formation of even transient nonlamellar lipid structures of geometries resembling the inverted hexagonal or inverted cubic phases must, of necessity, induce trans-bilayer or flip-flop movement of lipids . Flip-flopping would also occur if ACT-induced lysis followed the mechanism proposed by us for E . coli HlyA (37), namely, insertion of an increasing number of toxin monomers into the membrane outer monolayer until the increase in lateral pressure leads to collapse of the membrane architecture . The data in Fig. 6 indicate that lipid trans-bilayer movement occurs in parallel with ACT-mediated efflux, thus providing a hitherto unobserved aspect of ACT insertion into membranes . Thus, toxin insertion joins the number of pathophysiological events that may induce collapse of lipid asymmetry in cell membranes (6) .

 
This work was supported in part by European Union contract QLK2-1999-0.0556 and by grant A502907 from the Grant Agency of the Czech Academy of Sciences .

 
* Corresponding author . Mailing address: Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), Departamento de Bioquímica, Universidad del País Vasco, Aptdo . 644, 48080 Bilbao, Spain . Phone: 34 94 601 26 25 . Fax: 34 94 601 33 60 . E-mail: gbzoseth@lg.ehu.es.

C.M . and M.-A.R . contributed equally to this work .

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What Is Pcr?, What Is Biotechnology?, What Is Anthrax?, What Is MIC?, What is Food Microbiology?, i, Bacteria, c, Microorganisms, c, Microbes, r, Bacteriology, i, Microbe, n, Prokaryotes, n, Pasteurella, n, Schizosaccharomyces, i, Escherichia coli, r, Salmonella, s, Escherichia coli, e, Prokaryotes, c, Salmonella typhimurium, o, Escherichia coli, n, Microbial, s, Antibiotics, c, S. cerevisiae, i, Gram positive, n, Corynebacter, e, Streptococcal, s, Candida tropicalis, o, Bacillus subtilis, a, Yeasts, s, Biofilms, n, Campylobacter, e, Bacteria




 

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