Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.

Arc crust-magma interaction in the Andean Southern Volcanic Zone from thermobarometry, mineral composition, radiogenic isotope and rare earth element systematics of the Azufre-Planchon-Peteroa volcanic complex, Chile

The Andean Southern Volcanic Zone (SVZ) is a vast and complex continental arc that has been studied extensively to provide an understanding of arc-magma genesis, the origin and chemical evolution of the continental crust, and geochemical compositions of volcanic products. The present study focuses on distinguishing the magma/sub-arc crustal interaction of eruptive products from the Azufre-Planchon-Peteroa (APP 35°15'S) volcanic center and other major centers in the Central SVZ (CSVZ 37°S–42°S), Transitional SVZ (TSVZ 34.3–37.0°S), and Northern SVZ (NSVZ 33°S–34°30'S). New Hf and Nd isotopic and trace element data for SVZ centers are consistent with former studies that these magmas experienced variable depths of crystal fractionation, and that crustal assimilation is restricted to the lower crustal depths with an apparent role of garnet. Thermobarometric calculations applied to magma compositions constrain the depth of magma separation from mantle sources in all segments of the SVZ to(70-90 km). Magmatic separation at the APP complex occurs at an average depth of ~50 km which is confined to the mantle lithosphere and the base of the crust suggesting localized thermal abrasion both reservoirs. Thermobarometric calculations indicate that CSVZ primary magmas arise from a similar average depth of (~54 km) which confines magma separation to the asthenospheric mantle. The northwards along-arc Sr-Nd-Hf isotopic data and LREE enrichment accompanied with HREE depletion of SVZ mafic magmas correlates well with northward increasing crustal thickness and decreasing primary melt separation from mantle source regions indicating an increased involvement of lower crustal components in SVZ magma petrogenesis. ^ The study concludes that the development of mature subduction zones over millions of years of continuous magmatism requires that mafic arc derived melts stagnate at lower crustal levels due to density similarities and emplace at lower crustal depths. Basaltic underplating creates localized hot zone environments below major magmatic centers. These regions of high temperature/partial melting, and equilibration with underplated mafic rocks provides the mechanism that controls trace element and isotopic variability of primary magmas of the TSVZ and NSVZ from their baseline CSVZ-like precursors.^