UHN UT University of Toronto University Health Network


 

Bacterial Toxins

 

Lab Members: Arshiya Mohammed

 

1) Exotoxin A

Pseudomonas aeruginosa Exotoxin A (ETA) is known to bind to cell surfaces via a membrane receptor (point A), to be internalized by receptor mediated endocytosis (point B), and to kill susceptible cells by inactivating protein synthesis in the cytosol (point C). However, the mechanistic pathway ETA follows to get from point B to point C is still not well understood. Previous work in our lab has used the ETA translocation domain to greatly improve the delivery of cell penetrating peptides (CPPs) and their cargos into cells by allowing these constructs to route to the ER lumen, avoiding lysosomal degradation or recycling out of cells, and reach the cytosol where most intracellular targets reside (1). Results from this work suggest that ETA follows a retrograde transport mechanism to reach the cytosol. To further map out the routing of ETA in human cells, our research aims to determine the identity and function of eukaryotic protein binding partners to ETA. Currently, multiple proteomic and genetic analyses are underway to elucidate the downstream events that occur in ETA’s intracellular routing pathway prior to cell killing.

 

 

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Figure 1. The structure of Pseudomonas aeruginosa exotoxin A and a schematic of the polypeptide chain divided into its three domains. Image modified from: Wedekind et al. 2001. Journal of Molecular Biology. 314(4):823-837. Figure 2. The hypothetical routing pathway of Pseudomonas aeruginosa exotoxin A into susceptible cells.

 

Selected References

1) Mohammed AF, Abdul-Wahid A, Huang EH, Bolweska-Pedyczak E, Cydzik M, Broad AE, Gariépy J. (2012) The Pseudomonas aeruginosa exotoxin A translocation domain facilitates the routing of CPP-protein cargos to the cytosol of eukaryotic cells. J Control Release 164(1):58-64. MOHAMMED1

2) Wedekind JE, Trame CB, Dorywalska M, Koehl P, Raschke TM, McKee M, FitzGerald D, Collier RJ, McKay DB. (2001) Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity. J Mol Biol 314(4):823-37.

 

 

2) Shiga-like Toxins

Shiga-like toxins are type II ribosome inactivating proteins (RIPs) produced by pathogenic E. coli strain that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The A chain of Shiga-like toxin 1 (SLT-1) blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve interactions with eukaryotic ribosomal proteins. Molecular details leading up to the depurination event are lacking, despite a general understanding of the biochemical basis of SLT-1 toxicity. Our laboratory has employed functional assays to elucidate biological processes involving the toxin action.

Using tandem mass spectrometry and pull-down experiments, we have confirmed that the A chain of SLT-1 interacts with the ribosomal proteins via a conserved 17 amino acid present on the C-terminal tail in the ribosomal stalk protein. Furthermore, yeast-2-hybrid and surface plasmon resonance (SPR) have been used to determine the binding kinetics of the interaction. The SPR result demonstrated that SLT-1 rapidly binds and dissociates from the conserved C-terminal peptide on ribosomal protein, which is mediated by distinct charged (cationic) and hydrophobic surfaces on the SLT-1 A chain that are also essential for its catalytic activity. Moreover, mutagenesis analysis revealed a specific peptide motif within the ribosomal proteins which include key anchor residues recognized by the toxin.

These findings suggest that the nature of these interactions may play an important role in orienting the catalytic domain of the toxin upon docking to the ribosomal stalk protein.

 

Figure 1. Surface rendering of the SLT-1A chain (PBD# 1DM0) depicting the cationic (blue) and hydrophobic (yellow) residues essential for optimal binding to the conserved peptide sequence in the ribosomal. Structure is rotated by 140° to show the catalytic residues (green) on the SLT-1A.

 

 

Selected References

1) McCluskey AJ, Bolewska-Pedyczak E, Jarvik N, Chen G, Sidhu SS, Gariépy J. (2012) Charged and hydrophobic surfaces on the a chain of shiga-like toxin 1 recognize the C-terminal domain of ribosomal stalk proteins. PLoS One. 7(2):e31191. McCluskey1

2) McCluskey AJ, Poon GM, Bolewska-Pedyczak E, Srikumar T, Jeram SM, Raught B, Gariépy J. (2008) The catalytic subunit of shiga-like toxin 1 interacts with ribosomal stalk proteins and is inhibited by their conserved C-terminal domain. J. Mol. Biol. 378:375-386.

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