TitleCharacterization of the A subunit mutants of Stx1 and Stx2 in Saccharomyces cerevisiae
NameKyu, Eric (author), Di, Rong (chair), Tumer, Nilgun (internal member), Martin, Charles (outside member), Rutgers University, Graduate School - New Brunswick,
SubjectMicrobiology and Molecular Genetics,
DescriptionShiga toxins are a family of related toxins with two major groups, Stx1 and Stx2. The most common sources for Shiga toxin are the bacteria Shigella dysenteriae and the Shigatoxigenic group of Escherichia coli (STEC), which includes serotype O157:H7. Shiga-like toxin 1 and 2 (Stx1 and Stx2) are produced by some E. coli strains. Stx produced by S. dysenteriae differs from Stx1 by one amino acid. Stx1 and Stx2 have only 56% sequence homology.
E. coli O157:H7 has been the major cause of food-borne illnesses. Symptoms include abdominal cramping, bloody diarrhea, vomiting and a low-grade fever. Serious complications can lead to Hemolytic Uremic Syndrome (HUS).
Stx1 and Stx2 are categorized as and AB5 toxin. The A complex is responsible for toxicity and has enzymatic activity inside the host cell. The B subunits bind to a receptor on the surface of the host cell. Shiga toxin acts as an N-glycosidase. It cleaves a specific adenine residue in the conserved α- sarcin ricin loop in the 28S subunit of the eukaryotic rRNA. This interferes with the translocation step in protein synthesis and leads to inhibition of translation and cell death.
When Stx1A and Stx2A cDNA was cloned downstream of the GAL1 promoter and transformed into Saccharomyces cerevisiae, cells growth was greatly reduced when expression of the toxin was induced. To identify residues critical for cytotoxicity or Stx1A and Stx2A random and site-directed mutations were made. The ability of the mutant proteins to depurinate ribosomes in vivo in yeast was determined by dual primer extension analysis. The amount of translation was measured by incorporation of [35S]-methionine in the cell.
The results of these assays show mutations in N75, Y77, E167, R170, and R176 are critical for toxicity in both toxins. Also C-terminal deletions after L240 in Stx1A and I238 in Stx2A abolished toxicity. Several of these mutants, even though they are non-toxic still have the ability to depurinate ribosomes. This indicates cytotoxicity and depurination may not be directly related. This provides evidence that cytotoxicity is not necessarily a direct result of depurination, and other potential mechanisms may contribute in Stx1A and Stx2A cytotoxicity in yeast.
Noteby Eric Kyu
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work