Adenylate Cyclase Toxin (CyaA) of Bordetella pertussis

T H E JOURNAL OF BIOWGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 44, Issue of November 4, pp. 27231-27239, 1994 Printed in U.S.A.

Adenylate Cyclase Toxin (CyaA)of Bordetella pertussis EVIDENCE FOR THE FORMATION OF SMALL ION-PERMEABLE CHANNELS AND COMPARISON WITH HlyA OF ESCHERICHIA COLI* (Received forpublication, May 24, 1994, and in revised form, August 10, 1994)

Roland BenzSP, E k e MaierS, Daniel Ladantn, Agnes Ullmannn, and Peter Sebon From the jLehrstuh1 fur Biotechnologie, Theodor-Boueri-Institut (Biozentrum) der Universitat Wurzburg, A m Hubland, 0-97074 Wurzburg, Federal Republic of Germany, and the llUnit6 de Biochimie des Rkgulations Cellulaires, Znstitut Pasteur, 28, rue du Docteur Roux, F-75724 Paris Cedex 15,France

CyaA is the subject of a number of recent studies. It posThe interaction between the adenylate cyclase toxin (CyaA) ofBordetella pertussis and lipid was studied us- sesses both invasive adenylate cyclase (cytotoxic) and hemeoing the lipid bilayer assay.The addition of CyaA to the lytic (pore-forming) activities that strictly depend on a postaqueous phase bathing lipid bilayer membranes com- translational CyaC-mediated modification (Barry et al., 1991; posed of different lipids resulted in the increase of the Sebo et al., 1991). The purified form of CyaA, endowed with membrane conductance. This increase was rather smallcytotoxic and hemeolytic activities, is a protein of 177 kDa for membranes formedof pure lipids as compared with (Gordon et al., 1989; Hanski, 1989; Hewlett et al., 1989; Rogel lipid mixtures such as asolectin. The toxin formed in et al., 1989; Bellalou et al., 1990a; Gentile et al., 1990). Howasolectin membranessmalltransiention-permeable ever, lower molecular mass fragments of CyaA were also rechannels with a single-channel conductanceof 27 pS in ported t o exhibit some invasiveadenylate cyclase activity 1 M KCl, which is considerably smaller than that of the (Masure et al., 1989). The 1706-residue long CyaA consists of a-hemeolysin (HlyA) of Escherichia coli (1500 pS). Experiments with different salts suggested thatthe CyaA- two functionally separable and distinct parts. (i) Thefully acinduced channels were exclusively cation-selective be- tiveand calmodulin-activated adenylate cyclase enzyme is cause of negative chargeslocalized at the channel formed by the 400 amino-proximal residues (Glaseret al., 1989; mouth. The single-channel conductance of channels ini- Ladant et al., 19891, while (ii) the polypeptide comprising the tiated by CyaA was independent of whether the toxin last 1332 residues of CyaA can function independently as a was purified from B. pertussis or from recombinant E. hemeolysin (Sakamoto et al., 1992). Thus, thehemeolysin part coli. However, the channel-formingactivity of the CyaA of CyaA must containenough informationfor cell-targeting and expressed in B. pertussiswas substantially higher than pore-formation. This part itself is composed of two distinct domains. It harbors four hydrophobic segments with potentially that ofthe recombinant toxin. Experiments with mutant formsof CyaA suggested that both the activation of amphiphilicand hydrophobic a-helicalstructures(residues CyaA by CyaCand the hemeolytic part of the toxin, but 500-700) that are similar to the pore-forming region of the not the repeats and the cyclase activity, are required fora-hemeolysin (HlyA) of Escherichia coli (Ludwig et al., 1987; channel formation in lipid bilayer membranes. Oropeza-Wekerle et al., 1992). Internal deletions around this region (amino acids 623-780 and 827-887) abolish the invasive activity and severely reduce the hemeolytic activity of CyaA The secreted adenylate cyclase toxin (CyaA)’ is an essential (Bellalou et al., 1990b), indicating thatthis portion is involved in both the adenylatecyclase delivery and pore formation. virulence factor of Bordetella pertussis and exhibits several The second domain, between residues 913 and 1612, contains a calmodulin-activated and invasive unusual features. It is aspartate-rich nonapeptide repeats (W adenylate cyclase enzyme that bypasses the receptor-mediated 38 glycine and F)XGGXG(N/D)DXcharacteristic for all RTX toxins, which are endocytosis pathway (Wolff et al., 1980; Confer and Eaton, 1982; Gordon et al., 1989) and penetrates directly across the involved in calcium- and target cell-binding (Ludwig et al., cytoplasmic membrane of a variety of epithelial and immune 1988; Boehm et al., 1990a, 1990b; Baumann et al., 1993) (reeffector cells. Upon activation by the intracellularcalmodulin, viewed by Welch (1991) and Coote (1992)). The mechanism of the invadingenzyme catalyzes uncontrolled formation of CAMP, pore formation by CyaA is unknown. The intoxication of target and, as a result, the microbicidal capacities of the intoxicated cells by CyaA occurs only in thepresence of free calcium ions, cells are debilitated (Confer and Eaton, 1982; Pearson et al., at concentrations above 0.1 mM Ca2+(Hanski and Farfel,1985) that induce important conformational changes of CyaA leading 19871, (reviewed by Hanski and Coote (1991)). to a burst in toxin activity (Hewlett et al., 1991). However, * This work was supported by grants (to R. B.) from the Deutsche Rogel and Hanski (1992) showed that after single exposure to Forschungsgemeinschaft (Sonderforschungsbereich 176) and the Fonds calcium, the toxin becomes competent for membrane insertion der Chemischen Industrie and by grants (to A. U.) fromthe CNRS (URA and hemeolytic activity, even in the absence of free calcium 1129), INSERM (CRE 91.06.15), the DRET (91.048) and the Human ions. They also showed that translocation of the adenylate cyFrontier Science Program. The costs of publication of this article were clase domain canbe uncoupled frommembrane insertionof the defrayed in part by the payment of page charges. This article must therefore be herebymarked “aduertisement” in accordance with 18 toxin a t low temperature and can be restored by raising the temperature or adding freecalcium. Our recent results suggest U.S.C. Section 1734 solely to indicate this fact. 5 To whom correspondence should be addressed. Tel.: 49-0931-888- that differences intheposttranslational modification(s) of 4501; Fax: 49-0931-888-4509. CyaA may modulate the intrinsic ratio of the invasiveadenylThe abbreviations used are: Cy&, adenylate cyclase toxin of Bordetella pertussis; HlyA, a-hemeolysin of Escherichia coli;RTX, repeats ate cyclase and pore-forming activities of CyaA. Moreover, the in iozin; G , conductance (i.e. current divided by voltage); MES, 4-mor- internalization of adenylate cyclase seems tobe a monomolecupholineethanesulfonic acid; pS, picosiemens. lar process, while more than one molecule of CyaA appears to

27231

27232

Adenylate Cyclase Toxin of B. pertussis

4

205

4

116

4

97.4

4

66.7

4

45

FIG.1. SDS-polyacrylamide gel electrophoresis analysis of the purified wild-type and truncated CyaA constructs. The proteins were purified as described under "Experimental Procedures" and subjected to SDS-polyacrylamide gel electrophoresis, and the gel (7.5% acrylamide) was stained withCoomassie Blue.

be involved in the formation of a CyaA pore (Sebo etal., 1991; Betsou et al., 1993). Thus, these two processes may be independent. In order to establish whether hemeolytic the activity of CyaA is due to formation of pores or to some other mechanism, we used the artificial planar-lipid-bilayer system for examination of the pore-forming properties of CyaA and of its derivatives. We show that CyaA forms small ion-permeable channels in lipid bilayer membranes with an extremely low single-channel conductance, which is much lower than the conductance of channels formed by HlyA of E. coli and Morganella morganii (Benz et al., 1989; Ropele and Menestrina, 1989; Benz et al., 1994). The channel-forming activity of CyaA was affected by the source of CyaA and was strongly dependent on toxin activation. Our data suggest that the CyaA-mediated colloid-osmotic lysis of erythrocytes is very likely to be caused by formation of defined small CyaA channelsinthe erythrocyte membrane. Moreover, the estimated diameter of these channels appears to be too small to allow the translocation of an even fully unfolded invasive adenylate cyclase domain of the toxin.

Bacterial Strains, Plasmids and Growth Media-The E. coli strain Xl1-Blue (endA1, hsdRl7, supE44, thil,I-, recA1, gyrA96, relA1, A(lacproB)/F-, p r o m , lacIqZDMl5,TnlOftetR))(Stratagene)was used throughout this work for DNA manipulation and for expression of recombinant CyaA and its derivatives. pACT7 for expression of nonactivated CyaA, pPS4C for expressionof the toxin-activating proteinCyaC, pCACT3 for highlevel co-expression of CyaAand CyaC, pACTA385-828 for expression of the CyaAAH protein, and pACTAC1322 bearing the first 384 codons of cyaA followed by a cloning polylinker with stop codons in all three reading frames were already described (Sebo et al., 1991; Betsou etal., 1993; Sebo and Ladant, 1993). Transformants were selected on MacConkey or LB agar mediacontaining 100 mg of ampicillineAiter . Construction of Plasmids-The in vitro DNA manipulations were performed accordingtostandard protocols (Sambrooket al., 1989). When appropriate, thenoncohesive ends of restriction fragments were blunt-ended before ligation by the action of T4 DNA polymerase. (Detailed schemes of plasmid constructions are available upon request.) Unless otherwise stated, pACT7 (Sebo et al., 1991) was used for construction of deletions in the cyaA gene. pACTAC843, which was used to produce a polypeptide comprising the first 863 residues of CyaA, was generated by the replacement of the 2.6-kilobase EcoRI fragment of pACT7 by the KmR cassette KmO (Ladant et al., 1992). pACTAC698 was obtained by insertion of the 1.9-kilobase BstBI-XhoI fragment of pACT7 into the BstBI-PstI sites of pACTAC1322. This resulted in the interruption of the cyaA reading frame beyond the codon for Glu-1008 and in the additionof a carboxyl-terminal linker-encoded nonapeptide VDSRGSPGT. The mutation was than introduced intocyaA the gene on the pCACT3 plasmid by subcloning the respective BstBI-ScaI fragment of pACTA385-828, into BstBI-ScaI sitesof pCACT3, yielding plasmid pCACTA385-828. The mutant, M C , which lacks the amino-terminal adenylate cyclase (ACTAI-373) was constructed as hasbeen described previously (Sakamoto etal., 1992). The mutants used in this study are shown in Fig. 2 HlyA of E. coli-Extracellular HlyA of E. coli was isolated from the supernatants of an E. coli 5WpANN 202-812 culture (20 ml)grown a t 37 "C in double concentrated yeast tryptone broth (2 x Y T ) to a density of 5 x 10' cells/ml a s described previously (Benz etal., 1989). Lipid BilayerExperiments-The method of lipid bilayer experiments was essentially the same as has been described previously in detail (Benz etal., 1978; 1979).Briefly, the apparatusconsisted of a Teflon cell with a thin wall separating two aqueous compartments. The Teflon divider had a small circular hole with an area of about 0.5 mm'. Different lipids were applied a s a 1%(w/v) solution in n-decane. The following lipidswereused:diphytanoylphosphatidylcholine, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, phosphatidylserine (all obtained from Avanti Polar Lipids,Alabaster,AL), andasolectin (lecithin type 111s from soy beans were from Sigma). The aqueous salt solutions werebuffered with 5 mM MESXOH and adjusted pH to 6. The temperature was kept at 25 "C throughout. The membrane current was measured with a pairof calomel electrodes switched in series with a voltage source and a current-to-voltage converter manufactured on the basis of a Burr-Brown operational amplifier. The amplified signal was monitored with a storage oscilloscope and recorded with a strip chart recorder. RESULTS

EXPERIMENTAL PROCEDURES Standard TechniquesSDS-polyacrylamide gel electrophoresis analysis and determinationof protein concentration were performed according to standardprotocols (Sambrook etal., 1989). Production a n d Purification oftheCyaA-deriued Proteins-Wild-type CyaA protein was purified from B. pertussis 18323 a s described previously (Bellalou etal., 1990a). Full-length recombinantCyaA protein or the different truncated variants were produced with or without CyaC E. coli strain Xl1-Blue (Stratcoexpression (Sebo etal., 1991) using the agene), transformed by the respective plasmid(s). The proteins were extracted with8 M urea in50 mM Tris-HCI, pH 8.0,0.2 mM CaCl, (buffer A) from cell debris after sonication and purifiedby single-step affinity chromatography on calmodulin-agarose (Sigma)a s described previously inurea, 50 mM (Sebo etal., 1991). The purified proteins were eluted8 M Tris-HC1, pH 8.0,2mM EGTA(buffer B) and stored a t -20 "C. CyaA and the Cy& mutants (see below) were essentially free of contaminant proteins as illustrated inFig. 1. Assay ofAdenylateCyclase-Adenylate cyclase activities were measured a s described previously (Ladantal., et 1989). One unitof adenylate 1 mmol of CAMPformed per min at 30 "C, cyclase activity corresponds to pH 8.0.

Interaction of the CyaA with LipidBilayer Membranes-The interaction of CyaA with lipid bilayer wasinvestigated by conductance measurements on membranes formed from either a variety of pure lipids, such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, or from asolectin (a mixture of many different lipids isolated from soy beans). The membranes madeof pure lipids were rather inactive targets for the toxin, and their conductance was only little increased over that of control membranes (data not shown). Only in experiments with membranes formed of asolectin could a substantial increase of the specific membrane conductance be observed. As shown in Fig. 3, the conductance increased several orders of magnitude above that of asolectin membranes without CyaA. The reason for this apparent"lipid specificity" is not clear. It is noteworthy, however, that we have observed in previous studies with HlyA of E. coli and M. morganii that lipid bilayers made of pure lipids were alsorather inactive targets for both hemeolysins (Benz et al., 1989; 1994).

27233


PROTEIN:

PLASMID:

NAME:

Mr: Wa)

CyaA

177.3

AC698

106.13

AC843

97.5

AH

134.3

M C

151.3

Putative modification site

I

Hydrophobic region ”

AC-domain I

pACT7

I

Rep:

.1.1.1.

pACTAC698 3R4 500

pACTAC843

-

1

I

3R4

500

7On

913

10OR

701 863

FIG.2. Scheme of the structure of wild type and mutant CyaA-derivatives used in this study. The plasmids were constructed as described under “Experimental Procedures.” The numbers that follows the symbol AC represents the number of missing carboxyl-terminal residues from 1009 or 863 onwards. The numbersin the name of the plasmid pACTA385-828 are the numbersof the first and the last codons of the deleted internal portion of the c y d reading frame. The linker-coded sequence used for replacement of internal portions of CyaA is given in single letter code above the dashed line indicating the internal deletion. AC, adenylate cyclase.

I

mediated membrane conductance for the latter form in lipid bilayermembranes from asolectin was approximately 4-10 0 times higher thanfor the recombinantform of CyaA produced in E. coli. As both bacteria express the toxin from the same 10 genes, this difference in pore-forming activity may reflect a possible difference in the activationprocess of CyaA in thetwo 1.o organisms, e.g. that different activating moieties could be linked toCyaA during theactivation process in B. pertussis and in E. coli. In fact, this result is consistent with our previous 0.1 observations that CyaA purified from B. pertussis has severalfold higher specific hemeolytic activity than the same toxin expressed in E. coli (Sebo et al., 1991; Betsou et al., 1993). The 0.01 I , I effect of the activation by CyaC on the membrane activity of 0 10 20 30 40 CyaAis also illustrated Fig. in 3 (triangles).Addition of up to10 timelmin pg/ml of nonactivated CyaA increased the membraneconductof time after the ance by a factor of only about 10, which means that it was FIG.3. Increase of the current as a function addition of 2 p g / d cyclolysin expressed in E. coli to a black considerably less active in the bilayer assay than both the asolectidn-decanebathed in 0.15 M KC1 ( f u l l circles). The full toxin from B. squares represent analogous experiment on another membrane with 2 activated CyaA expressed in E. coli and the same pg/ml of CyaA purified from B. pertussis. The triangZes represent the pertussis. Similarly, nonactivated HlyA of E. coli was shown to effect of 10 pg/ml of nonactivated recombinant CyaA on a third mem- have a rather low channel-forming activity in planar lipid bibrane. The applied voltage was 50 mV; temperature = 25 “C. Thetoxin was added in all cases 10 min (corresponding to the start of the record) layers (Ludwig et al., 1992). Single-channel Analysis-The addition of lower concentraaRer the membrane was in the black state. tions of CyaA (50-200 ng/ml) to asolectidn-decane membranes The conductance increase in the presence of CyaA was not allowed the resolution of stepwise conductanceincreases. Fig. 4 sudden (Fig. 3) but was a function of time after the additionof shows a single-channel recording in the presence of 100 ng/ml the toxin to asolectin membranes in the black state. A mem- of CyaA expressed in B. pertussis. The toxin was added 5 min black state and only small brane wasformed fromasolectidn-decane insolutions contain- after the membrane was in the black conductance steps, which frequently switched off again after a ing 1 M KCl, and 10 min after the membrane was in the short time,could be observed. After some time, the membrane state, 2 pg/ml of CyaA produced in E. coli was added to the aqueousphase, while stirring,to allow equilibration (full contained so many small channels that it became further impossible to distinguish the single channels. It is noteworthy circles). Approximately 2 min after addition, the membrane conductance started to rise and increased by more than 2 or- that the single-channel conductance of the channelsformed by ders of magnitude in approximately 30min. Only a small fur- CyaA were much smaller than thoseformed by HlyA of E. coli ther increase, as compared with the initialone, occurred after under otherwise identical conditions (27 pS as compared with that time. The same effect was observed irrespective of whether 1500 pS;see Fig. 4, inset 1. Fig. 5 shows a single-channelrecord the toxin was added toone or both sides of the membranes. a t very small protein concentration (10 ng/ml); only one or two I t is noteworthy that under otherwise identical conditions, channels are formed under these conditions. Fig. 6 shows a the toxin produced by B. pertussis had a higher membrane histogram of the conductance fluctuations observed under the activity as compared with CyaA expressed in E. coli, and re- conditions of the experiments shown in Figs. 4 and 5 (1 M KC1 peatedly induced higher membrane Conductance. A conductand 50 mV membrane potential). Theconductance increments ance-time curve measured for 2 pg/ml of CyaAfrom B. pertussis were fairly homogeneous, and a single-channel conductance of is also shown in Fig. 3 (full squares). In general, the CyaA- 27 pS at 1M KC1 was observed for 452 single events. loo G/pScm-2

2 pglml CyaA

B.

pertussis 2 pglml CyaA E. c o l ~

I

27234


30 s FIG.4. Single-channel recording of an asolectidn-decane membrane in the presence of 100 ng/ml cyclolysin expressed in B. pertussis. The aqueousphase contained 1 M KCl, pH 6. The applied membrane potential was 50 mV; temperature = 25 "C.The inset shows a singlechannel recording in the presence of HlyA of E. coli under otherwise identical conditions.

lmin

FIG.5. Single-channel recordingof an asolectidn-decanemembrane in the presence of 10 ndml CyaA expressed inB. pertussis. The aqueous phase contained 1 M KCl, pH 6. The applied membrane potential was 50 mV; temperature = 25 "C.

Single-channel experiments were also performed with salts other than KC1 to obtain some information on the size of the CyaA channel and its ion selectivity. The results are summarized in Table I. Replacement of chloride by the less mobile acetate hadonly a little, if any, influence on the conductance of the CyaA channels, Theinfluence of the cations on the singlechannel conductance was more substantial (Table I) and suggested that the channelsformed by CyaA are cation-selective. This is supported by the finding that we were not able to detect any single channels in 1 M Tris-HC1, pH 6, and this again suggested that the diameter of the CyaA channel was much smaller than that made by HlyAof E. coli. Also divalent cations such as Ca2+were permeable through the channelat approximately the same rate aspotassium ions (Table I). It is important to note, that the channels formed byCyaA expressed

singleeither inE. coli or in B. pertussis had virtually the same channel conductance. This result is consistent with the assumption that the higher channel-forming activityof the toxin, expressed by B. pertussis, is caused by a difference in theposttranslational activation process and has nothing to do with the conductance of the CyaA channel itself. Table I also shows the average single-channel conductance, G, as a function of the KC1 concentration in theaqueous phase. Measurements were performed for 100,300,and 3000 mM KC1. Surprisingly, we did not observe a 1:l relationship between conductance and KC1 concentration as would be expected for wide water-filled channels similar to those formed by general diffusion porins (Benz, 1988).Instead, theslope of the conductance uersus concentration curves on a double logarithmic scale was about0.5, which suggested negative surfacecharge effects

Adenylate Cyclase

Toxin of B. pertussis

27235

the zero currentmembrane potential usingthe GoldmanHodgkin-Katz equation (Benz et al., 1979) suggestedthat chlo0.4 ride could have a certain permeability through theCyaA channel, since the permeability ratio P,/P,, was between 9 and 11. P(GJ On the other hand, is it also possible that the CyaA channel is ideally selective for cations becauseof point charges attachedto 0.3 the channels (see “Discussion”). A quantitative description of the effect of the point charges on the single-channel conductance has been previously described in detail for HlyA of M. 0.2 morganii (Benz et al., 1994) and was calculated on the basisof the Debye-Hiickel-Theory or by the treatment of Nelson and McQuarrie (1975). 0.1 Effect of Mutations in CyaA on Its Channel-forming Properties-We performed lipid bilayer experiments with mutant CyaAs to investigate which part of the toxin molecule is responsible for the formation of channels inlipid bilayer mem0 branes. In a first set of experiments, we studied the channel40 50 30 0 10 20 formation by a mutant (AC698) in which 698 amino acids were G/pS removed at the carboxyl-terminal end beginning with residue FIG.6. Histogram of the probability for the occurrence of a 1009. This truncated toxin is nonhemeolytic’ and lacks the given conductivity unit observed with membranes formed of repeat region that is supposed t o be responsible for the recogasolectidn-decane in thepresence of 10-100 ng/ml CyaA purified from B. pertussis. P, is the probability that a given conductance nition of target cell surface, while the putativeactivation site of Fig. 7 showsa single-channel recordingon increment G is observed in the single-channel experiments. It was cal- CyaAis still present. culated by dividing the number of fluctuations with a given conductance an asolectin membrane measured in the presence of 100 ng/ml by the total number of conductance fluctuations. The aqueous phase of this mutant protein. The AC698 channels were indistincontained 1 M KC1. The applied membrane potential was 50 mV; temguishable from those formed by wild-type toxin (compare Figs. perature = 25 “C. The average single-channelconductance was 27 p s for 4 and 7; and Tables Iand 11). This resultsstrongly suggest that 452 single-channel events measured on 10 different membranes. the repeatregion by itself is not involved in theformation of the CyaA channel. It seems, therefore, that the repeats are reTABLE I quired for the recognition and binding of CyaA to the surface of Average single-channel conductance, G, of the cyclolysin channel the targetcells but notfor its insertion intolipid bilayer memin different salt solutions The membranes were formed of asolectin dissolved in n-decane. The branes. This meansalso that calcium ions are not required for aqueous solutions were unbuffered and had a pH of 6 unless otherwise membrane activity in lipid bilayer membranes. indicated. The applied voltage was 50 mV, and the temperature was In another set of experiments, we studied the formation of 20 “C. The average single-channel conductance, G , was calculated from channels by a mutant AC843 in which the putative activation at least 80 single events. c is the concentration of the aqeous salt site (between residues 913 and 1000) was also deleted. As ilsolutions. The data for HlyA of E. coli were taken in part from Benz et al. (1989);ND means not detectable. The single-channel conductance of lustrated inTable 11,even though thisconstruct was expressed HlyA of Escherichia coli is given for comparison. in a strain producing the toxin-activating protein CyaC, its channel-forming activity was very low. In fact, the membrane G Salt C activity of AC843, i.e. the numberof channels formed at a given CY& HlyA protein concentration, wassimilartothat of nonactivated M PS CyaA. However, the few channels formed at high concentraLiCl 1.0 15 700 tions of AC843 (10 pg/ml) were again indistinguishable from NaCl 1.0 18 1200 those formed by theactivated wild-type toxin. Thisresult KC1 310 0.10 4.8 0.3 11 720 strongly suggests that the AC843 construct did not undergo the 1.0 27 1500 CyaC-mediated modification during its synthesis, in spite of 3900 3.0 48 the presence of CyaC, and that thislack of activation accounts RbCl 1.0 29 1700 for its low channel-forming activity. KCH,COO (pH 7) 1.0 25 1400 Tris-HC1(pH 6 ) 1.0 ND 240 To get some insight on the portion of the toxin that is inCaCl, 0.15 8.5 volved in formation of the CyaA channels, we studied the for1.0 24 mation of channels by a mutant (AH) lacking the hydrophobic region of CyaA, located between residues 500 and 700. In AH, on the CyaA channel (see “Discussion”). the portion corresponding to residues 385-828 of CyaA was Selectivity of the CyaA Channel-In order to obtain further deleted and replaced by the linker-coded sequence CRSTinformation on the selectivity of the CyaA channel, we per- LEDPR. This mutant (AH) was not able to increase the conformed zero-current membrane potential measurements. The ductance of lipid bilayer membrane to any appreciable extent asolectin membranes were formed in 50 m~ KC1 solution, and even at very highprotein concentration. Simultaneously we did the toxin was added t o the aqueous phase when the membranes not observe current fluctuations similar to those observed with were in theblack state. After incorporation of 100-1000 chan- wild-type CyaA or with the mutants AC698 or AC843. Fig. 8 nels into the membrane, 10-fold salt gradientswere established showsasingle-channel record in the presence of 10 pg/ml by the addition of small amountsof concentrated KC1 solution ACTA385-828. Even at this high toxin concentration, we did to one side of the membrane. In all experiments, the more not observe much influence on the membrane current. In andiluted side of the membranebecame positive, which indicated other set of experiments, we investigated the channel formapreferential movement of potassium through the CyaA chan- tion by a mutant toxin (AAC) in which the adenylate cyclase nel, i.e. the channel is cation-selective as suggested from the part at the amino terminus (amino acids1to 373) were deleted. single-channel data. The zero current membrane potential for 10-fold KC1 gradients was between 39 and 41 mV. Analysis of P. Sebo, unpublished results.

1

27236


]

loops 5PA

30 s FIG.7. Single-channel recordingof an asolectin membrane in the presenceof 100 ng/ml of the mutant protein AC698,expressed in E. coli, in which residues 1009-1706 were deleted.The aqueous phase contained 1 M KC1, pH 6. The applied membrane potential was 50 mV; temperature = 25 "C.

TABLEI1 Average single-channel conductance, G, of different CyaA mutants in 1 M KC1 The membranes were formed of asolectin dissolved in n-decane. The IM KC1 solution was unbuffered and hada pH of 6. The applied voltage was 50 mV, and the temperature was 25 "C. The average single-channel conductance, G , was calculated from at least 100 single events. The activated and nonactivated wild-type CyaA were expressed from plasmids pCACT3 and pACT7, respectively. Constructs used for expression of the mutant proteins are indicated in Fig. 2; NDmeans notdetectable.

channel conductances of HlyA of E. coli (Benz et al., 1989; this study) and M . morganii (Benz et al., 1994) are about 50-times higher than those of CyaA under identical conditions (see Table I). This result is consistent with the several hundred-fold lower hemeolytic activity of CyaAwhen comparedwith HlyA. A lower single-channel conductance means that the flux of ions and other hydrophilicmolecules through the channel is much smaller, and formation of a higher number of CyaAchannels in the target cell membrane is required to achieve the same perCyaA mutant Deletion G Membrane meability change than with channels of high conductance. activity It is important to note that the formation of channels by PS activated CyaA was not a rare event. Addition of 1 pg of Wild-type (B. pertussis) 27 ++++ CyaA/ml of aqueous phase bathing an artificial membrane was Wild-type (E. coli) 28 +++ able to considerably increase the conductance of the lipid biNon-activated wild-type 28 + (E. coli) layer membranes, and more than 10,000 channels couldbe AC698 +++ 25 formed in a membrane with a surface area of only 0.5 mm2. AC843 26 + Larger toxin concentrations led to the formation of even more MC 1-373 +++ 25 channels. Several lines of evidence demonstrate thatthe AH 385-828 ND formed channels were specifically due to the activity of the toxin and that the observed formation of channels was not an unspecific artifact caused by contaminating proteins. First, the This mutant has stillhemeolytic activity and formed channels current fluctuations were not caused by porins of B. pertussis or in lipid bilayer membranes that were indistinguishable from E.coli that could be present in the purified toxin preparations. those of wild-type CyaA (see Table 11). The CyaA channel had amuch smaller single-channel conductance than theporins (Benz, 1994).Secondly,the only difference DISCUSSION observed between the toxins purified from the two distantlyThe adenylate cyclase toxin (CyaA) of B. pertussis exhibits a related organisms E. coli and B . pertussis, was their overall low hemeolytic activity,and we show here that itforms defined membrane activity, while their single-channel properties were channels in lipid bilayer membranes. The hemeolytic activity of identical. Thirdly, properties other than the observed conductCyaA was shown to be comprised in the'hemeolysin portion'of ance of the CyaA channel were also different from those of the the molecule (Sakamoto et al., 19921, which exhibits a struc- porin channels, which usually have a long lifetime and only a tural homology to many other RTX-toxins, such as those of E. limited selectivity for ions (Benz, 1988). Finally, controlexpericoli (Felmlee et al., 1985;Hess et al., 1986; Ludwiget al.,19911, ments with the nonhemeolytic mutant AH, for whichthe puriPasteurellahemeolytica (Lo et al., 1987) and Actinobacillus fication procedure was identical to that of the active mutants pleuropleunomiae (Devenish et al., 1989; Frey et al., 1991). and/or wild-type CyaA, and which did not exhibit any appreMost of these toxins were shownto possess both channel-form- ciable channel-forming activity, demonstrate that our protein ing activity on target cells and on artificial membranes (Bhakdi samples were essentially free of any contaminating porins or et al., 1986, 1989; Menestrina et al., 1987; Benz et al., 1989; other nonspecific channel-forming compounds. Lalonde et al., 1989). Here we report that CyaA forms defined Interestingly, the single-channel conductance of the CyaAchannels with a rather low single-channel conductance com- channel showed a 2-fold variation, since it was somewhat difpared with those of other RTX-toxins. In particular, the single- ficult to separate the single events one from another. This re-

27237


'loops

5 PA

-

lmin FIG.8. Single-channel recordingof an asolectin membrane in the presenceof 10 ng/d of the mutant proteinAH, expressed inE. CRSTLEDPR. The aqueous phase contained1M KC1, pH 6. coli, in which residues386-828 were replaced by the linker-coded sequence The applied membrane potential was50 mV; temperature = 25 "C.

sult could mean that the fluctuations represent monomers and dimers of the CyaA channel. Such interpretation would be consistent with our previous results on CyaA-mediated lysis of erythrocytes, which suggested thatmore than one CyaA molecule may be required for the formation of a functional hemeolytic pore (Betsou et al., 1993). Furthermore, it haspreviously been shown that the channels formed by a homologous toxin HlyA of E. coli are made by protein oligomers (Ludwig et al., 1993). Nevertheless, on the basis of the present data, itis not yet clear whether the CyaA channel is also formed by an oligomer, and we consider this as less likely, as far as the rather low CyaA-mediated single-channel conductance is concerned. The reason why the membranes madeof pure phospholipids are so insensitive to the actionof CyaA, when compared with thesensitivity (CyaA-inducedconductance) of membranes made fromasolectin, is not clear. One plausible hypothesis would be that asolectin, which is a mixture of lipids extracted from soy bean, may contain some sort of a receptor molecule for of the toxin into the the toxin that may facilitate the integration membrane. The target cell receptor(s) for CyaA is not known. However, it is most likely to be an abundant and ubiquitos molecule, such asa particular lipid or glycolipid, because of its resistance toproteolysis and because of the difficulty t o observe saturation in bindingof CyaA t o the target cells and artificial membranes (Gordon et al., 1989).2 Requirements for Formation of CyaA Channels-We performed lipid bilayer experiments with several mutant forms of CyaA and found that deletion of a large portion of the CyaA molecule, comprising 36 of the 38 of the glycin and aspartaterich calcium-binding repeats, had only small, if any, influence on its channel-forming properties. This result can be understood on the basisof the close analogy of channel formationby CyaA and HlyA of E. coli. The deletion of several repeatsfrom a homologous toxin, the HlyA of E. coli, also lead to the complete inhibition of its hemeolytic activity but did not alter its channel-forming properties in lipid bilayer membranes (Ludwig et al., 1988). Hence,the calcium-promoted and presumably repeat-mediated binding of these toxins to the targetcell surface appears tobe essential for lysis of red blood cells, but not for channel formation in lipid bilayer membranes (Ludwig et

al., 1988). This difference is likely to be due to thesmooth and sufficiently hydrophobic surface of these membranes that allows a close contact between toxin and lipid as a prerequisite for channel formation. This would further imply that the presence of calcium is only required for successful binding of the toxin to the target cell surface but not for insertion into lipid bilayer membranes. On the other hand, the activation of CyaA and HlyA by posttranslational modification appears to be required for both, effective channel-formation in artificial membranes andhemeolytic activity of these toxins. Nevertheless, formation of some few channels could be observed in thepresence of high concentrations of nonactivated HlyA (Ludwig et al., 1992), and we observed here some few CyaA channels in thepresence of high concentrations (10 pg/ml) of nonactivated toxin. However, its channel-forming activity was low. It was similar for both the nonactivated CyaA and for the mutantAC843 produced in the presence of the activating proteinCyaC, but lacking the putative modification site. Collectively, these observationsshow that theCyaC-mediated activation of the toxin is essential not only for its hemeolytic activity (Barry et al., 1991; Sebo et al., 1991), but also for formation of the CyaA channels in lipid bilayers. Hence, the activation process obviously provides to the RTX-toxin molecules some sort of lipophilic membrane anchor, possibly by fatty-acylation (Issartel et al., 19911, which may serve to increase the surface concentration of the toxin, formathereby facilitatingits membrane insertion and channel tion. It is conceivable that the membrane anchor of CyaA expressed in B. pertussis and inE. coli may be different and that such a difference may account for the higher channel forming activity of the former in the lipid bilayer membranes observed here aswell as it may account for its higherspecific hemeolytic activity on erythrocytes (Sebo et al., 1991; Betsou et al., 1993). We have recently demonstrated (Sakamotoet al., 1992) that the deletion of the catalytic adenylate cyclase domain of the toxin, AAC, had no influence whatsoever on the CyaA-induced hemeolysis. Similarly we observed in thelipid bilayer assay no difference in channel-formation between wild-type andthe AAC-mutant. Also, the channel-forming activity was virtually the same. On the other hand, deletion of the hydrophobic region

27238


of the hemeolysin portion of the toxin completely abolished its allow the passage of even a fully unfolded polypeptide chain. capacity to form channels. Thissuggested that thehydrophobic Instead, we propose that an alternative model for adenylate region is both the hemeolytic and the channel-forming part of cyclase delivery into cells should be considered in which the CyaA. Similar conclusions have recently been derivedfrom the catalytic domain undergoes a calcium-induced conformational studies with mutantHlyA of E. coli (Ludwig et al., 1991). change (Masure et al., 1989; Hewlett et al. 19911, which may Effectof Point Chargesat the Channel Mouth on Properties of result in an amphipathic structure, somehow directly interacts the C y d channel-The concentration dependenceof the single- with lipid bilayer of target cell membrane and slides across it, channel conductance of the CyaAchannel(Table I) was found to perhaps, in association with the channel-forming portion of be a nonlinear function of the bulk aqueous concentration. In- CyaA. deed, a slope of less than0.5 on a double-logarithmic scale was Finally, our data are consistent withtheresults of the observed for the conductance versus concentration curve. This measurements of the hemeolytic activity of CyaA, which is very result indicates thatsurface charge effects influence the prop- weak as compared with thatof the HlyA of E. coli (Ehrmann et erties of the CyaA channel similarly as hasbeen found for HlyA al., 1991), and we show that thislow hemeolytic activity is not of E. coli (Ropele and Menestzna, 1989; Benz et al., 1989), M. due toa low probability of formation of CyaA channels. In fact, morganii, and Proteus vulgaris (Benz et al., 1994). Negative the channel-forming probability of CyaA is even higher than charges at the pore mouth result in substantial ionic strength- that of HlyA. However, the conductance of the CyaA channels dependent surface potentials at thepore mouth, which attract and thus its permeability for small ions is considerably lower than thatof the HlyA channels. This is most likely the primary cations and repelanions. Accordingly, they influenceboth single-channel conductance and zero current membranepoten- cause of the low hemeolytic activity of CyaA. tial. The chargeeffects are a function of the channel diameter REFERENCES and suggest that it is below 0.8 nm. This is again in good agreement with the observed small single-channel conductance Barry, E. M., Weiss, A. A,, Ehrmann, I. E., Gray, M. C.,Hewlett, E. L., and Goodwin, M. S. (1991)J. Bacteriol. 173, 720-726 and the osmotic protection data (Ehrmann et al., 1991). U.,Wu, S., Flaherty, K.M., and McKay,D.B. (1993)EMBO J. 12, The point charge effects are probably also responsiblefor the Baumann, 3357-3364 permeability of the CyaA channel toward divalentcations. We Bellalou, J., Ladant, D., and Sakamoto, H. (1990a)Infect. Zmmun. 68, 1195-1200 have demonstrated here that calcium ions are able to penetrate Bellalou, J.,Sakamoto, H., Ladant, D., Geofioy, C., andUllmann,A. (1990b)Znfect. Zmmun. 68,3242-3247 the cy&-channel at approximately the same rateas potassium Benz, R. (1988)Annu. Reu. Microbiol. 42,359393 ions. This result makesit very likelythat calcium ions can also Benz, R. (1994)inBacterial Cell Wall (Ghuysen J.-M., and Hakenbeck R., eds.) pp. 397423,Elsevier Science Publishers B. V., Amsterdam permeate theCyaA channel in viuo. The influx of calcium in the Benz, R., Janko, K, Boos, W., and Lauger, P. (1978)Biochim. Biophys. Acta 611, target cells may contribute to theinfection of the cells since it 305319 has been demonstrated that calcium is somehow involved in Benz, R., Janko K , and Lauger P. (1979)Biochim. Biophys. Acta 661,23%247 Benz, R., Schmid, A,, Wagner, W., and Goebel, W. (1989)Infect. Zmmun. 67, 887the uptake of the catalytic domain into the cells (see below). 895 Size of the C y d channel andIts Physiological Implica- Benz, R. Hardie, K. R., and Hughes, C. (1994)Eur. J . Biochem. 220,339-347 Betsou, F., Sebo, P., and Guiso, N. (1993)Infect. Zmmun. 61, 3583-3589 tions-The single-channelconductance of the CyaA-channel Bhakdi, S., Mackman, N., Nicaud, J.-M., and Holland, I. B. (1986)Infect. Zmmun. 52,6349 was found to be considerably lower than that of a-hemeolysin S., Greulich, S., Muhly, M., Eberspacher, B., Becker, H., Thiele, A,, and (HlyA) of E. coli under identical conditions (see Table I). This Bhakdi, Hugo, F. (1989)J . Exp. Med. 169, 737-754 strongly suggests that thecross-section of the CyaA channel is Boehm, D. F., Welch, R. A., and Snyder,1. S . (1990a)Znfict.Zmmun. 68,1951-1958 considerably smaller than thatof the HlyA channel. For small Boehm, D. F., Welch, R. A., and Snyder, I. S . (1990b)Infect. Zmmun. 68,1959-1964 Confer, D. L., and Eaton, J. W. (1982)Science 217,948-950 channels, however, precise estimations of the channel radii Coote, J. G. (1992)FEMS Microbiol. Reu. 88, 137-162 from the single-channelconductance are difficult to derive, Devenish, J., Rosendal, S., Johnson, R., and Hubler, S . (1989)Infect. Zmmun. 67, 3210-3213 since their conductance is not proportional t o their cross-sec- Ehrmann, I. E., Gray, M. C., Gordon, V. M., Gray, L. S., and Hewlett, E. L. (1991) tion. This is because the ions are partially dehydrated during FEBS Lett. 278,79-83 Felmlee, T., Pellett, S., and Welch, R. A. (1985)J. Bacteriol. 163, 94-105 the movement through the channels and interact with the J., Meier, R., Gygi, D., and Nicolet, J . (1991)Infect. Zmmun. 69, 30264032 channel walls. On the other hand, the observed CyaA-mediated Frey, Gentile, F., Knipling, L. G., Sackett, D. L., and Wolff J. (1990)J. Biol. Chem. 265, single-channel conductance is similar to that of the potassium 10686-10692 channels of nerve and muscle tissues, which argues infavor of Glaser, P., Elmaoglou-Lazaridou, A., Krin, E., Ladant,D., Blrzu, O., and Danchin, A,. (1989)EMBO J . 8, 967-972 a diameter of less than 0.6-0.8 nm. Such a diameter would be Gordon, V. M., Young, W.W., Jr., Lechler, S. M., Gray, M. C., Leppla, S. H., and Hewlett, E. L. (1989)J. Biol. Chem. 264, 14792-14796 consistent with the observed charge effects on the channel (as E. (1989)Dends Biochem. Sci. 14,459463 described above), the experimental observation that we could Hanski, Hanski, E., and Coote, J. G. (1991)in Sourcebook ofBacterial Toxins (Alouf, J. E., not detect channels inTris-HC1 and also with the resultsshowand Freer, J. H., eds.) pp. 349-366,Academic Press, London ingthatsheep erythrocytes can be osmotically protected Hanski, E., and Farfel, Z. (1985)J. Biol. Chem. 260, 5526-5532 Hess, J., Wels, W., Vogel, M., and Goebel, W. (1986)FEMS Microbiol. Lett. 34, 1-11 against CyaA-promoted lysis by addition small sugars such Hewlett, E. L., Gordon, V. M., McCaf€ery, J. D., Sutherland, W. M., and Gray, M. C. media (1989)J . Biol. Chem. 264, 19379-19384 as sucrose, mannitol, or arabinosetotheexternal E. L., Gray, L., Allietta, M., Ehrmann, I., Gordon, V. M., and Gray, M. C. (Ehrmann et al., 1991). Indeed, the channel diameter estimatedHewlett, (1991)J. B i d . Chem. 266,17503-17508 from the diameter of these protecting sugars fits well with a Issartel, J.-P., Koronakis, V., and Hughes, C. (1991)Nature 361, 759-761 Ladant, D., Michelson, S., Sarfati, R., Gilles, A,"., Predeleanu, R., and BBrzu, 0. diameter of the CyaA channel between 0.6 and 0.8 nm. (1989)J. Biol. Chem. 264,40154020 This makes rather unlikely a recently proposed hypothesis of Ladant, D., Glaser, P., and Ullmann, A. (1992)J. Biol. Chem. 267,2244-2250 Rogel and Hanski (1992), in which the hemeolysin portion of Lalonde, G., McDonald, T. V., Gardner, P., and O'Hanley, P. D. (1989)J. Biol. Chem. 13559-13564 CyaA is directly involved in the internalizationof the catalytic Lo,264, R. Y. C., Strathdee, C. A,, and Shewen, P. E. (1987)Infect. Zmmun. 66,1987adenylate cyclase domain into the target cells by creating a 1996 A,, Vogel, M., and Goebel, W. (1987)Mol. & Gen. Genet. 206,238-245 channel throughwhich this amino-terminaldomain is translo- Ludwig, Ludwig, A., Jarchau, T., Benz, R., and Goebel, W. (1988)Mol. & Gen. Genet. 214, cated. The results presented here with wild-type CyaA and a 553-561 number of mutants, including the mutant in which the adenyl- Ludwig, A,, Schmid, A,, Benz, R., and Goebel, W. (1991)Mol. & Gen. Genet. 226, 198-208 ate cyclase activity (AAC) was removed, suggest that the cross- Ludwig, A., Garcia, F., Benz, R., Jarchau, T., Oropeza-Wekerle, R.-L., and Goebel, W. (1992)in Bacterial Protein Toxins (Witholt, B., Alouf, J. E., Boulnois, G. J . , section of the hemeolytic channel of CyaA would be too small to

Adenylate Cyclase Toxin of B. pertussis Cossant, P., Dijkstra, B. W., Falmagne, P., Fehrenbach, F. J.,Freer, J., Niemann, H., Rappuoli, R., and Wadstriim, T.,eds) pp. 450-460, Gustav Fischer Verlag, stuttgart Ludwig, A,, Benz, R., and Goebel, W.(1993)Mol. & Gen. Genet. 241,8%96 Masure, H. R., and Storm, D. R. (1989)Biochemistry 28,438442 Menestrina, G., Mackman, N., Holland, I. B., and Bhakdi, S. (1987)Biochim. Biophys. Acta 905, 109-117 Nelson, A. P., and McQuame, D. A. (1975)J. Theor. Biol. 55, 13-27 Oropeza-Wekerle, R.-L., Muller, S., Briand, J.-P., Benz, R., Schmid, A,, and Goebel, W. (1992)Mol. Microbiol. 6, 115-121 Pearson, R. D., Symes, P., Conboy, M., Weiss, A. A., and Hewlett, E. L. (1987)J . Immunol. 139,2749-2754 Rogel, A., and Hanski, E.(1992)J. Biol. Chem. 267, 22599-22605

27239

Rogel, A,, Schultz, J., Brownlie, R. M., Coote, J. G., Parton, R., and Hanski, E. (1989)EMBO J. 8,2755-2760 Ropele, M., and Menestrina, G. (1989)Biochirn. Biophys. Acta 985, 9-18 Sakamoto, H., Bellalou, J., Sebo, P., and Ladant, D. (1992)J. Biol. Chem. 267, 13598-13602 Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989)Molecular C1oning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY Sebo, P., Glaser, P., Sakamoto, H., and Ullmann, A. (1991)Gene (Amst.)104,1%24 Sebo, P., and Ladant, D. (1993)Mol. Microbiol. 9,999-1009 Welch, R. A. (1991)Mol. Microbiol. 5,521528 Wolff, J., Cook, G. H., Goldhammer, A. R., and Berkowitz, S. A. (1980)Proc. Natl. Acad. Sci. U. S. A. 77, 3841-3844