4410 total record number 100 records this year

Simultaneous vaccination and therapy against anthrax: a dominant-negative inhibitor of the protective antigen of B. anthracis is. at the same time a potent and safe antigen for a vaccine

Aulinger, BA;

As already described above, the toxin effect of _B. anthracis_ of crucial importance for the full pathogenicity of the pathogen. Numerous studies have attempted to decipher the cellular mechanism of intoxication. The anthrax toxin is currently one of the best-studied exotoxins in bacteriology. The steps from the binding of PA to the membrane-bound receptor to the infiltration of the enzyme domains of EF and LF into the cytosol of the affected cell are now relatively well understood. Analysis of the structure of PA (from the amino acid sequence to the crystallographic structure of the PA heptamer) has allowed the individual domains of PA to be assigned a function in the toxin’s action. It could be shown that certain amino acids of domain 2 are crucial for the conformational changes during pore formation. Sellman et al. demonstrated how targeted mutations of individual amino acids, or a combination of different mutations in this area, lead to a loss of effectiveness of LeTx, since the enzyme component can no longer be transported into the cytosol (Sellman et al., 2001b). However, most of these mutant PA variants continue to associate with the cellular receptor, bind both LF and EF, and form heptamers. In another study, Sellmann et al. show that some of these PA mutants also co-oligomerize with native PA, thereby preventing the translocation of LF into the cytosol mediated by wild-type PA (wtPA) (Sellman et al., 2001a). In particular, the double mutation Lys397÷Asp397, Asp425÷Lys425 (K397D, D425K) showed particularly strong inhibitory activity. Further analysis suggests that just one such mutated PA variant per heptamer is sufficient to prevent toxin translocation. This property corresponds to that of a so-called dominant-negative inhibitor (DNI), i.e. an inhibitor that not only competes with the native PA for receptor binding, but also binds wtPA and LF and EF to itself and thereby prevents the toxin effect. Accordingly, the simultaneous administration of the DNI double mutant with an otherwise lethal dose of LeTx in mice prevented the symptoms of intoxication and all tested animals survived (Sellman et al., 2001a). Further characterization of the double mutant PA (K397D, D425K) and other mutants with three or four point mutations showed that the double mutant described has a very strong inhibitory effect with sufficient stability of the heptamer complexes (Yan and Collier, 2003). Therefore, only this dominant-negative PA mutant was used for the experiments described here. From now on, this will only be referred to as DNI. Another advantage for the experiments described below is that in DNI only two amino acids are reversed compared to wtPA and the antigenicity should therefore not be significantly different compared to PA.