Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells.

Pertussis toxin (PT) comprises an active subunit (S1), which ADP-ribosylates the alpha subunit of several mammalian G proteins, and the B oligomer (S2-S5), which binds glycoconjugate receptors on cells. In a previous report, expression of S1 in Cos cells resulted in no observable cytotoxicity, and it was hypothesized that either S1 failed to locate its target proteins or the B oligomer was also necessary for cytotoxicity. To address this, we stably transfected S1 with and without a signal peptide into mammalian cells. Immunofluorescence analysis confirmed the function of the signal peptide. Surprisingly, we found that S1 was active in both transfectants, as determined by clustering of transfected Chinese hamster ovary (CHO) cells and ADP-ribosylation of G proteins. Constructs with a cysteine-to-serine change at residue 201 or a truncated S1 (residues 1-181) were also active when transfected into cells. Constructs with an inactive mutant S1 had no activity, confirming that the observed results were due to the activity of the toxin subunit. We conclude that S1 is active when expressed in mammalian cells without the B oligomer, that secretion into the endoplasmic reticulum does not prevent this activity and that the C-terminal portion of S1 is not required for its activity in cells.

Intracellular delivery of a cytolytic T-lymphocyte epitope peptide by pertussis toxin to major histocompatibility complex class I without involvement of the cytosolic class I antigen processing pathway.

A CD8(+) cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion by Bordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.