Structure-Function Analysis Of Shiga-Like Toxin
The long range goals of this project are to understand thestructure-function relationships of the Shiga toxin family, at themolecular level. All members of this class of toxins act by catalyzingthe removal of a single adenine at position 4324 from ribosomal RNA.This N-glycosidic depurination inactivates the ribosome, inhibits proteinsynthesis, and causes target cell death. Holotoxins in this familyconsist of a single enzymatically active A subunit and multiple identicalB subunits that bind the toxin to susceptible cells. The work describedis focused on Shiga-like toxin type I (SLT-1) which our laboratory -usesas a representative model of this larger group of proteins. SLT isproduced by the enterohemorrhagic Escherichia coli (EHEC) and has beenstrongly associated with human diarrhea syndromes, hemorrhagic colitis,and two extra-colonic complications, the hemolytic uremic syndrome, andthrombotic thrombocytopenic purpura. Outlined here, are three approachesto characterize the active site of SLT-1 A subunit (SLT-IA) by (i)oligonucleotide-directed mutagenesis, which will be guided by sequencesimilarity among toxins in the ricin and Shiga family and computermodeling of the SLT-IA structure; (ii) random point mutagenesis of SLT-IAand selection for reduced toxicity in yeast, a eukaryotic host sensitiveto wild-type toxin; and (iii) determining the 3-dimensional structure ofenzymatically active SLT-IA and mutants of interest, which will requirethe purification and crystallization of recombinant SLT-IA and mutants.The fourth aim is to use SLT-specific monoclonal antibodies, syntheticpeptides and site-directed mutation to identify and characterize theamino acid residues of SLT-IA and SLT-IB involved in holotoxin assembly.The fifth aim is to develop a murine model, using recombinant Citrobacterfreundii which produce SLT, to determine the mechanism of pathogenesis indiseases associated with EHEC infection. Detailed structure-functioninformation about SLT-1 is pertinent to bacterial pathogenesis, vaccineproduction, design of new therapies, and insights into endogenousregulatory enzymes yet to be characterized.