Activation of ExoU phospholipase activity requires specific C-terminal regions.

Journal of Bacteriology
Katherine M SchmalzerDara W Frank

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that utilizes a type III secretion system to subvert host innate immunity. Of the 4 known effector proteins injected into eukaryotic cells, ExoS and ExoU are cytotoxic. The cytotoxic phenotype of ExoU depends on the enzymatic activity of the patatin-like phospholipase A(2) domain localized to the N-terminal half of the protein. Amino acid residues located within the C-terminal region of ExoU are postulated to be required for trafficking or localization to the plasma membrane of eukaryotic cells. This report describes the characterization of a transposon-based linker insertion library in ExoU. Utilizing an unbiased screening approach and sensitive methods for measuring enzymatic activity, we identified regions of ExoU that are critical for activation of the phospholipase activity by the only known cofactor, SOD1. Insertions at D572 and L618 reduced the rate of substrate cleavage. Enzymatic activity could be restored to almost parental levels when SOD1 concentrations were increased, suggesting that the linker insertion disrupted the interaction between ExoU and SOD1. An enzyme-linked immunosorbent assay (ELISA)-based binding test was developed to measure ExoU-SOD1 b...Continue Reading

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Citations

May 13, 2011·American Journal of Respiratory and Critical Care Medicine·Matthew J WargoDeborah A Hogan
Jan 12, 2013·Current Protein & Peptide Science·Marlies GalleRudi Beyaert
Jan 1, 2013·Molecular and Biochemical Parasitology·María Laura BelaunzaránElvira Luisa Durante de Isola
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Nov 19, 2014·Journal of Bacteriology·David M AndersonDara W Frank
Aug 3, 2013·The Journal of Biological Chemistry·David M AndersonDara W Frank
Jan 19, 2021·The Biochemical Journal·Daniel M FoulkesStephen B Kaye

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