Evaluating the nuclease activity of Pseudomonas exotoxin A

Abstract

Pseudomonas exotoxin A (PE) is a protein toxin secreted by Pseudomonas aeruginosa. The toxin is remarkably potent, and believed to be the major virulence factor of P. aeruginosa. PE inhibits protein synthesis by inactivating elongation factor 2 (EF2), the protein that advances the ribosome along the mRNA during translation. Cell with inactive EF2 are unable to synthesize proteins, which leads to apoptosis. Recent studies have indicated that, in addition to inhibition of protein synthesis, PE has also functions as a nuclease. The purpose of the experiments described here is to verify this unusual claim and characterize the nuclease activity of PE using only the catalytic fragment of the toxin. We have conducted basic time‐course and concentration assays, and have confirmed a low level of nuclease activity from PE. We are currently examining cleavage in the presence of different divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ca2+, Mg2+ and Zn2+). We have also tested a fluorescence‐based assay to observe cleavage of DNA in real‐time. Initial experiments using DNase I have been successful, and we hope to use this method to study PE in the near future.Support or Funding InformationResearch reported in this poster was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R25GM058264. Additional support for this research comes from Towson University, the Jess and Mildred Fisher College of Science and Mathematics, Graduate Studies and the Department of Biological Sciences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.