Cytidylyl and uridylyl cyclase activity of bacillus anthracis edema factor and Bordetella pertussis CyaA

Abstract

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers for numerous mammalian cell functions. The natural occurrence and synthesis of a third cyclic nucleotide (cNMP), cyclic cytidine 3',5'-monophosphate (cCMP), is a matter of controversy, and almost nothing is known about cyclic uridine 3',5'-monophosphate (cUMP). Bacillus anthracis and Bordetella pertussis secrete the adenylyl cyclase (AC) toxins edema factor (EF) and CyaA, respectively, weakening immune responses and facilitating bacterial proliferation. A cell-permeable cCMP analogue inhibits human neutrophil superoxide production. Here, we report that EF and CyaA also possess cytidylyl cyclase (CC) and uridylyl cyclase (UC) activity. CC and UC activity was determined by a radiometric assay, using [alpha-(32)P]CTP and [alpha-(32)P]UTP as substrates, respectively, and by a high-performance liquid chromatography method. The identity of cNMPs was confirmed by mass spectrometry. On the basis of available crystal structures, we developed a model illustrating conversion of CTP to cCMP by bacterial toxins. In conclusion, we have shown both EF and CyaA have a rather broad substrate specificity and exhibit cytidylyl and uridylyl cyclase activity. Both cCMP and cUMP may contribute to toxin actions.

Fig. 1. Signals obtained using the isotopic EF cytidylyl cyclase activity assay

Reactions were carried out for 20 min at 37 °C as described in “Materials and Methods”. A: Time course experiment using 20 pM EF. Reaction mixtures contained the following components to yield the given final concentrations: 100 mM KCl, 5 mM Mn²⁺, 10 µM free Ca²⁺, 100 µM EGTA, 10 µM CTP, [α–³²P]CTP (0.4 µCi/tube), 100 nM CaM. B: Signal dependence on EF protein concentration. Reaction mixtures contained EF at various concentrations, the components listed under A and additionally 100 µM cAMP. C: Dependence of CC activity on pH. Reaction mixtures contained 80 pM EF and the components listed under B. Buffering systems were 30 mM sodium acetate·HCl (pH 5.0 and pH 6.0), 30 mM Hepes·NaOH (pH 7.4) and 30 mM Tris·HCl (pH 8.0 and pH 9.0). D: Activation of CC activity by CaM. Reaction mixtures contained 5 mM Mn²⁺, 10 µM free Ca²⁺, 100 µM EGTA, 10 µM CTP, [α–³²P]CTP (0.4 µCi/tube), 40 pM EF and CaM at various concentrations. Data shown are means ± SD of representative experiments performed in duplicates; similar results were obtained in at least 3 independent experiments.

Fig. 2. HPLC chromatograms of nucleotide mixtures and eluates from solid phase extraction (SPE) using alumina

SPE and HPLC analytics were performed as described in “Materials and Methods”. A: Chromatogram of a mixture of 15 µM CMP, CDP, CTP and 3′:5′-cCMP prior to SPE (dashed line) and chromatogram after SPE (solid line). B: Chromatogram of a mixture of 15 µM UMP, UDP, UTP and 3′:5′-cUMP prior to SPE (dashed line) and chromatogram after SPE (solid line). C: Chromatograms of 15 µM 2′:3′-cCMP and 3′:5′-cCMP prior to SPE (dashed line) and chromatogram after SPE (solid line). The same results were obtained in at least 3 independent experiments.

Fig. 3. Michaelis-Menten kinetics of CC and UC activity of EF and CyaA using the isotopic NC activity

Reactions were carried out as described in “Materials and Methods”. Reaction mixtures contained the following components to yield the given final concentrations: 100 mM KCl, 10 µM free Ca²⁺, 100 µM EGTA, [α–³²P]CTP (0.8 µCi/tube) or [α–³²P]UTP (0.8 µCi/tube), 100 nM CaM and non-labeled cyclic nucleotides as described in “Materials and Methods”. CTP/Mn²⁺ or UTP/Mn²⁺ (1 µM to 1 mM) plus 5 mM of free Mn²⁺ were added. The final protein concentration was 40 pM EF or CyaA in case of CC activity (A and B) and 400 pM EF or CyaA in case of UC activity (C and D). Reactions were carried out at 37 °C for 20 min in case of CC activity and for 30 min in case of UC activity. Data shown are means ± SD of representative experiments performed in duplicates; similar results were obtained in at least 5 independent experiments.

Fig. 4. Linear correlation of the K_i values of AC and CC inhibition by MANT-nucleotides

Data shown in Tab. 2 provide the basis for the correlations. Dashed lines represent 95% confidence intervals of the linear regression lines. A: Inhibition of EF; slope, 1.224 ± 0.1604; r², 0.9510; p, 0.0047; significant. B: Inhibition of CyaA; slope, 0.9847 ± 0.1226; r², 0.9555; p, 0.0040; significant. Linear regression analysis was performed using the Prism 4.02 software (Graphpad, San Diego, CA).

Fig. 5. HPLC chromatograms of reaction mixtures from the non-isotopic NC assay

The non-isotopic NC assay, sample preparation and HPLC analytics were carried out as described in “Materials and Methods”. Reaction mixtures contained 5 mM Mn²⁺, 5 µM Ca²⁺ and 100 µM CTP (A), UTP (B) or ITP (C). Protein concentrations were 20 nM EF and 20 nM CaM (A), 120 nM EF and 120 nM CaM (B) and 300 nM EF and 300 nM CaM (C). Samples were withdrawn at the indicated reaction times (colored solid lines). Inosine (20 µM) was added as internal standard (IS); cCMP, cUMP and cIMP were also added as standard substances (black, dotted lines). In order to prevent overlapping of the lines, the chromatograms of the standard substances were moved vertically. D: Chromatograms of reaction samples containing 20 nM EF, 20 nM CaM and 100 µM CTP after 60 min reaction time. Results for active enzyme (solid line) and heat-inactivated enzyme (dotted line). Similar results were obtained in at least 3 independent experiments.

Fig. 6. Turnover of NTPs to cNMPs by EF using the non-isotopic NC assay

Nucleotide concentrations were calculated by evaluating the chromatograms shown in Fig. 5. CTP (A), UTP (B) or ITP (C), 100 µM each, were converted to the corresponding cyclic nucleotides by the indicated concentrations of EF and equivalent concentrations of CaM ensuring 1:1 stoichiometry of EF and CaM. Experiments were performed as described in “Materials and Methods”. Similar results were obtained in at least 3 independent experiments.

Fig. 7. Verification of the identities of the cyclic nucleotides formed in the non-isotopic NC assay by mass spectrometry

HPLC-MS analytics were performed as described in “Materials and Methods”. Shown are fragmentation patterns of the standard cyclic nucleotides, chromatograms from standard cyclic nucleotides and chromatograms from reaction samples for cCMP (A–C), cUMP (D–F) and cIMP (G–I). Similar results were obtained in at least 3 independent experiments.

Fig. 8. Suggested interaction of nucleotides with EF and CyaA

The minimized models are based on the crystal structures of EF in complex with 3'-deoxy-ATP (28) and CyaA in complex with PMEApp (16), respectively. Colors of atoms, unless otherwise indicated: P – orange, O – red, N – blue, C, H –grey, Mg²⁺ – purple spheres. A, Interaction of EF with ATP. B, Interaction of EF with CTP. C, Alignment of the nucleotide binding sites of EF and CyaA in complex with CTP (represented as MOLCAD surfaces, bound to EF – black lines, bound to CyaA – grey opaque). Enzyme models: cylinders – helices, ribbons – β-sheets, tubes – loops, EF domain C_A – green, domain C_B – greenblue, switch B – yellow, switch C – blue, CyaA domain C_A – pink, domain B – purple, switch C_B – orange. D, Interaction of CTP with CyaA. In panels A, B and D, the side chains of the amino acids of the binding sites are drawn as sticks and labeled. Colors of C atoms and Cα-traces: domain A – green, domain B – greenblue, switch B – yellow. The backbone oxygen atoms suggested to form hydrogen bonds with the amino groups of ATP and CTP are marked as balls.