Molecular and cellular pathogenesis of Pseudomonas type III cytotoxins, ExoS/ExoT

Bacterial pathogens utilize toxins to modify or kill host cells. The bacterial ADP-ribosyltransferases are a family of protein toxins that covalently transfer the ADP-ribose portion of NAD to host proteins. Each bacterial ADP-ribosyltransferases toxin modifies specific host protein(s) that yields a unique pathology. ExoS/ExoT are type III cytotoxins of Pseudomonas aeruginosa that possess ADP-ribosyltransferase activity.;P. aeruginosa is a Gram negative, opportunistic pathogen that primarily affects immuno compromised patients. ExoT, a type III delivered cytotoxin by P. aeruginosa, is a bi-functional cytotoxin possessing both RhoGAP and ADP-ribosyltransferase activities. ADP-ribosyltransferase activity elicits anti-internalization activity independent of RhoGAP function. ExoT ADP-ribosylates CT10 regulator of kinase I and II (Crk I and Crk II), which are SH2 and SH3 domain containing eukaryotic adaptor proteins involved in the intergrin signaling pathway leading to phagocytosis. Mass spectroscopic analysis identified R20 as the site of ADP-ribosylation by ExoT. R20 is a conserved residue located within the SH2 domain that is required for interactions with phosphotyrosine on upstream signaling molecules, such as Paxillin and p130-C&barbelow;rk A&barbelow;ssociated S&barbelow;ubstrate (p130Cas). GST-pull down and Far Western assays showed that ADP-ribosylated-Crk or Crk (R20K) failed to bind p130Cas or Paxillin, indicating that ADP-ribosylation inhibited the direct interaction of Crk with these focal adhesion proteins. Over-expression of wild-type Crk I or CA Rac1 reduced cell rounding caused by ExoT. Thus, the ADP-ribosylation of Crk uncouples intergin signaling by direct inhibition of the binding of Crk to focal adhesion proteins.;P. aeruginosa ExoS is a bi-functional type III cytotoxin that disrupts Ras- and Rho-signaling pathways in mammalian cells. A hydrophobic region (residues 51-77, termed the Membrane Localization Domain (MLD) targeted ExoS to the plasma membrane (PM) and late endosomes of host cells. Metabolic inhibitors and dominant negative proteins that disrupt known vesicle trafficking pathways were used to define the intracellular trafficking of ExoS. Release of type III delivered ExoS from the PM was independent of dynamin and Arf 6, but was inhibited by methyl-beta-cyclodextrin (MbetaCD), a cholesterol depleting reagent. Perinuclear localization of ExoS was disrupted by nocodazole, while p50 dynamintin, a dynein inhibitor, partially disrupted perinuclear localization of ExoS. MbetaCD and nocodazole inhibited the ability of type III delivered ExoS to ADP-ribosylate Golgi/ER resident Ras. MbetaCD also relocated ExoS from the perinuclear region to the PM, indicating that ExoS can cycle through anteriograde as well as retrograde trafficking pathways. These findings show that ExoS endocytosis is cholesterol dependent and ExoS utilizes host microtubules for intracellular trafficking.;The RhoGAP and ADPr activities of ExoS were tested for the ability to disrupt mammalian epithelial cell physiology. RhoGAP inhibited internalization/phagocytosis of bacteria, while ADPr inhibited vesicle trafficking, including both general fluid phase uptake and epidermal growth factor (EGF)-activated EGF receptor (EGFR) degradation. In ADPr-intoxicated cells, upon EGF activation, EGFR co-localized with clathrin coated vesicles (CCV) which did not mature into Rab5 positive early endosomes. Constitutively active Rab5 recruited EGFR from CCV to early endosomes. Consistent with the inhibition of Rab5 function by ADPr, several Rab proteins including Rab5 and 9, but not Rab4, were ADP-ribosylated by ExoS. Thus, the two enzymatic activities of ExoS have different effects on epithelial cells. The ability of ADP-ribosylation to inhibit mammalian vesicle trafficking provides a new mechanism for bacterial toxin-mediated virulence.;Understanding the mode of action of ExoS/ExoT may provide novel therapies to treat human disease, and may be used as tools to dissect host cell physiology.