Pore‐formation by adenylate cyclase toxoid activates dendritic cells to prime CD8⁺ and CD4⁺ T cells

Abstract

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

“…In the second line, the cAMP-mediated downregulation of pro-inflammatory IL-12 and TNF-α production and the upregulation of anti-inflammatory IL-10 cytokine release by myeloid cells, would skew the adaptive immune responses to infection and protract bacterial colonization. In contrast, the pore-forming activity of CyaA was shown to elicit potassium efflux and thereby contributes activation of the p38 and JNK kinases in toxin-permeabilized monocyte-derived cells59. This then synergizes with TLR-mediated signaling of bacterial components towards promoting NALP3 inflammasome assembly and induction of pro-inflammatory IL-1β cytokine release13.…”

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity

“…3B). Such synergy would have been expected to result from the impact of toxin-provoked Ca 2+ influx on PKC activity and from perturbation of ion homeostasis upon cell permeabilization by the inserted CyaA pores, which activates MAPK signaling (46)(47)(48). The impact of cAMP signaling thus overrode the impact of the poreforming activity.…”

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

“…In parallel, the Hly forms small cation-selective pores that permeabilize the cytoplasmic membrane of target cells and account for the moderate hemolytic activity of CyaA on erythrocytes (25,26). Cell permeabilization by Hly also elicits efflux of cytosolic K ϩ ions and activates the p38 and Jun N-terminal protein kinase (JNK) kinases (27), thus contributing to Toll-like receptor (TLR)-induced NALP3 inflammasome formation and interleukin-1␤ (IL-1␤) secretion by dendritic cells (DCs) (28). The pore-forming activity of CyaA further synergizes with cAMP signaling elicited by the AC enzyme in bringing about the overall cytotoxic (cytolytic) potency of CyaA (29,30).…”