This project uses biochemical, genetic, and cell culture methods to study the structure and function of protein toxins involved in bacterial pathogenesis. Current studies focus on the three protein components of anthrax toxin. Prior work defined three domains in the Protective Antigen protein, an N-terminal region needed only to maintain solubility, a C-terminal domain involved in receptor binding, and a central domain required for binding of the other two components, lethal factor (LF) and edema factor (EF). We have now used site specific mutagenesis to show that deletion of twelve amino acids from the C-terminus of PA destroys receptor-binding activity. Mutagenesis was also used to replace twelve selected serine or threonine residues in PA with cysteine. One cysteine mutant was found which has normal binding properties, but appears unable to translocate EF or LF to the cytosol of eukaryotic cells. All of these mutants were expressed in Bacillus species, and the secreted mutant proteins were purified from culture supernatants. LF has been mutagenized at 15 different sites with an oligonucleotide linker that inserts two or four amino acids. Insertions that inactivated the protein were clustered in the N-terminal region considered to be involved in binding to PA and in a C-terminal region of 150 amino acids considered to be the putative catalytic domain. The methods developed with anthrax toxin will later be applied to secreted proteins considered to be virulence factors of oral bacteria.
