Pseudomonas aeruginosa ExoS: Characterization of in vivo targets of ADP-ribosylation

P. aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in cystic fibrosis, immunocompromised, and burn patients. Pathogenesis stems from a number of virulence factors, including four type-III delivered cytotoxins: ExoS, ExoT, ExoY, and ExoU. ExoS contains an N-terminal Rho GTPase Activating Protein (GAP) activity which inactivates the Rho GTPases Rho, Rac, and Cdc42 and a C-terminal polysubstrate specific ADP-ribosyltransferase activity which covalently transfers an ADP-ribose moiety onto numerous host proteins, including Ras. The ADP-ribosylation domain is cytotoxic to mammalian cells. This thesis focuses on three aspects of ExoS function:; ExoS contains a Membrane Localization Domain (MLD) (residues 51--72), which targets intracellular localization to the Golgi/ER of mammalian cells. The MLD is responsible for the aggregative properties of ExoS. Deletion of the MLD yields soluble, non-aggregative and enzymatically active ExoS, which can be purified to homogeneity in E. coli. ExoS(DeltaMLD) is a useful reagent for enzymatic, structural, and biophysical studies.; ExoS focuses with pI heterogeneity. The nature of this modification was characterized by Mass spectrometry and gel elution analysis. ExoS heterogeneity was not caused by a mass addition, but was an intrinsic charge variance of the native protein. ExoU and ExoT from P. aeruginosa, and YopE from Yersinia, also display charge conformers, suggesting that this is a general property of type-III toxins.; ExoS induces cell rounding and death in HeLa cells. ExoS(DeltaMLD), ADP-ribosylates Ras inefficiently, yet is also cytotoxic in HeLa cells, indicating the ADP-ribosylation of Ras was not responsible for cytotoxicity. This prompted the identification and characterization of host proteins that are early targets for ADP-ribosylation by ExoS. Five proteins, moesin, ExoS, ubiquilin 1, Hsp27, and Ras, were early and efficient substrates of ADP-ribosylation by ExoS. Over expression of Hsp27, an anti-apoptotic cell survival protein, delayed ExoS-mediated cell death, suggesting that ADP-ribosylation of Hsp27 may contribute to the cytotoxic action of ExoS. ExoS ADP-ribosylated Moesin, Ezrin, and Radixin (ERMs) with a Km ∼100-fold lower than Ras. ADP-ribosylation inhibited ERM phosphorylation in vivo and in vitro. Inhibition of the phosphorylation of ERMs may down-modulate cytoskeleton function, preventing the activation of Rho and/or stabilization of actin filaments.; The identification and characterization of physiologic substrates of ExoS ADP-ribosyltransferase has utility not only in the development of therapeutics aimed at P. aeruginosa virulence factors but also as guides for using ExoS as a tool to study the interworkings of mammalian protein function.