Subunit S1 of pertussis toxin: mapping of the regions essential for ADP-ribosyltransferase activity.

Abstract

The toxicity of pertussis toxin is mediated by the ADP-ribosyltransferase activity of subunit S1. ADP-ribosylation of the target substrates in eukaryotic cells is a common mechanism of action of many bacterial protein toxins (1-3). The best-studied molecules that adopt this mechanism are diphtheria toxin (4-7), Pseudomonas exotoxin A (8-10), cholera toxin (11-13), and pertussis toxin (14-16), but several other toxins possessing ADP-ribosyltransferase activity have also been described (17,18). The toxins usually contain two functional moieties: A, which is enzymatically active, and B-, which recognizes and binds the receptors on the cell surface facilitating the entry of the enzymatically active subunit into the target cells.The proteins that are ADP-ribosylated by diphtheria, (20)(21)(22).For diphtheria and Pseudomonas toxins, a series of studies, which include nitrosoguanidine mutagenesis of the genes (23), characterization of the mutant genes (5) and their products (24-26), photoaffinity labeling of the toxins with NAD (27, 28), site-directed mutagenesis (29, 30), and crystallographic structure of the Pseudomonas exotoxin A (31) have led to the identification of amino acids essential to the enzymatic activity of the proteins. For example, Glu-148 of diphtheria toxin and Glu-553 of Pseudomonas -exotoxin A, located within the catalytic site of the two enzymes, cannot be replaced even with an aspartic acid without abolishing enzymatic activity (29,30).We used a similar approach to study the structure-function relationship of the S1 subunit ofpertussis toxin. By carboxyland amino-terminal deletion analysis and site-directed mutagenesis, we identified at least three regions of the S1 subunit that are essential for enzyme function. Substitutions within these regions produce enzymatically inactive molecules. MATERIALS AND METHODSConstruction of the Si Deletion Mutants. The S1 subunit of pertussis toxin was expressed in Escherichia coli fused to the 98 amino-terminal amino acids of the MS2 polymerase. This fusion protein (PTE255) contains amino acids 2-235 of the S1 subunit and is enzymatically active (32). The gene coding for the amino acids 2-235 is contained within a BamHI-Xba I fragment flanked by an EcoRI site at the 5' end and a HindIII site at the 3' end. To obtain the plasmids expressing carboxylterminal deletion mutants of the S1 protein, the plasmid pTE255 was digested first with Xba I and then with Nco I, Nru I, Bal I, Sal I, and Sph I, respectively.' The sticky ends generated by the restriction enzymes were then repaired by the large fragment of DNA polymerase, and the plasmids were circularized by DNA ligase. During this process, the natural stop codon of the S1 subunit was lost, and therefore the new proteins contained a few amino acids fused at the carboxyl terminus: NCO, NRU, and SPH proteins had the following carboxyl-terminal unrelated amino acids: Leu-ProArg-Ala-Phe-Arg. BAL and SAL proteins contained, respectively, 50 and 16 amino acids deriving from the sequence of pBR322 (33) fused at th...

Pertussis Toxin Utilizes Proximal Components of the T-Cell Receptor Complex To Initiate Signal Transduction Events in T Cells

“…Thus, the biological consequences of PTx intoxication would vary depending on the cell type encountered and the proximity to the bacteria. It appears that most cells would be susceptible to altered G-protein signaling as a result of ADP ribosylation of G alpha subunits and that PTx holotoxin-mediated toxicity could occur at many sites in the body (34). Our data predict that T lymphocytes will not only exhibit inhibited G-protein signaling but will also exhibit strong activation of a TCR-"like" pathway: however, only T cells in close proximity to the site of bacterial infection will be targeted.…”

“…First, the mobilization of the catalytic A subunit into recipient cells leads to the ADP ribosylation of G-protein alpha subunits, which robustly inactivates signaling via G i and G o proteins (34,43). Second, the binding of PTx (via its B subunit) to the cell surface of T lymphocytes initiates a series of rapid signaling events analogous to that of TCR activation.…”

“…The A subunit (S1) has catalytic activity and functions to ADP ribosylate and inhibit cellular G␣ proteins, while the five B subunits (S2, S3, two copies of S4, and S5) are responsible for binding and delivery of the A subunit to mammalian cells (34). Traditionally, the biological effects of PTx have been attributed to the enzymatic activity of the A subunit; however, recent work demonstrates that PTx has biological activities that cannot be accounted for by the enzymatic activity of the A subunit alone (11,28,39,43).…”

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

“…To determine whether the route of PTx exposure, to native PTx during an infection or to PTd during vaccination, could explain the differences in 1B7-and 11E6-like titers in exposed and vaccinated adults, we prepared PTd with procedures similar to those used during vaccine production using formaldehyde. A genetically detoxified variant of PTx, containing two amino acid changes in the S1 subunit (R9K and E129G; PTg) was developed in the late 1980s as an alternative to chemical toxoiding (7,26) but was not included in the acellular vaccine due to patent issues.…”

Expression of a C Terminally Truncated Form of Pertussis Toxin S1 Subunit Effectively Induces Protection against Pertussis Toxin following DNA-Based Immunization

“…1C), C160 and N40 polypeptides might be degraded in the immunized mice. The enzymatic domain of S1, associated with ADP-ribosyltransferase activity, is located in residues 2 to 179, and a recombinant fragment (residues 1 to 123) of S1 is known to lose enzymatic activity (16). Therefore, expression of the enzymatic domain may be essential in the induction of protective immunity with DNAbased immunization.…”