Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis

Microbial Pathogenesis 46 (2009) 36–42

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Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis Gordon Y.C. Cheung a,1, Sharon M. Kelly b, Thomas J. Jess b, Sandra Prior c, Nicholas C. Price b, Roger Parton a, John G. Coote a, * a b c

Division of Infection and Immunity, University of Glasgow, Institute of Biomedical and Life Sciences, Glasgow Biomedical Research Centre, 120 University Place, Glasgow G12 8TA, UK Division of Biochemistry and Molecular Biology, Joseph Black Building, University of Glasgow, G12 8QQ, UK Division of Bacteriology, National Institute of Biological Standards and Control, South Mimms, Hertfordshire EN6 3QG, UK

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 June 2008 Received in revised form 2 October 2008 Accepted 7 October 2008 Available online 1 November 2008

A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration >1 mg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations <0.5 mg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 mg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 mg/ml, whereas a w500fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high b-sheet content or a b-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Bordetella pertussis Adenylate cyclase toxin CyaA Cytotoxicity Secondary structure Circular dichroism

1. Introduction Bordetella pertussis, the aetiological agent of whooping cough, secretes an adenylate cyclase toxin (CyaA) that belongs to the Repeats in ToXin (RTX) family [1,2]. CyaA, a 177 kDa protein, is synthesised as a protoxin (proCyaA) that is post-translationally acylated by a separate protein, CyaC. CyaA has two functional domains: the C-terminal domain (of about 1300 amino acids) which has membrane-targeting and pore-forming activity; and the 400 amino acid N-terminal domain which has adenylate cyclase (AC) enzymatic activity. Interaction with, and invasion of, mammalian target cells, such as monocytes and neutrophils, that express the CD11b/CD18 (CR3) receptor [3], is facilitated by * Corresponding author. Tel.: þ44 (0) 141 330 5845; fax: þ44 (0) 141 330 4600. E-mail address: [email protected] (J.G. Coote). 1 Present address: Laboratory of Bacterial Diseases, National Institutes of Health, 33 North Drive, Building 33, Room 1W20, Bethesda, MD 20892, USA. 0882-4010/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2008.10.005

acylation of CyaA. Binding of calcium ions to the C-terminal glycine/ aspartate repeats causes a significant conformational change [4–7] and is a prerequisite for toxin activity [4,6,8]. Upon entry into the cell, host calmodulin activates the N-terminal AC enzymatic moiety by interacting with several helical structural elements of the AC domain [9] to produce supraphysiological levels of cyclic AMP (cAMP) from adenosine triphosphate (ATP), a process referred to as intoxication [10]. In immune effector cells, this impairs their phagocytic and bactericidal capacities and induces apoptosis, features that are assumed to assist the survival of the bacterium in the initial stages of respiratory tract colonisation [11]. CyaA forms small (0.6–0.8 nm diameter), transient, ionpermeable channels in target membranes [12–15]. CyaA-induced haemolysis requires higher toxin concentrations and occurs more slowly than intoxication [16]. Dose–response experiments indicate that intoxication can be triggered by CyaA monomers at low toxin concentrations, whereas cytolytic activity is a co-operative event, mediated by an oligomeric structure consisting of two or more

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toxin monomers responsible for the production of the pores or channels at higher toxin concentrations [17–20]. Other studies have shown that, at high toxin concentrations, non-acylated CyaA can intoxicate macrophages by delivery of the catalytic domain [21,22]. The reduced intoxication by proCyaA may be explained by the reduced capacity of non-acylated CyaA to bind to cells expressing the CR3 receptor [8]. There have been contrasting reports on the cytotoxic activities of non-enzymatically active (AC) forms of CyaA, equivalent to CyaA* used here. Hewlett et al. [22] reported that an AC CyaA with a Cys/Thr insertion at amino acids 188/189 was unable to kill J774 mouse macrophage-like cells at a concentration as high as 10 mg/ml, yet lytic activity towards erythrocytes was retained. Binding of a monoclonal antibody distal to the inactivated catalytic site restored cytotoxicity towards J774 cells and further enhanced haemolytic activity. Basler et al. [23], using a similar construct, reported that the AC toxin possessed about 10% of the specific cytolytic activity of the intact CyaA towards J774 cells whereas Boyd et al. [21] reported that another AC construct (possessing H63A, K65A and S66G substitutions) had w50% cytotoxic activity at 10 mg/ml with J774 cells. In this report we have investigated whether the different cytotoxic activities of CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*, could be explained by secondary structural features of the toxins determined by far and near UV CD and fluorescence spectra analyses. 2. Results 2.1. Cytotoxicity of the different CyaA forms By the MTT assay, both CyaA and CyaA* caused approximately 100% cytotoxicity of J774.2 cells after 2 h at toxin concentrations approaching 1.0 mg/ml but, at <0.5 mg/ml, CyaA* exhibited less cytotoxicity and, in contrast to CyaA, at 0.1 mg/ml little activity was detected (Fig. 1A). The non-acylated proCyaA and proCyaA* forms did not exhibit detectable cytotoxicity towards J774.2 cells, up to the highest toxin concentration tested (10 mg/ml) (data not shown). By the LDH release assay, for CyaA, approximately 10-fold more toxin was needed for 50% cytotoxicity of J774.2 cells compared to the MTT assay (Fig. 1A,B), whereas CyaA* was more active and required only approximately 2-fold more protein than in the MTT assay to achieve the 50% cytotoxic effect. At a concentration of 1.25 mg/ml, CyaA* promoted cell killing at a faster rate than CyaA, achieving >80% cytotoxicity in both assays after 60 min (Fig. 2A,B). Cytotoxicity of both CyaA and CyaA* measured by the MTT assay took place more quickly (Fig. 2A) than that measured in the LDH assay (Fig. 2B). These data are in keeping with the apparent poorer activity of the toxins, especially CyaA, noted after incubation for 2 h in the LDH assay (Fig. 1B) compared with the MTT assay (Fig. 1A).

Fig. 1. Cytotoxicity of CyaA and CyaA* for J774.2 cells after incubation for 2 h, as measured by the (A) MTT or (B) LDH release assays. Results represent the means of assays performed in duplicate, with standard errors of the means (SEM) (bars).

at a concentration >0.1 mg/ml but, at 10 mg/ml, little caspase 3/7 activity was detected (Fig. 3). The non-acylated proCyaA and proCyaA*, up to a concentration of 10 mg/ml, failed to induce caspase 3/ 7 activity (data not shown). Thus, induction of apoptosis was dependent on both AC enzymatic activity and acylation of the toxin.

2.2. Other cytotoxicity assays

2.3. Effect of calcium on the secondary structure of the different forms of CyaA

The different CyaA forms were investigated for their ability to inhibit the zymosan-stimulated oxidative burst in J774.2 cells. CyaA caused 50% inhibition at a dose of 0.003  0.0012 mg/ml whereas a w500-fold greater concentration of CyaA* (1.71  0.11 mg/ml) was required for 50% inhibition. No activity was detected with proCyaA*, but proCyaA had an activity comparable to that of CyaA* (1.48  0.065 mg/ml required for 50% inhibition). Thus, efficient inhibition of the oxidative burst was dependent on both AC enzymatic activity and acylation of the toxin. The four CyaA forms were also tested for their ability to induce caspase 3/7 activity, an indicator of apoptosis, in J774.2 cells after incubation for 2 h. CyaA, but not CyaA*, induced caspase 3/7 activity

The capacity of CyaA* to elicit loss of cell viability at a faster rate than CyaA (Fig. 2) prompted an investigation of the structural features of the toxin derivatives in the presence of Caþþ ions which are needed for the invasive activity of the toxin. The far UV CD spectra of dialysed CyaA and CyaA*, at 0.5 mg/ml in different CaCl2 concentrations from 0 mM CaCl2 to 7.5 mM CaCl2 (Fig. 4), showed that both proteins exhibited a change in the spectrum whereby a negative peak at 207 nm with a shoulder around 222 nm changed to one in which there is a negative peak at 220 nm with a shoulder around 208 nm (Fig. 4A,B). The CD spectra of CyaA and CyaA* at 0.5–5 mM CaCl2 were essentially superimposable but, at 7.5 mM CaCl2, the spectra altered (Fig. 4A,B) in a way that was indicative of

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Fig. 3. Effect of different concentrations of CyaA or CyaA* on the induction of caspase 3/7 activity in J774.2 cells. Results, presented in arbitrary fluorescence units (RFLU), are representative of 2 separate experiments.

any reliable analysis of the spectra in terms of secondary structural content. Similar effects of concentration on the far UV CD spectra were observed using CyaA*, proCyaA and proCyaA* (data not shown). This dependence of spectral changes on protein concentration implied an interaction between individual molecules that was promoted at higher protein concentrations. 2.4. Effect of calcium on the tertiary structure of the different forms of CyaA

Fig. 2. Time course of cytotoxicity of CyaA and CyaA* for J774.2 cells. Cytotoxicity was measured over 2 h by (A) MTT and (B) LDH release assays using 1.25 mg/ml of either CyaA or CyaA*. Results represent the means of assays performed in duplicate, with SEM (bars).

high b-sheet content or a b-aggregated state. This was more apparent with CyaA. ProCyaA or proCyaA* showed similar spectral trends to those observed for CyaA and CyaA*, respectively, in the presence and absence of CaCl2 (data not shown). The CDSSTR program in Dichroweb was used to analyse the far UV CD spectra of CyaA at 0.5 mg/ml over the wavelength range from 185 nm to 240 nm in terms of the secondary structure content. Notwithstanding the slight differences detected in proCyaA in the absence of calcium, the data in Table 1 suggest that all the forms of CyaA have similar overall structures and show similar responses upon the addition of CaCl2. A comparison of the far UV spectra of CyaA, at 0.5 mg/ml and 0.05 mg/ml, in the presence and absence of 0.3 mM CaCl2 showed that there was a marked dependence of the spectrum of CyaA on protein concentration (Fig. 4C). In the absence of calcium, the normalised molar ellipticity value at 220 nm was reduced by a factor of approximately 2 at the lower concentration. In addition, the response to CaCl2 was different at the lower protein concentration (Fig. 4C) with the reduction in amplitude being approximately 3 fold. Because of the high level of noise below 195 nm in the spectra of CyaA at 0.05 mg/ml, it was not possible to undertake

Conformational changes in CyaA in the presence of 1 mM CaCl2 were also obtained from near UV CD studies, where increased spectral intensities at 293 nm and in the 270–285 nm region were observed for CyaA in the presence of 1 mM CaCl2 compared to the spectrum obtained in the absence of 1 mM CaCl2 (Fig. 4D), consistent with the placing of aromatic side chains into a more rigid environment. The results of fluorescence studies also indicated that a conformational change had occurred in CyaA in the presence of 1 mM CaCl2, with a blue shift of 2 nm and a 30% increase in emission intensity (data not shown), consistent with a more pronounced burial of one or more tryptophan side chains in the interior of the protein. CyaA*, proCyaA and proCyaA* showed near UV CD and fluorescence spectra similar to those produced by CyaA (data not shown). 3. Discussion The concentration of CyaA required for 50% LDH release from J774.2 cells after incubation for 2 h was greater than the concentration required for 50% killing in the MTT assay (Fig. 1A,B). This in part may be explained by the lag period required for LDH release compared to the loss of viability measured by the MTT assay where the cytotoxic effect occurs without a lag period (Fig. 2A,B). It is known that, upon exposure of cells to CyaA, cAMP accumulation is almost immediate and the accumulation of intracellular CyaA proceeds without any noticeable lag period [24–26]. Killing, measured by the MTT assay, must therefore reflect accumulation of the toxin within the cell and the rapid onset of cell death in this assay may well be due to the ability of CyaA to uncouple oxidative phosphorylation of mitochondria [27] which is believed to be the basis for the MTT assay [28]. The release of LDH is an indication of loss of cell membrane integrity, assumed to be the result of pore formation by the toxins. Neither toxin below 0.1 mg/ml caused LDH

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Fig. 4. Far UV CD spectra of (A) CyaA and (B) CyaA* at 0.5 mg/ml in the absence (black lines) or in the presence of increasing concentrations of CaCl2. (C) Far UV CD spectra of CyaA at 0.5 mg/ml or 0.05 mg/ml in the absence or in the presence of 0.3 mM CaCl2. Results represent the means of titrations performed in duplicate. Each spectrum represents the average of 8 scans, carried out at 20  C between 190 and 260 nm, using 0.1-nm steps and a time constant of 2 s. (D) Near UV CD spectra of CyaA (at 1.5 mg/ml) in the absence and presence of 1 mM CaCl2 (thick and thin solid line, respectively). Each spectrum represents the average of 8 scans, carried out at 20  C between 260 and 320 nm, using 0.2-nm steps with a time constant of 2 s.

release (Fig. 1B), yet, unlike CyaA*, CyaA was active in the MTT assay at concentrations <0.04 mg/ml (Fig. 1A). Hewlett et al. [22] showed that, at low toxin concentration, CyaA killed cells mainly by apoptosis but, at higher toxin concentrations, it lysed cells efficiently by the production of pores causing the loss of cell membrane integrity. Our data support this as there was a decline in caspase 3/7 activity induced by CyaA at a concentration above 2 mg/ ml (Fig. 3), presumably reflecting a loss of cell viability due to lysis (Fig. 1A,B). CyaA* was unable to induce caspase 3/7 activity (Fig. 3) which would explain its lack of cytotoxicity at low concentrations. Our data show greater cytotoxicity of CyaA* at concentrations above 0.5 mg/ml than that reported for other AC derivatives [21–23]. This may be related to the nature of the amino acid insertions used to create the AC defective proteins. It has been shown recently that structural alteration of the N-terminal AC domain by oligopeptide insertion affects the rate of CyaA-mediated influx of calcium into J774.2 cells [29]. In addition, hyper-lytic forms

of CyaA with amino acid insertions in the N-terminal domain have been described [23]. CyaA* promoted greater LDH release than CyaA at concentrations above 0.5 mg/ml (Fig. 1B) which could be explained by a greater cytolytic capacity. This is borne out by the data in Fig. 2 where, at 1.25 mg/ml, a more rapid onset of LDH release (Fig. 2B) and loss of cell viability (Fig. 2A) were seen in the presence of CyaA* compared to CyaA, which would be in keeping with a greater cytolytic activity. Although proCyaA and proCyaA* were inactive at concentrations up to 10 mg/ml in the cytotoxicity and caspase assays, proCyaA was able to inhibit the zymosan-stimulated oxidative burst by J774.2 cells, albeit with a 50% inhibition concentration of 1.48 mg/ ml. In our hands, this assay was the most sensitive, with CyaA giving 50% inhibition at 0.003 mg/ml. ProCyaA* was ineffective and CyaA* was 500-fold less effective than CyaA. However, CyaA* may inhibit the zymosan-stimulated oxidative burst at high concentrations by causing cell lysis. The data are in accordance with other

Table 1 Secondary structure composition of the different CyaA forms in the presence or absence of CaCl2. CyaA form CaCl2 (1 mM)

Helix total Strand total Turns Unordered NRMSD

CyaA

CyaA*

proCyaA

proCyaA*



þ



þ



þ



þ

22 24 21 32 0.03

22 23 24 31 0.028

20 26 21 32 0.029

24 25 21 30 0.026

32 17 25 27 0.033

32 20 20 29 0.026

25 23 21 32 0.064

28 22 20 30 0.015

Deconvolution of spectra like that shown in Fig. 4A,B was performed using CDSSTR, as described in ‘‘Materials and methods’’. Numbers represent percentage of predicted structural motifs within the protein in the absence () or presence (þ) of 1 mM CaCl2. NRMSD, Normalised Root Mean Squared Deviation.

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work [21,22] where it was shown that, at toxin concentrations greater than 1 mg/ml, non-acylated proCyaA was able to increase cAMP levels in J774.2 cells. The fact that proCyaA* was ineffective and CyaA* was 500-fold less effective than CyaA also agrees with previous work indicating that this inhibition is mainly cAMPdependent [30]. CyaA and CyaA* exhibited a change in the far UV CD spectrum in the presence of calcium (Fig. 4A,B). The spectra of CyaA observed in the presence and absence of 1 mM CaCl2 were comparable to previous CD spectra with regard to spectral intensities [6,7]. CD spectrum analysis of the full-length protein suggested that there were, in general, only small changes in the secondary structure content in the presence of 1 mM CaCl2 (Table 1). This is in agreement with the work of Rhodes et al. [7] who reported <1% change in secondary structure upon addition of 1 mM CaCl2 to CyaA. In an earlier report, Rose et al. [6] indicated a 5% increase in alpha helical content upon addition of Caþþ to CyaA, but we consider our analysis more reliable because of (a) the use of a more robust algorithm with a larger database of reference proteins and (b) the significantly lower root mean square errors. Thus, changes in the CD spectra on addition of Caþþ may arise from rearrangements of secondary structural elements rather than changes in the actual proportions of such elements. This would be consistent with the observations of changes at the tertiary structure level detected by near UV CD and fluorescence analysis. CD spectra of CyaA* and the two nonacylated forms had not previously been determined, but showed changes in spectra similar to those seen with CyaA, suggesting that they were all similar in structure and that the addition of 1 mM CaCl2 induced the same type of changes. Thus, CD spectra at 1 mM CaCl2 were not sufficiently sensitive to distinguish between the various forms of CyaA in a way that could be related to their different biological activities. However, far UV CD analysis showed that CyaA changed structure markedly upon the addition of 7.5 mM CaCl2 (Fig. 4A) (w37% b-strands or b-aggregated state, as predicted by CDSSTR from the far UV CD spectrum, compared to 20–25% at 1 mM CaCl2, Table 1) with a significant loss of CD spectral amplitude between 200 and 220 nm, unlike CyaA* which showed only a small decrease of CD spectral amplitude between 200 and 220 nm (Fig. 4B). It was of interest to note in this respect that, at 1 mM CaCl2, the haemolytic activities of CyaA and CyaA* for sheep erythrocytes were similar but, in agreement with previous reports [12,13], the haemolytic activity of CyaA declined progressively at concentrations of CaCl2 up to 4 mM (data not shown). It was suggested that high calcium concentrations may interfere with the oligomerisation process by promoting aggregation [6]. In contrast, CyaA* exhibited progressively enhanced haemolytic activity at concentrations of CaCl2 between 1 and 4 mM (data not shown) which may have been related to the less significant alteration in secondary structure shown by CyaA* compared to CyaA in the presence of 7.5 mM CaCl2 (Fig. 4A,B). Thus, the far UV CD spectrum analysis did indicate an underlying structural difference between CyaA and CyaA*, presumably related to the amino acid insertions in the N-terminal domain of CyaA*, which may explain in part the more rapid onset of cytotoxicity against J774 cells in the presence of CyaA* compared to CyaA (Fig. 2A,B) and the differences in haemolytic behaviour exhibited by the two forms at high Caþþ concentrations. Near UV CD spectral changes on addition of CaCl2 to the CyaA forms have not been reported previously, but these, and the fluorescence data, pointed to a calcium-induced conformational change in CyaA which resulted in partial burial and immobilisation of aromatic side chains. It has been demonstrated that small backbone conformational distortions can lead to marked changes in CD signal [31]. Indeed the ratio of ellipticities at 222 nm and 208 nm moves from a value of 0.73–1.10 for all CyaA forms on addition of CaCl2. Such a change would be consistent with isolated helices interacting

to form structures of a coiled-coil type [31-33] or could indicate the rearrangement of b-structures into a helical-like pattern [34] of the type proposed for a parallel b-roll motif within CyaA [6]. Studies on the alkaline protease from Pseudomonas aeruginosa containing nonapeptide sequence repeats at the C-terminal end similar to those of CyaA have shown that these repeats are involved in calcium binding and give rise to a parallel a-helix or parallel b-roll structure [35]. Similarly, chemically-synthesised proteins containing 6 glycine-rich repeats have been shown to give rise to a marked increase in the ellipticity value obtained at 220 nm following calcium binding and CD analysis [36]. An interesting observation from the far UV CD spectra was that there was a 2–3 fold decrease in the normalised molar spectral amplitude at 220 nm of the CyaA forms when they were diluted from 0.5 mg/ml to 0.05 mg/ml, both in the absence or presence of calcium (Fig. 4C). This type of spectral change has been observed previously in the case of dilution of a solution of a 43-residue model peptide derived from the N-terminal domain of tropomyosin [37], and has been interpreted as reflecting dissociation of a coiled-coil structure to form isolated helices. In the case of CyaA, the far UV CD data may indicate that CyaA can assume both monomeric and oligomeric forms in solution, depending on the protein concentration. Lee et al. [18] demonstrated the self-association of CyaA monomers, a process that they indicated was dependent on acylation and not calcium, but our spectra of CyaA*, proCyaA and proCyaA* were almost identical to that of native CyaA which might suggest that non-acylated CyaA could also be oligomeric in solution, but this needs to be addressed in future studies.

4. Materials and methods 4.1. Expression and purification of different CyaA forms Escherichia coli BL21/DE3 (F ompT rB mB) was used as the host strain for the production of recombinant CyaA. The construction of plasmids pGW44, pGW44/188 and pGW54 has been described previously [38,39]. Co-expression of pGW44 or pGW44/ 188 with pGW54, generates fully-active, acylated CyaA or an enzymatically-inactive, acylated CyaA (CyaA*) carrying a Leu-Gln di-peptide insertion between codons 188 and 189, respectively. Expression of pGW44 or pGW44/188 alone produces non-acylated protoxins with enzymatic activity (proCyaA) or without enzymatic activity (proCyaA*), respectively. The purification and characterisation of CyaA, CyaA*, proCyaA and proCyaA* have been described elsewhere [40]. Purified preparations were stored at 80  C in 8 M urea, 50 mM Tris–HCl (pH 8.0) and contained <0.1 endotoxin units/ mg protein, determined using a Kinetic-QCLÔ (Biowhittaker) Limulus amoebocyte lysate (LAL) assay.

4.2. Estimation of protein and endotoxin Protein concentrations were determined by the Bradford assay (BioRad) according to the manufacturer’s instructions. Endotoxin was determined using a Kinetic-QCL (Biowhittaker) Limulus Amoebocyte Lysate (LAL) assay.

4.3. Mammalian cell culture The murine J774.2 macrophage cell line (ECACC number 91051511) was grown in DMEM (Gibco) tissue culture medium supplemented with 2 mM L-glutamine (Gibco), 1  antibiotics/ antimycotics (Sigma), and 10% (v/v) foetal bovine serum (FBS) (Gibco).

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4.4. Adenylate cyclase activity This was determined in the presence of 1 mM calmodulin using a rapid conductimetric procedure [41]. Only proCyaA and CyaA were enzymatically active with specific activities of 760  110 and 854  47 mmol cAMP/min/mg protein, respectively. 4.5. Haemolysis The haemolytic activities of the different CyaA forms were measured by incubating 50 ml of serial dilutions of toxin in Hanks HEPES (HH) buffer [150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM D-glucose, 10 mM HEPES, 0.2 M urea, 1 mM CaCl2 (pH 7.4)] with 50 ml of a 0.7% (v/v) sheep erythrocyte suspension in HH buffer, at 37  C for 24 h, as described previously [42]. 4.6. Cytotoxicity assays 4.6.1. Dye reduction (MTT) assay This assay was done using the CellTiter 96Ò kit (Promega) treatment according to the manufacturer’s instructions. J774.2 cells (50 ml of 5  105 cells/ml) were mixed with toxin (50 ml), serially diluted in DMEM tissue culture media supplemented with 10% (v/v) FBS, 2 mM L-glutamine and antibiotics/antimycotics, in 96-well tissue culture plates (Costar) and incubated for 2 h at 37  C in a humidified 5% (v/v) CO2 atmosphere. Percentage cytotoxicity was calculated using the formula: 100  (((sample OD  positive OD)/ (negative OD  positive OD))  100). Cells incubated in the presence or absence of 1% (v/v) Triton-X100 (Sigma) served as a positive and negative controls, respectively. This assay measures the ability of the toxin to inhibit the capacity of living cells to reduce the yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyl tetrazolium bromide) dye to an insoluble purple formazan product. 4.6.2. Lactate dehydrogenase (LDH) release assay This assay was done using the CytoTox 96Ò kit (Promega) according to the manufacturer’s instructions. J774.2 cells, prepared as above, and serial dilutions of toxin were incubated in round-bottomed 96-well plates (Costar) for 2 h at 37  C in a humidified 5% (v/v) CO2 atmosphere. Percentage cytotoxicity, in terms of LDH release, was calculated using the formula: ((sample OD  negative OD)/ (positive OD  negative OD))  100. Cells incubated in the presence or absence of 1% (v/v) Triton-X100 (Sigma) served as positive and negative controls, respectively. This assay is based on the conversion of lactate to pyruvate by LDH released from cells as a measure of loss of cell membrane integrity. 4.6.3. Apoptosis assay This was done using the Apo-ONEÒ Homogeneous caspase 3/7 assay (Promega). J774.2 cells, prepared as above, and serial dilutions of toxin were incubated in 96-well black tissue culture plates (with clear bottom) (Labtech) for 2 h at 37  C in a humidified 5% (v/v) CO2 atmosphere. After addition of 100 ml of substrate and incubation for a further 18 h, fluorescence was recorded over an excitation wavelength range from 465 to 505 nm and over an emission wavelength range from 505 to 555 nm in a spectrofluorimeter (LS 55, Perkin Elmer). Fluorescence was measured in arbitrary relative fluorescence units (RLFU). 4.6.4. Inhibition of the zymosan-stimulated oxidative burst This was assessed in J774.2 cells by chemiluminescence as described previously [43]. Briefly, 100 ml of 2  106 cells/ml were incubated with 100 ml of dilutions of toxin in DMEM for 1 h at 37  C in 5% (v/v) CO2, followed by incubation for 30 min with 100 ml of 1.2 mg/ml zymosan A (Fluka). Chemiluminescence was measured in a luminometer (TD-20/20, Turner Designs) after the addition of

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100 ml of 0.2 mg/ml lucigenin (N,N0 -dimethyl-9,90 -biacridinium dinitrate, Fluka). Cells incubated in the absence of toxin served as the control (0% inhibition). 4.7. Circular dichroism (CD) CD is based on the differential absorbance, of the left and right circularly polarised components of polarised light, by chiral chromophores such as the amide bonds in the regular secondary structural elements of proteins (a-helices, b-sheets, etc.). For these studies, 1 ml of CyaA preparation (at 3–4 mg/ml) in 8 M urea was dialysed twice for 1 h at 4  C against 10 mM Tris–HCl (pH 8.0). Protein concentrations were calculated from the A280 nm values determined using a spectrophotometer (V550, Jasco). The residual urea concentration in the dialysed samples was determined using a refractometer (ABBE 60/70, Bellingham & Stanley) and calculated using a table of refractive indices [44]. The concentration of urea in the samples was lower than the detection limit of this assay (0.58 mM). The AC enzymatic specific activities of CyaA and proCyaA were reduced by w50% in the dialysed samples (data not shown), indicating that some of the protein had aggregated in the absence of high concentrations of urea. 4.7.1. Far UV CD For assessing the effects of calcium on CyaA structure, dialysed samples were diluted to 0.5 mg/ml in 10 mM Tris–HCl (pH 8.0) with or without different concentrations of CaCl2. The blanks were 10 mM Tris–HCl (pH 8.0) plus the corresponding concentration of CaCl2. For assessing the effect of a reduced protein concentration on CyaA structure, dialysed CyaA, diluted to 0.05 mg/ml with 10 mM Tris–HCl (pH 8.0), was incubated with or without 0.3 mM CaCl2. The blank was 10 mM Tris–HCl  0.3 mM CaCl2 (pH 8.0). Spectra were acquired using a spectropolarimeter (J810, Jasco) with 0.02 or 0.2 cm path length cells (Hellma) for CyaA at 0.5 or 0.05 mg/ml, respectively; each spectrum represented the average of 8 scans at a scan rate of 10 nm/min over the range 180–260 nm, with a time constant of 2 s. All samples were stored at 4  C for 24 h before spectra were recorded. The CDSSTR program in Dichroweb [45,46] was used to analyse the far UV CD spectra of CyaA at 0.5 mg/ml over the wavelength range from 185 nm to 240 nm in terms of the secondary structure content. It was found that each of the other procedures such as VARSELC, SELCON and CONTIN in Dichroweb did not give satisfactory analyses for spectra in both the absence and presence of CaCl2. For each sample, CDSSTR fitted both spectra satisfactorily as judged by 2 criteria:- (i) the Normalised Root Mean Square Deviation (NRMSD) values were low, in the range of 0.015–0.064 for all samples, and (ii) the reconstructed spectra using CDSSTR were essentially superimposable on the experimental data over the wavelength range 185–240 nm. 4.7.2. Near UV CD Dialysed samples were diluted to 1.5 mg/ml in 10 mM Tris–HCl (pH 8.0)  1 mM CaCl2 and analysed in a 0.5 cm path length cell (Hellma) over the range 260–320 nm at a scan rate of 10 nm/min, using a 2 s time constant; again 8 scans were averaged. The blank was 10 mM Tris–HCl (pH 8.0)  1 mM CaCl2. All samples were stored at 4  C for 24 h before spectra were recorded. 4.8. Fluorescence Dialysed samples were diluted to 0.1 mg/ml in 10 mM Tris–HCl (pH 8.0)  1 mM CaCl2. The blank was 10 mM Tris–HCl (pH 8.0)  1 mM CaCl2. Spectra were recorded after incubation for 24 h at 4  C in a rectangular quartz cell of internal dimensions 1  0.4 cm (Hellma) over the range 300–400 nm with excitation at 295 nm.

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