Pseudomonas exotoxin A (ETA), one of several potential virulence products produced by Pseudomonas aeruginosa is a three domain bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inhibiting protein synthesis. The toxin binds to a surface receptor on susceptible cells, is internalized via coated pits and endosomes, and enters the cytosol. During this journey, the toxin is cleaved to generate an enzymatically active fragment which ADP-ribosylates elongation factor 2. The pathway ETA takes to reach its target is only partially elucidated. Recently the ETA receptor has been isolated and shown to be similar to the alpha2macroglobulin receptor/LDL related protein (alpha2MR/LRP) The long-term goals of this research are to fully understand the cellular action of ETA, including how it is processed, and to determine its interaction with its receptor on toxin-sensitive cells.
The specific aims of this proposal are: 10 Confirm the relationship between the ETA receptor and the alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP receptor that correspond to ETA binding sites; 2) Determine the basis for cellular susceptibility to toxins. This includes a) determining ETA receptor level in toxin sensitive (LM and Chang) and resistant cells (OVCAR and Hela); b) establishing the level of alpha2MR/LRP receptor associated protein (RAP) in various mammalian cells; c) identifying and localizing the compartments where ETA is proteolytically processed, is activated, and enters the cytosol. Where feasible, these studies will include diphtheria toxin (DT), to determine if a second bacterial toxin is processed in a manner similar to ETA. 3) Define the intracellular pathway taken by the ETA receptor in sensitive cells. This work is significant because it will increase understanding of the pathophysiology of pseudomonas infections, help in the construction of efficient chimeric toxins, and help to answer the question of how toxins have adapted to take advantage of well conserved cellular components such as the alpha2MR/LRP.
