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

Jakub Novák,Peter Šebo,Ondřej Černý,Irena Linhartová,Marek Link,Ivo Fabrik,Jiří Stulík

doi:10.1038/s41598-017-14501-x

Abstract

The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the αMβ2 integrin (CD11b/CD18, CR3 or Mac-1). CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.

Highlights

The Gram-negative coccobacillus Bordetella pertussis excels in sophistication of its immunomodulatory action
We report here the first phosphoproteome-wide analysis of the signaling impact of the cyclic adenosine monophosphate (cAMP)-elevating activity of B. pertussis adenylate cyclase toxin in primary myeloid cells
The adenylyl cyclase (AC) domain of the CyaA toxin binds calmodulin in the cytosol of cells and acquires an extremely high specific enzymatic activity of about 2,000 molecules of ATP converted to cAMP per second per AC enzyme molecule[61]

Summary

Introduction

The Gram-negative coccobacillus Bordetella pertussis excels in sophistication of its immunomodulatory action. Among the first cells of the immune system that respond to B. pertussis infection are the myeloid phagocytic cells that bear the complement receptor 3 (CR3, the αMβ2 integrin CD11b/CD18 or Mac-1). This includes macrophages, neutrophils and dendritic cells (DCs)[3]. The high specific activity of the CyaA-delivered adenylyl cyclase (AC) enzyme represents, a unique tool for analysis of the impact of cAMP signaling on myeloid cell function in general. We have used stable isotope labelling by amino acids in cell culture (SILAC)[10] for quantitative shotgun phosphoproteomic analysis of cAMP signaling resulting from CyaA toxin action on primary mouse bone marrow derived dendritic cells (BMDC). The results reveal that CyaA action causes alteration of phosphorylation of a number of proteins involved in regulation of actin cytoskeleton homeostasis, phagocytosis, translation, chromatin remodeling, IL-10 secretion and tolerogenic DC shaping

Methods

Results

Conclusion