Quantification of diphtheria toxin mediated ADP-ribosylation in a solid-phase assay.
BACKGROUND
Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADP-ribosylation by DT.


METHODS
Solid phase-bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD(+) as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System.


RESULTS
The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase.


CONCLUSIONS
The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.
Bachran
Christopher
C
Zentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.
Sutherland
Mark
M
Bachran
Diana
D
Fuchs
Hendrik
H
D036002:ADP Ribose Transferases / Q000032:analysis; D000246:Adenosine Diphosphate Ribose / Q000032:analysis*; D001427:Bacterial Toxins / Q000032:analysis; D015153:Blotting, Western; D020134:Catalytic Domain; D003124:Colorimetry; D004167:Diphtheria Toxin / Q000032:analysis*; D005098:Exotoxins / Q000032:analysis; D020652:Peptide Elongation Factor 2 / Q000032:analysis; D011549:Pseudomonas / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis; D037521:Virulence Factors / Q000032:analysis; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D005098:Exotoxins; D020652:Peptide Elongation Factor 2; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A