The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, ␣ M ␤ 2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b ϩ ) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b Ϫ cells. The nonhemolytic AC ϩ Hly Ϫ bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC ϩ Hly Ϫ mutant also infected mouse lungs as efficiently as the parental AC ϩ Hly ϩ strain. Hence, elevation of cAMP in CD11b Ϫ cells and/or the poreforming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (Ͼ10 7 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.KEYWORDS Bordetella pertussis, adenylate cyclase toxin-hemolysin, cAMP intoxication, lung colonization, pore-forming activity, virulence T he adenylate cyclase toxin-hemolysin is produced by all three Bordetella species pathogenic to mammals, and it plays a prominent role in the early phases of respiratory tract infection by the whooping cough agent, Bordetella pertussis (1-3). The toxin specifically binds the CD11b subunit of the complement receptor 3 (CR3) (4, 5) and exerts an array of immunosubversive and cytotoxic activities on myeloid phagocytes. CyaA delivers a cell-invasive adenylyl cyclase (AC) enzyme domain into cytosol of CD11b ϩ cells, where the AC is activated by calmodulin and converts cytosolic ATP to the signaling molecule cyclic AMP (cAMP). The generated supraphysiological levels of cAMP then nearly instantly ablate the bactericidal oxidative burst and opsonophago-

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4+ and CD8+ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4+ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4+CD25+Foxp3+ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8+ T cell proliferation and limited the induction of IFN-γ producing CD8+ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.

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.

The metabolic and immunological indicators were determined in the blood of laying hens of ISa broWn hybrid divided into three groups, the control group and two experimental groups. the administration of n-3 polyunsaturated fatty acids (PuFa) in the form of linseed (Group 1) and fish oil (Group 2) and α-tocopherol as antioxidant to laying hens resulted in a significant increase in concentrations of high density lipoproteins (HDL) cholesterol (P < 0.05), eicosapentaenoic (ePa), docosahexaenoic (DHa) and α-linolenic acids (aa) in blood in comparison to the control group. Significantly lower levels of cholesterol (CHoL) were determined in both experimental groups at the third sampling (P < 0.05) and arachidonic acid (aa) in the fish group (P < 0.01). The metabolic activity of phagocytes and polyclonal activation of lymphocytes showed no significant differences and remained within the physiological range. oral administration of n-3 PuFa showed no significant increase of the immune response of experimental animals.