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Abstract
Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins.
Highlights
Tandem repeats that are characteristic of a large family of bacterial cytolysins known as RTX (Repeat-in-ToXin) toxins[22,23]
The holo-CyaA monomers (hCyaAm) refolded in the presence of 2 mM calcium was first equilibrated by G25 chromatography in buffer A (20 mM Hepes, 150 mM NaCl, pH 7.4) containing various calcium concentrations (2, 0.5, or 0.2 mM CaCl2) or without supplemented calcium – in that latter case, the contaminating levels of free calcium in the buffers were in the range of a few μM
The data show that hCyaAm in the presence of 0.2 to 2 mM calcium is very stable at 25 °C, as the protein largely remains in a monomeric form, even after 72 hours of incubation at room temperature
Summary
Tandem repeats that are characteristic of a large family of bacterial cytolysins known as RTX (Repeat-in-ToXin) toxins[22,23]. Toxin refolding into a urea-free, holo-CyaA monomeric and functional state (hCyaAm), was critically dependent upon molecular confinement, the presence of calcium and post-translational acylation[20]. This procedure of monomeric toxin refolding does not prevent, the formation of a population of multimers of CyaA (M-CyaA). The structure and functions of the monomeric toxin can be preserved on a short time basis (a few dozen minutes) even in a milieu completely depleted of calcium These results suggest that within hCyaAm, the RTX-containing RD domain is somehow stabilized in a high-affinity calcium-binding state by the N-terminal region (1–1000) of CyaA. CAMP accumulation in cell exposed to hCyaAm was observed only in the presence of sub-millimolar free calcium concentrations (>0.1 mM) in the milieu, indicating that calcium ions are actively involved in the translocation process of the CyaA toxin catalytic domain
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