Abstract
Bordetella pertussis, the causative agent of whooping cough, secretes among other virulence factors an adenylate cyclase (AC) toxin that is able to enter into eukaryotic cells where it is activated upon binding to endogenous calmodulin (CaM) and synthesizes supraphysiological cAMP levels. In vivo, the AC toxin, through its specific interaction with the CD11b/CD18 integrin, primarily targets phagocytic cells such as neutrophils and macrophages. Because neutrophil priming and activation result in the production of reactive oxygen species that may cause intracellular oxidation, we have examined the biological consequences of the oxidation of CaM methionines upon its interaction with AC. We show here that the interaction of CaM with AC is dependent on the reduced state of methionines, because oxidation of all methionine residues of CaM dramatically decreases its affinity for AC. Peptide methionine sulfoxide reductases A (MsrA) and B (MsrB) were able to partially reduce the oxidized CaM, and these partially "repaired" forms could interact with AC nearly as efficiently as the native protein. We further showed that the CaM.AC complex is resistant to oxidation with tert-butylhydroperoxide, and we identified methionine residues 109, 124, and 145 as critical for binding to AC. The resistance of the AC.CaM complex to oxidation and the ability of AC to be efficiently activated by partially oxidized CaM molecules should allow the toxin to exert its cytotoxic effects on activated neutrophils and contribute to the host colonization.
Highlights
Proteins are sensitive to reactive oxygen species that lead to the oxidation of certain amino acid residues
1 The abbreviations used are: CaM, calmodulin; MetO, methionine sulfoxide; AC, adenylate cyclase; methionine sulfoxide reductases A (MsrA), peptide methionine sulfoxide reductase A; MsrB, peptide methionine sulfoxide reductase B; CaM: native (CaMN), native CaM; CaMox, oxidized CaM; CaM/MsrA, oxidized calmodulin reduced with MsrA; CaM/MsrB, oxidized calmodulin reduced with MsrB; CaM/MsrA ϩ MsrB, oxidized calmodulin reduced with MsrA and MsrB; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; nanoESI-Q-TOF, nanoelectrospray ionization quadrupole time-of-flight; LC-MS/MS, liquid chromatography/tandem mass spectrometry; tBHP, tert-butylhydroperoxide; Mes, 4-morpholineethanesulfonic acid; DTT, dithiothreitol; NOS, nitric-oxide synthase
To study the influence of calmodulin (CaM) oxidation on interaction between mammalian CaM and B. pertussis adenylate cyclase (AC), we analyzed the interaction of a truncated form of B. pertussis AC, encompassing the residues 1–384 of wild-type toxin, with various forms of CaM: native (CaMN), oxidized (CaMox) and CaMox reduced by treatment with either MsrA (CaM/MsrA), MsrB (CaM/MsrB), or both MsrA and MsrB (CaM/MsrA ϩ MsrB)
Summary
CaM, calmodulin; MetO, methionine sulfoxide; AC, adenylate cyclase; MsrA, peptide methionine sulfoxide reductase A; MsrB, peptide methionine sulfoxide reductase B; CaMN, native CaM; CaMox, oxidized CaM; CaM/MsrA, oxidized calmodulin reduced with MsrA; CaM/MsrB, oxidized calmodulin reduced with MsrB; CaM/MsrA ϩ MsrB, oxidized calmodulin reduced with MsrA and MsrB; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; nanoESI-Q-TOF, nanoelectrospray ionization quadrupole time-of-flight; LC-MS/MS, liquid chromatography/tandem mass spectrometry; tBHP, tert-butylhydroperoxide; Mes, 4-morpholineethanesulfonic acid; DTT, dithiothreitol; NOS, nitric-oxide synthase. After treatment with either MsrA or MsrB, oxidized CaM was partially “repaired,” exhibiting a maximum of seven reduced methionines out of a total of nine This partially repaired CaM could interact with AC almost as efficiently as the native protein as monitored by binding and activity assays. The ability of AC to be fully activated by partially oxidized CaM molecules, as documented here, might contribute to the pathophysiological role of this toxin by allowing it to exert its cytotoxic effects in activated neutrophils
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