Shiga-like toxin 1 (SLT-1) is produced by Escherichia coli strains like the pathogenic strain O157:H7. These bacterial strains are responsible for worldwide cases of food poisoning and water contamination, and the toxin is a major cause of hemorrhagic colitis and the hemolytic uremia syndrome. SLT-1 is defined as a type II ribosome-inactivating protein (RIP) and belongs to a family of plant and bacterial AB toxins. The A1 chain blocks protein synthesis in eukaryotic cells by depurinating a single adenine base in 28S rRNA. The mechanisms by which the A chain of SLT-1 interacts with the host components to route itself to the cytosol remains largely unknown. This thesis project identified a list of putative cellular proteins that interact with the SLT-1 A1 chain by the use of yeast-2-hybrid (Y2H) screens and HeLa lysate pull down/mass spectrometry analyses. Further assessment of the top 8 host interactors did not yield true interactions.

Shiga-like toxin 1 (SLT-1) is produced by Escherichia coli strains like the pathogenic strain O157:H7. These bacterial strains are responsible for worldwide cases of food poisoning and water contamination, and the toxin is a major cause of hemorrhagic colitis and the hemolytic uremia syndrome. SLT-1 is defined as a type II ribosome-inactivating protein (RIP) and belongs to a family of plant and bacterial AB toxins. The A1 chain blocks protein synthesis in eukaryotic cells by depurinating a single adenine base in 28S rRNA. The mechanisms by which the A chain of SLT-1 interacts with the host components to route itself to the cytosol remains largely unknown. This thesis project identified a list of putative cellular proteins that interact with the SLT-1 A1 chain by the use of yeast-2-hybrid (Y2H) screens and HeLa lysate pull down/mass spectrometry analyses. Further assessment of the top 8 host interactors did not yield true interactions.

Pathogenic bacteria use an arsenal of toxic protein virulence factors to cause disease in host cells. The mono-ADP-ribosyltransferase (mART) toxins are a family of exotoxins produced by pathogens which contribute to a wide range of diseases including cholera, diphtheria and whooping cough. Specifically, mART toxins act by transferring ADP-ribose from NAD+ to target proteins in host cells, altering or inhibiting target activity with deleterious downstream effects. Recently, in silico analyses have revealed two novel mARTs, Photox and Certhrax, from pathogenic organisms. Photox, from Photorhabdus luminescens was successfully expressed and purified from E. coli and was shown to target actin, specifically at Arg177. This covalent modification inhibits actin polymerization and leads to observed cytotoxicity in yeast cells. Photox has 35% identity with SpvB from Salmonella enterica, which allowed for a structural model to be built, showing the location of all characteristic mART active site components, and the binding site for potential inhibitors. Certhrax originates from Bacillus cereus G9241, implicated in a number of severe pneumonia cases. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we demonstrated that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. In vivo tests employing toxin gene expression in yeast, and receptor-mediated infection of mammalian, cells showed the extreme cytotoxicity of Certhrax (LD50 = 100 pg/mL against mouse macrophage cells), making it 60 times more toxic than its infamous counterpart, anthrax lethal factor. In vitro analysis indicated that Certhrax possesses NAD+ glycohydrolase activity, characteristic of many mART toxins, but we continue to search for the natural host protein target of this toxic enzyme. We determined the crystal structure of Certhrax to 2.2 Å, which illustrates a close structural similarity with anthrax lethal factor. Furthermore, we identified several small molecule inhibitors which show protection against Certhrax both in vitro and in vivo. We determined a 1.9 Å crystal structure of one inhibitor in complex with Certhrax. Through identification and characterization of novel mART enzymes, we seek to better understand this family of toxic enzymes to aid in the discovery and development of more potent therapeutics. / National Sciences and Engineering Research Council, Canadian Institutes of Health Research