Abstract
The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine- and aspartate-rich nonapeptide repeats (residues 1006-1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel beta-helix or beta-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.
Highlights
The adenylate cyclase toxin (CyaA)2 is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough (1–3)
CyaA is constructed in a modular fashion; the calmodulinactivated catalytic domain is located in the 400-amino-proximal residues, whereas the C-terminal moiety is endowed with hemolytic activity (4, 5, 15, 16), which results from its ability to form cation-selective channels in membranes (17, 18)
The RTX motif is a structural motif found in a number of cytolysins produced by Gram-negative bacteria as well as in several other potential bacterial pathogenic proteins (20, 21)
Summary
Construction of the CyaA-derived Proteins—DNA manipulations were performed according to standard protocols (34) in the Escherichia coli XL1-Blue strain (Stratagene, Amsterdam, The Netherlands) as host cells. The plasmid pTRCyaA⌬1– 1006⌬1490 –1706, coding for the truncated CyaA RTX domain (CyaA1006–1490), is a derivative of pTRCyaA⌬1–1006, in which the sequence coding for the last block of repeated sequences (between the SmaI and BspE1 sites) has been replaced by an appropriate synthetic double-stranded oligonucleotides encoding a termination sequence. The plasmid pTRCyaA⌬1491–1706 encoding the acylated truncated protein (CyaA1–1490) is a derivative of pTRCAG (37), in which the molecule C-terminal DNA sequence (encompassing the RTX last block of repeated sequences and the secretion signal, located between the SmaI and BamH1 sites) was deleted and replaced by an appropriate synthetic double-stranded oligonucleotide encoding an amino acid termination sequence. EGTA was added to a final concentration of 5 mM to record the fluorescence spectra of calcium-free proteins. Proteolysis was initiated by the addition of 3 nM (final concentration) TPCK-treated trypsin, and the kinetics were recorded for 2000 s
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