Delivery of CD8⁺T-Cell Epitopes into Major Histocompatibility Complex Class I Antigen Presentation Pathway byBordetella pertussisAdenylate Cyclase: Delineation of Cell Invasive Structures and Permissive Insertion Sites

Abstract

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8(+) T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8(+) T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8(+) T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.

Phospholipase A activity of adenylate cyclase toxin?

“…The modest, if any, effect of the S606A substitution on toxin activities on cells (see, for example, figures 5-8 and table S1 of ref. 1) then goes well with a previous report that insertion of a VYT tripeptide between residues 606 and 607 has no impact on toxin activity of ACT (7). Finally, incubation of J774A.1 cells with 20 nM (3.5 μg/mL) ACT leads to rapid ATP depletion in cells within minutes (8) and this is known to provoke arachidonate release by cellular enzymes (9).…”

cAMP signalling ofBordetellaadenylate cyclase toxin through the SHP‐1 phosphatase activates the BimEL‐Bax pro‐apoptotic cascade in phagocytes

“…The cells were monitored in time by flow cytometry, and Hoechst 33258 exclusion capacity was used as a measure of cell viability, while early manifestations of apoptosis were detected by Annexin V staining for phosphatidylserine (PS) surface exposure. As a control of specificity of cAMP-induced cytopathic effects of CyaA toxin action, we employed the pore-forming but enzymatically inactive CyaA-AC À toxoid that is unable to convert cellular ATP to cAMP because of disruption of the ATPbinding site of the AC enzyme (Osicka et al, 2000).…”

“…The CyaA, CyaA-AC À or CyaAΔAC endotoxin-free proteins were produced in the Escherichia coli strain XL1-Blue (Stratagene, La Jolla, CA) and purified by a combination of ion exchange chromatography on DEAE-Sepharose and hydrophobic chromatography on Phenyl-Sepharose (Osicka et al, 2000).…”

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

“…The CyaA-AC − toxoid is devoid of adenylate cyclase enzyme activity due to insertion of the GlySer dipeptide between residues 188 and 189 in the ATPbinding site of the AC domain. 47 The toxoid variants used in this study were CyaA-E570Q-K860R-AC − (abbreviated CyaA-QR-AC − ), a mutant with strongly reduced pore-forming activity and CyaA-E509K-E516K-AC − (abbreviated CyaA-KK-AC − ) a mutant with an increased pore-forming activity. 5,18,19,21 CyaA-AC − and low-permeabilizing CyaA-QR-AC − toxoids carrying MHC class I epitope from chicken egg ovalbumin SIINFEKL (CyaA-SIINFEKL-AC -) inserted in position of 233 of AC domain are described in.…”