The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The 'AC to Hly-linking segment' thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins.

Faculty of Science Charles University Prague Czech Republic

Institute of Microbiology of the CAS v v i Prague Czech Republic

Zobrazit více v PubMed

Linhartova I. et al. RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol Rev 34, 1076–1112, 10.1111/j.1574-6976.2010.00231.x (2010). PubMed DOI PMC

Guermonprez P. et al. The adenylate cyclase toxin of PubMed PMC

Osicka R. et al. adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3. Elife 4, 10.7554/eLife.10766 (2015). PubMed DOI PMC

Wald T. et al. Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of PubMed DOI

Gordon V. M., Leppla S. H. & Hewlett E. L. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of PubMed PMC

Rogel A. & Hanski E. Distinct steps in the penetration of adenylate cyclase toxin of PubMed

Glaser P., Sakamoto H., Bellalou J., Ullmann A. & Danchin A. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of PubMed PMC

Ahmad J. N. et al. cAMP signalling of PubMed DOI

Bellalou J., Sakamoto H., Ladant D., Geoffroy C. & Ullmann A. Deletions affecting hemolytic and toxin activities of PubMed PMC

Wolff J., Cook G. H., Goldhammer A. R. & Berkowitz S. A. Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci USA 77, 3841–3844 (1980). PubMed PMC

Holubova J. et al. Delivery of large heterologous polypeptides across the cytoplasmic membrane of antigen-presenting cells by the PubMed DOI PMC

Benz R., Maier E., Ladant D., Ullmann A. & Sebo P. Adenylate cyclase toxin (CyaA) of PubMed

Dunne A. et al. Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis. J Immunol 185, 1711–1719, 10.4049/jimmunol.1000105 (2010). PubMed DOI

Gray M., Szabo G., Otero A. S., Gray L. & Hewlett E. Distinct mechanisms for K+ efflux, intoxication, and hemolysis by PubMed

Wald T. et al. Quantification of potassium levels in cells treated with PubMed DOI

Barry E. M. et al. PubMed PMC

Basar T. et al. The conserved lysine 860 in the additional fatty-acylation site of PubMed

Hackett M., Guo L., Shabanowitz J., Hunt D. F. & Hewlett E. L. Internal lysine palmitoylation in adenylate cyclase toxin from PubMed

Hackett M. et al. Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in PubMed

Masin J. et al. Acylation of lysine 860 allows tight binding and cytotoxicity of PubMed

Baumann U., Wu S., Flaherty K. M. & McKay D. B. Three-dimensional structure of the alkaline protease of PubMed PMC

Knapp O. et al. Channel formation in model membranes by the adenylate cyclase toxin of PubMed

Rose T., Sebo P., Bellalou J. & Ladant D. Interaction of calcium with PubMed

Basler M. et al. Segments Crucial for Membrane Translocation and Pore-forming Activity of PubMed

Osickova A. et al. Adenylate cyclase toxin translocates across target cell membrane without forming a pore. Mol Microbiol 75, 1550–1562, 10.1111/j.1365-2958.2010.07077.x (2010). PubMed DOI

Osickova A., Osicka R., Maier E., Benz R. & Sebo P. An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels. J Biol Chem 274, 37644–37650 (1999). PubMed

Gray M. C. et al. Translocation-specific conformation of adenylate cyclase toxin from PubMed DOI PMC

Otero A. S., Yi X. B., Gray M. C., Szabo G. & Hewlett E. L. Membrane depolarization prevents cell invasion by PubMed

Veneziano R. et al. PubMed DOI PMC

Guo Q. et al. Structural basis for the interaction of PubMed DOI PMC

Chenal A., Guijarro J. I., Raynal B., Delepierre M. & Ladant D. RTX calcium binding motifs are intrinsically disordered in the absence of calcium: implication for protein secretion. J Biol Chem 284, 1781–1789, 10.1074/jbc.M807312200 (2009). PubMed DOI

Osicka R. et al. Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by PubMed PMC

Sotomayor Perez A. C. et al. Characterization of the regions involved in the calcium-induced folding of the intrinsically disordered RTX motifs from the PubMed DOI

Juntapremjit S. et al. Functional importance of the Gly cluster in transmembrane helix 2 of the PubMed DOI

O’Brien D. P. et al. Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion. Sci Rep 5, 14223, 10.1038/srep14223 (2015). PubMed DOI PMC

Bumba L. et al. Calcium-Driven Folding of RTX Domain beta-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts. Mol Cell 62, 47–62, 10.1016/j.molcel.2016.03.018 (2016). PubMed DOI

Shur O. & Banta S. Rearranging and concatenating a native RTX domain to understand sequence modularity. Protein Eng Des Sel 26, 171–180, 10.1093/protein/gzs092 (2013). PubMed DOI

Sotomayor-Perez A. C., Subrini O., Hessel A., Ladant D. & Chenal A. Molecular crowding stabilizes both the intrinsically disordered calcium-free state and the folded calcium-bound state of a repeat in toxin (RTX) protein. J Am Chem Soc 135, 11929–11934, 10.1021/ja404790f (2013). PubMed DOI

Zhang L., Morrison A. J. & Thibodeau P. H. Interdomain Contacts and the Stability of Serralysin Protease from PubMed PMC

Karst J. C. et al. Identification of a region that assists membrane insertion and translocation of the catalytic domain of PubMed DOI PMC

Subrini O. et al. Characterization of a Membrane-Active Peptide from the PubMed DOI PMC

Masin J. et al. Differences in purinergic amplification of osmotic cell lysis by the pore-forming RTX toxins PubMed DOI PMC

Geourjon C. & Deleage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11, 681–684 (1995). PubMed

Basler M., Masin J., Osicka R. & Sebo P. Pore-forming and enzymatic activities of PubMed PMC

Hewlett E. L., Donato G. M. & Gray M. C. Macrophage cytotoxicity produced by adenylate cyclase toxin from PubMed DOI

Ziolo K. J. et al. PubMed DOI PMC

Karst J. C. et al. Calcium, acylation, and molecular confinement favor folding of PubMed DOI PMC

Masin J., Osicka R., Bumba L. & Sebo P. PubMed DOI PMC

Fiser R. et al. Third activity of PubMed DOI

Karimova G. et al. Charge-dependent translocation of PubMed PMC

Hervas-Stubbs S. et al. High frequency of CD4+ T cells specific for the TB10.4 protein correlates with protection against PubMed DOI PMC

Tartz S. et al. Immunization with a circumsporozoite epitope fused to PubMed DOI PMC

Fujiki Y., Hubbard A. L., Fowler S. & Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93, 97–102 (1982). PubMed PMC

Masin J., Konopasek I., Svobodova J. & Sebo P. Different structural requirements for adenylate cyclase toxin interactions with erythrocyte and liposome membranes. Biochim Biophys Acta 1660, 144–154 (2004). PubMed

Kumar S. & Nussinov R. Salt bridge stability in monomeric proteins. J Mol Biol 293, 1241–1255, (1999). PubMed

Musafia B., Buchner V. & Arad D. Complex salt bridges in proteins: statistical analysis of structure and function. J Mol Biol 254, 761–770, 10.1006/jmbi.1995.0653 (1995). PubMed DOI

Rogel A., Meller R. & Hanski E. Adenylate cyclase toxin from PubMed

Vojtova J., Kamanova J. & Sebo P. PubMed DOI

Svedova M. et al. Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8 and CD4 T cells. Immunol Cell Biol, 10.1038/icb.2015.87 (2016). PubMed DOI

Kuban V., Novacek J., Bumba L. & Zidek L. NMR assignment of intrinsically disordered self-processing module of the FrpC protein of PubMed DOI

Ladant D. Interaction of PubMed

Iwaki M., Ullmann A. & Sebo P. Identification by PubMed

Micsonai A. et al. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci USA 112, E3095–3103, 10.1073/pnas.1500851112 (2015). PubMed DOI PMC

A conserved tryptophan in the acylated segment of RTX toxins controls their β2 integrin-independent cell penetration

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

Selective Enhancement of the Cell-Permeabilizing Activity of Adenylate Cyclase Toxin Does Not Increase Virulence of Bordetella pertussis

Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin

Acyltransferase-mediated selection of the length of the fatty acyl chain and of the acylation site governs activation of bacterial RTX toxins

Retargeting from the CR3 to the LFA-1 receptor uncovers the adenylyl cyclase enzyme-translocating segment of Bordetella adenylate cyclase toxin

Colicin U from Shigella boydii Forms Voltage-Dependent Pores

Rapid Purification of Endotoxin-Free RTX Toxins

Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin

Bordetella Pertussis Adenylate Cyclase Toxin Does Not Possess a Phospholipase A Activity; Serine 606 and Aspartate 1079 Residues Are Not Involved in Target Cell Delivery of the Adenylyl Cyclase Enzyme Domain

Phosphoproteomics of cAMP signaling of Bordetella adenylate cyclase toxin in mouse dendritic cells

Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes

The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin