Intracellular trafficking of Pseudomonas aeruginosa ExoS within mammalian cells

This thesis is focused on the intracellular localization and trafficking of type III cytotoxin ExoS.; 1. Mapping the MLD and motif recognition. Deletion mapping showed that the MLD included a symmetrical leucine rich motif within residues 51--77 of ExoS. The terminal di-leucines and internal leucines and an isoleucine within the MLD, but not charged or other hydrophobic residues, targeted a reporter protein to the Golgi/ER region of HeLa cells. Mutations to the leucines within the MLD did not affect type-III secretion or translocation into HeLa cells, but limited the ability of ExoS to ADP-ribosylate Ras GTPases. Mutations to charged residues within the MLD did not affect type-III secretion, delivery into HeLa cells, or the ability of ExoS to ADP-ribosylate Ras GTPases. Deletion of either end of the MLD did not affect membrane association, but decreased membrane binding affinity. The organization of the leucines within the MLD of ExoS is different from classical leucine rich motifs, but is present in several other bacterial proteins. This implicates a role for intracellular targeting in the efficient targeting of mammalian cells by type-III cytotoxins.; 2. Intracellular localization and trafficking pathways of ExoS. ExoS is injected directly into host cells by type III secretion apparatus. At steady state ExoS localized to a peri-nuclear region, but how ExoS traffics to the perinuclear region is not known. Immuno-staining of type III delivered ExoS indicated an early and transient plasma membrane accumulation. In chase experiments, the turnover of ExoS plasma membrane association was ∼ 30--45 min. Compared with known cellular markers, ExoS at steady state accumulated in ER/Golgi region. Type III delivered ExoS ADP-ribosylated plasma membrane bound Ras and Golgi/ER bound Ras.; A co-immunoprecipitation experiment was used to gain insight into the cellular basis for the steady state association of ExoS with intracellular membranes. Tip47 and Rab9, as well as Rab6, co-immunoprecipitated with type-III delivered intracellular ExoS. Localization of Rab9 and Rab6 also partially overlapped with localization of type III delivered intracellular ExoS. This indicated that ExoS was localized on a late endosome vesicle. However, single knockdown of either Rab9A or Rab6A did not block any trafficking or steady state localization of ExoS, indicating vesicle-mediated multiple trafficking routes of ExoS to the Golgi/ER.; An inhibitor screening has been conducted to further elucidate intracellular trafficking of the ExoS. Nocodazole, which disrupts microtubles, partially inhibited ExoS ADP-ribosylation activity, whereas Cytochalasin D, an inhibitor of actin cytoskeleton, did not affect localization or activity of ExoS. Wortmannin also partially attenuated ExoS activity for Golgi/ER bound Ras, showing the physical delivery of ExoS to the plasma membrane and Golgi/ER.; 3. How the MLD affects ExoS RhoGAP activity and specificity. Previous studies showed that membrane localization was essential for ExoS to ADP-ribosylate Ras GTPases. Next, the relationship between intracellular localization and expression of RhoGAP activity was investigated. Loss of MLD function abolished expression of ExoS RhoGAP activity in HeLa cells. One mutation within the MLD, (RRD→3N), changed several properties of ExoS, including loss of detectable plasma membrane localization and altered the cell rounding phenotype elicited by Rho GAP to that elicited by Yersinia YopE. Cell rounding caused by ExoS MLD-(RRD→3N) was reversed by Dominant Active Rac1, but not Dominant Active Cdc42, a phenotype identical to YopE, indicating a switch in RhoGAP specificity. The C-terminal polybasic region mutation abolished the ability of Dominant Active Rac1 to protect cells for ExoSMLD (RRD→3N) mediated Rho GAP activity. Together, ExoS MLD contributes to the ability of RhoGAP to express Rho GAP activity within cultured cells, and charge residues within MLD influence the abili...