The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding. However, helix-breaking proline substitutions in these segments uncovered a structural role of the Y632, Y658, Y725 and Y738 residues in AC domain delivery and pore formation by CyaA. Substitutions of Y940 of the fifth motif, conserved in the acylated domains of related RTX toxins, did not impact on fatty-acylation of CyaA by CyaC and the CyaA-Y940F mutant was intact for toxin activities on erythrocytes and myeloid cells. However, the Y940A or Y940P substitutions disrupted the capacity of CyaA to insert into artificial lipid bilayers or target cell membranes. The aromatic ring of tyrosine 940 side chain thus appears to play a key structural role in molecular interactions that initiate CyaA penetration into target membranes.

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 Microbiology Reviews. 2010;34:1076–1112. doi: 10.1111/j.1574-6976.2010.00231.x. PubMed DOI PMC

Masin J, Osicka R, Bumba L, Sebo P. Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme. Pathog Dis. 2015;73 doi: 10.1093/femspd/ftv075. PubMed DOI PMC

Masin J, et al. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin. Sci Rep. 2016;6 doi: 10.1038/srep29137. PubMed DOI PMC

Subrini O, et al. Characterization of a membrane-active peptide from the Bordetella pertussis CyaA toxin. J Biol Chem. 2013;288:32585–32598. doi: 10.1074/jbc.M113.508838. 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. Molecular Cell. 2016;62:47–62. doi: 10.1016/j.molcel.2016.03.018. PubMed DOI

Bellalou J, Sakamoto H, Ladant D, Geoffroy C, Ullmann A. Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase. Infect Immun. 1990;58:3242–3247. PubMed PMC

Wolff J, Cook GH, Goldhammer AR, Berkowitz SA. Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci USA. 1980;77:3841–3844. doi: 10.1073/pnas.77.7.3841. PubMed DOI PMC

Fiser R, et al. Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores. PLoS Pathog. 2012;8 doi: 10.1371/journal.ppat.1002580. PubMed DOI PMC

Gray M, Szabo G, Otero AS, Gray L, Hewlett E. Distinct mechanisms for K+ efflux, intoxication, and hemolysis by Bordetella pertussis AC toxin. J Biol Chem. 1998;273:18260–18267. doi: 10.1074/jbc.273.29.18260. PubMed DOI

Vojtova-Vodolanova J, et al. Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin. Faseb J. 2009;23:2831–2843. doi: 10.1096/fj.09-131250. PubMed DOI

Wald T, et al. Quantification of potassium levels in cells treated with Bordetella adenylate cyclase toxin. Anal Biochem. 2014;450:57–62. doi: 10.1016/j.ab.2013.10.039. PubMed DOI

Basar T, Havlicek V, Bezouskova S, Hackett M, Sebo P. Acylation of lysine 983 is sufficient for toxin activity of Bordetella pertussis adenylate cyclase. Substitutions of alanine 140 modulate acylation site selectivity of the toxin acyltransferase CyaC. J Biol Chem. 2001;276:348–354. doi: 10.1074/jbc.M006463200. PubMed DOI

Hackett M, Guo L, Shabanowitz J, Hunt DF, Hewlett EL. Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science. 1994;266:433–435. doi: 10.1126/science.7939682. PubMed DOI

Hackett M, et al. Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in Escherichia coli. J Biol Chem. 1995;270:20250–20253. doi: 10.1074/jbc.270.35.20250. PubMed DOI

Masin J, et al. Acylation of lysine 860 allows tight binding and cytotoxicity of Bordetella adenylate cyclase on CD11b-expressing cells. Biochemistry. 2005;44:12759–12766. doi: 10.1021/bi050459b. PubMed DOI

Basler M, Masin J, Osicka R, Sebo P. Pore-forming and enzymatic activities of Bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes. Infect Immun. 2006;74:2207–2214. doi: 10.1128/IAI.74.4.2207-2214.2006. PubMed DOI PMC

Cerny O, Anderson KE, Stephens LR, Hawkins PT, Sebo P. cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity. Journal of Immunology. 2017;198:1285–1296. doi: 10.4049/jimmunol.1601309. PubMed DOI

Cerny O, et al. Bordetella pertussis Adenylate Cyclase Toxin Blocks Induction of Bactericidal Nitric Oxide in Macrophages through cAMP-Dependent Activation of the SHP-1 Phosphatase. Journal of Immunology. 2015;194:4901–4913. doi: 10.4049/jimmunol.1402941. PubMed DOI

Confer DL, Eaton JW. Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science. 1982;217:948–950. doi: 10.1126/science.6287574. PubMed DOI

Hewlett EL, Donato GM, Gray MC. Macrophage cytotoxicity produced by adenylate cyclase toxin from Bordetella pertussis: more than just making cyclic AMP! Mol Microbiol. 2006;59:447–459. doi: 10.1111/j.1365-2958.2005.04958.x. PubMed DOI

Kamanova J, et al. Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling. Journal of immunology. 2008;181:5587–5597. doi: 10.4049/jimmunol.181.8.5587. PubMed DOI

Ahmad JN, et al. cAMP signalling of Bordetella adenylate cyclase toxin through the SHP-1 phosphatase activates the BimEL-Bax pro-apoptotic cascade in phagocytes. Cellular Microbiology. 2016;18:384–398. doi: 10.1111/cmi.12519. PubMed DOI

Pearson RD, Symes P, Conboy M, Weiss AA, Hewlett EL. Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin. Journal of Immunology. 1987;139:2749–2754. PubMed

Guermonprez P, et al. The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18) J Exp Med. 2001;193:1035–1044. doi: 10.1084/jem.193.9.1035. PubMed DOI PMC

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

Morova, J., Osicka, R., Masin, J. & Sebo, P. RTX cytotoxins recognize {beta}2 integrin receptors through N-linked oligosaccharides. Proc Natl Acad Sci USA (2008). PubMed PMC

Hasan S, et al. Interaction of Bordetella adenylate cyclase toxin with complement receptor 3 involves multivalent glycan binding. FEBS Lett. 2015;589:374–379. doi: 10.1016/j.febslet.2014.12.023. PubMed DOI

Eby JC, et al. Selective translocation of the Bordetella pertussis adenylate cyclase toxin across the basolateral membranes of polarized epithelial cells. J Biol Chem. 2010;285:10662–10670. doi: 10.1074/jbc.M109.089219. PubMed DOI PMC

Gordon VM, Leppla SH, Hewlett EL. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin. Infect Immun. 1988;56:1066–1069. PubMed PMC

Martin C, et al. Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family. J Bacteriol. 2004;186:3760–3765. doi: 10.1128/JB.186.12.3760-3765.2004. PubMed DOI PMC

Masin J, Konopasek I, Svobodova J, Sebo P. Different structural requirements for adenylate cyclase toxin interactions with erythrocyte and liposome membranes. Biochimica et Biophysica Acta. 2004;1660:144–154. doi: 10.1016/j.bbamem.2003.11.008. PubMed DOI

Basler M, et al. Segments crucial for membrane translocation and pore-forming activity of Bordetella adenylate cyclase toxin. J Biol Chem. 2007;282:12419–12429. doi: 10.1074/jbc.M611226200. PubMed DOI

Gray MC, et al. Translocation-specific conformation of adenylate cyclase toxin from Bordetella pertussis inhibits toxin-mediated hemolysis. J Bacteriol. 2001;183:5904–5910. doi: 10.1128/JB.183.20.5904-5910.2001. PubMed DOI PMC

Osickova A, et al. Adenylate cyclase toxin translocates across target cell membrane without forming a pore. Mol Microbiol. 2010;75:1550–1562. doi: 10.1111/j.1365-2958.2010.07077.x. 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. 1999;274:37644–37650. PubMed

Otero AS, Yi XB, Gray MC, Szabo G, Hewlett EL. Membrane depolarization prevents cell invasion by Bordetella pertussis adenylate cyclase toxin. J Biol Chem. 1995;270:9695–9697. doi: 10.1074/jbc.270.17.9695. PubMed DOI

Veneziano R, et al. Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer. Proc Natl Acad Sci USA. 2013;110:20473–20478. doi: 10.1073/pnas.1312975110. PubMed DOI PMC

Fiser R, et al. Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities. J Biol Chem. 2007;282:2808–2820. doi: 10.1074/jbc.M609979200. PubMed DOI

Bumba L, Masin J, Fiser R, Sebo P. Bordetella adenylate cyclase toxin mobilizes its beta2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. PLoS Pathog. 2010;6 doi: 10.1371/journal.ppat.1000901. PubMed DOI PMC

Vojtova J, Kofronova O, Sebo P, Benada O. Bordetella adenylate cyclase toxin induces a cascade of morphological changes of sheep erythrocytes and localizes into clusters in erythrocyte membranes. Microsc Res Tech. 2006;69:119–129. doi: 10.1002/jemt.20277. PubMed DOI

Brown AC, et al. Aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition/amino acid consensus site for membrane association. J Biol Chem. 2013;288:23607–23621. doi: 10.1074/jbc.M113.486654. PubMed DOI PMC

Brown AC, Koufos E, Balashova NV, Boesze-Battaglia K, Lally ET. Inhibition of LtxA toxicity by blocking cholesterol binding with peptides. Mol Oral Microbiol. 2015;31:94–105. doi: 10.1111/omi.12133. PubMed DOI PMC

Vazquez RF, et al. Novel evidence for the specific interaction between cholesterol and alpha-haemolysin of Escherichia coli. Biochem J. 2014;458:481–489. doi: 10.1042/BJ20131432. PubMed DOI

Li H, Papadopoulos V. Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology. 1998;139:4991–4997. PubMed

Epand RF, et al. Juxtamembrane protein segments that contribute to recruitment of cholesterol into domains. Biochemistry. 2006;45:6105–6114. doi: 10.1021/bi060245+. PubMed DOI PMC

Jamin N, et al. Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor. Mol Endocrinol. 2005;19:588–594. doi: 10.1210/me.2004-0308. PubMed DOI

Epand RM. Cholesterol and the interaction of proteins with membrane domains. Progress in Lipid Research. 2006;45:279–294. doi: 10.1016/j.plipres.2006.02.001. PubMed DOI

Rosendal S, et al. Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae. Am J Vet Res. 1988;49:1053–1058. PubMed

Osicka R, et al. Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites. Infect Immun. 2000;68:247–256. doi: 10.1128/IAI.68.1.247-256.2000. PubMed DOI PMC

Powthongchin B, Angsuthanasombat C. Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment. Arch Microbiol. 2009;191:1–9. doi: 10.1007/s00203-008-0421-3. PubMed DOI

Basar T, et al. The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independently of its acylation status. J Biol Chem. 1999;274:10777–10783. doi: 10.1074/jbc.274.16.10777. PubMed DOI

Koufos E, Chang EH, Rasti ES, Krueger E, Brown AC. Use of a Cholesterol Recognition Amino Acid Consensus Peptide To Inhibit Binding of a Bacterial Toxin to Cholesterol. Biochemistry. 2016;55:4787–4797. doi: 10.1021/acs.biochem.6b00430. PubMed DOI PMC

Levitan I, Singh DK, Rosenhouse-Dantsker A. Cholesterol binding to ion channels. Front Physiol. 2014;5 doi: 10.3389/fphys.2014.00065. PubMed DOI PMC

Fantini J, Barrantes FJ. How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains. Front Physiol. 2013;4 PubMed PMC

Fantini, J., Di Scala, C., Baier, C. J. & Barrantes, F. J. Molecular mechanisms of protein-cholesterol interactions in plasma membranes: Functional distinction between topological (tilted) and consensus (CARC/CRAC) domains. Chem Phys Lipids (2016). PubMed

Ahmed SN, Brown DA, London E. On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry. 1997;36:10944–10953. doi: 10.1021/bi971167g. PubMed DOI

Silvius JR, del Giudice D, Lafleur M. Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length. Biochemistry. 1996;35:15198–15208. doi: 10.1021/bi9615506. PubMed DOI

Juntapremjit S, et al. Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation. Toxicon. 2015;106:14–19. doi: 10.1016/j.toxicon.2015.09.006. PubMed DOI

Karst JC, et al. Calcium, acylation, and molecular confinement favor folding of Bordetella pertussis adenylate cyclase CyaA toxin into a monomeric and cytotoxic form. J Biol Chem. 2014;289:30702–30716. doi: 10.1074/jbc.M114.580852. PubMed DOI PMC

Meetum K, Imtong C, Katzenmeier G, Angsuthanasombat C. Acylation of the Bordetella pertussis CyaA-hemolysin: Functional implications for efficient membrane insertion and pore formation. Biochimica et Biophysica Acta. 2017;1859:312–318. doi: 10.1016/j.bbamem.2016.12.011. PubMed DOI

Masin J, et al. Differences in purinergic amplification of osmotic cell lysis by the pore-forming RTX toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the role of pore size. Infect Immun. 2013;81:4571–4582. doi: 10.1128/IAI.00711-13. PubMed DOI PMC

Ladant D. Interaction of Bordetella pertussis adenylate cyclase with calmodulin. Identification of two separated calmodulin-binding domains. J Biol Chem. 1988;263:2612–2618. PubMed

Geourjon C, Deleage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Computer applications in the biosciences:CABIOS. 1995;11:681–684. PubMed

Eisenberg D, Schwarz E, Komaromy M, Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984;179:125–142. doi: 10.1016/0022-2836(84)90309-7. PubMed DOI

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

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

Colicin U from Shigella boydii Forms Voltage-Dependent Pores

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

Cytotoxic activity of Kingella kingae RtxA toxin depends on post-translational acylation of lysine residues and cholesterol binding

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

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