Infections with Shigella dysenteriae, producing Shiga toxin and Diarrheagenic E. coli, producing Shiga-like toxins (Stx) are responsible for widespread disease and death. Bacteria producing Shiga-like toxins are the most common cause of hemolytic euremic syndrome (HUS), for which there is no vaccine or an effective treatment. Due to the relative ease of production and the lethality of Stx, Shiga-toxin producing E. coli is a major threat as an agent of bioterrorism and has been classified as NIAID Category B Priority for biodefense. Recent outbreaks due to contaminated food and the increased threat of bioterrorism underscore the importance of research to identify the ribosomal targets of Shiga-like toxins. Both Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) belong to the group of ribosome inactivating proteins (RIPs), which includes ricin and pokeweed antiviral protein (PAP). The A-chains of all RIPs have RNA N-glycosidase activity that catalytically removes a specific adenine from the highly conserved sarcin/ricin loop (SRL) of the large rRNA, resulting in irreversible inhibition of protein synthesis. Our studies established for the first time that binding of RIPs to ribosomal proteins is essential for depurination of ribosomes. Exactly which ribosomal proteins are involved in interaction of Shiga-like toxins with ribosomes and the contribution of these interactions to ribosome depurination are not known. Our preliminary results indicate that expression of a N-terminal fragment of ribosomal protein L3 prevents the cytotoxicity of Stx2 in yeast. The primary goal of this exploratory R21 application is to test the hypothesis that Shiga-like toxins bind to ribosomal protein L3 and to evaluate the feasibility of using the N-terminal fragment of L3 to provide resistance to these toxins in human cells.
The Specific Aims are: 1. We will analyze ribosome interactions of Shiga-like toxins 1 and 2 to determine if they bind to ribosomal protein L3 to depurinate the SRL. 2. We will use mutagenesis analysis to determine if the mRNA or the amino acid sequence of the N-terminal fragment of L3 is critical for providing resistance to Shiga-like toxins. 3. We will introduce the N-terminal fragment of L3 into human cells to determine if it will eliminate the cytotoxic effects of Shiga-like toxins. The findings from the proposed studies will represent an investment in the development of new technologies designed to counteract the potential use of these toxins as agents of bioterror. ? ? ? ?
| Di, Rong; Kyu, Eric; Shete, Varsha et al. (2011) Identification of amino acids critical for the cytotoxicity of Shiga toxin 1 and 2 in Saccharomyces cerevisiae. Toxicon 57:525-39 |
