Adenylate cyclase toxin

The toxin is secreted by the Type I secretion system, which spans both membranes and periplasm space, allowing the toxin to be secreted from the cytoplasm straight outside the cell.[1][2] A large proportion of the toxin remains associated with the bacterium exterior proteins, mainly filamentous haemagglutinin, but these toxin molecules are not active.[5][1] Besides attachment to bacterial proteins, aggregation also inactivates the toxin.[5] This quick inactivation highlights the necessity of close contact between secreting bacterium and target cell.[1]

Adenylate cyclase toxin binds to target cells by the complement receptor 3 (CD11b/CD18, or Mac-1).[7] Target cell are therefore myeloid lineage cells, mainly phagocytes, such as neutrophils.[1] Binding to cells without the CR3 also happens, but at a much lower rate.[5] The portion responsible for binding to the receptor is inside the calcium binding repeats, from residues 1166 to 1287.[3] The hemolysin portion of the protein then binds to the target membrane and inserts itself into the bilayer.[3][5] The adenylate cyclase (AC) domain is then translocated across the cytoplasmic membrane into the cytoplasm. Translocation of the AC domain is independent of cytotoxic pore-forming activity, as these two activities require to toxin to adopt different conformations.[5][2] The transiently opened pores do, however, contribute to AC domain function by potassium leakage and calcium influx into the target cell, which slows endocytosis of CR3/adenylate cyclase toxin clusters,[2] also, the CR3/toxin complex is mobilized by detachment from the cytoskeleton. The complex is then recruited into cholesterol-rich lipid rafts.[3] Calcium influx by itself has many negative effects on target cells, such as deregulation of cellular signalling.[5]

The adenylate cyclase domain has intrinsic enzymatic activity. Translocation of the AC domain into the cell starts the main process by which this toxin influences target cells: the AC domain binds calmodulin, and catalyzes unregulated production of cAMP from ATP.[7] cAMP is an important second messenger molecule and its massive overproduction affects many cellular processes. In phagocytes, most of the bactericidal functions are stopped by cAMP-mediated activation of PKA and Epac.[7]

The above described effects of the adenylate cyclase toxin, mainly the cAMP overproduction, have a profound effect on target cells. Although phagocytic immune cells migrate to the site of infection in the lungs, they are not able to mount an effective response. Not only the phagocytic uptake of bacteria is blocked, but subsequent production of ROS by neutrophils and monocytes, NETs by neutrophils, and NO by macrophages, is also inhibited.[5][7][3] The effect on neutrophils is most important in early infection with Bordetella, impairing most of their antimicrobial functions.[3] Intoxication with the adenylate cyclase toxin leads to shift in polarization of macrophages from M1 (proinflammatory) phenotype to M2 (immunoregulatory) phenotype and may lead to macrophage apoptosis.[7][3] cAMP accumulation after adenylate cyclase intoxication also interferes with IRF signalling in dendritic cells, which leads to lower IL-12 production. IL-12 is important for T-cell response polarization.[7] Other effects of cAMP on dendritic cell interaction with T-cells are also detrimental to the immune response. Although cAMP induces dendritic cell migration into lymph nodes, it lowers their capacity to interact with T-cells and present antigen. This has a tolerogenic effect on the T-cell population.[7]