Anchors Away : Determining the Role of Outer Membrane Proteins in Pseudomonas aeruginosa Vesicle Formation

ii DEDICATION First of all, I would like to thank Marvin for allowing me, back in January 2008, to work in his lab. This experience has enriched my undergraduate career by bringing textbook concepts to life. I am also grateful for the opportunity to have met and worked with such a great group of people. Thanks for this project go out to: Aimee Wessel, for her overall guidance and support (and coffee); Aishwarya Korgaonkar, for help with the TEM and MV preps; Holly Huse, for help with MV preps and data analysis; Tina Shay and Jeff Schertzer, for help with the lipid assay; and Greg Palmer, for providing criticism on the first draft of this thesis. Additionally, Stacie Brown has provided me with not only continued support, but also excellent baked goods. I would also like to thank Matt Ramsey and Megan Boulette for their time commitment while working with me on earlier projects. Lauren Warren, the originator of this project, deserves a big shout-out for her help and support from her faraway post in windy Chicago. Thanks, also, to my friends (especially Kathryn Busby and Katy Loeffler, who get the honor of being mentioned in both of my theses) and family, for understanding that a tad of madness is a necessary component of the scientific method. iii ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes chronic infections in the lungs of individuals with cystic fibrosis (CF). Like many Gram-negatives, P. aeruginosa produces outer membrane vesicles (MVs), which have been shown to package numerous factors including antimicrobial quinolone molecules, toxins, DNA, antibiotic resistance determinants, and cell-cell signaling molecules. The mechanism for the formation of MVs has not been fully elucidated. The Gram-negative outer membrane (OM) contains associated proteins, which anchor it to the peptidoglycan, and keep the OM stable. We hypothesized that peptidoglycan-associated outer membrane lipoproteins OprF, OprL, and OprI contribute to MV formation in P. aeruginosa. In this study, we quantified MVs harvested from oprF, oprL, and oprI mutants. The MV levels produced by the oprL and oprI mutants were not significantly different from those produced by the wild type; however, the oprF mutant showed a threefold increase in MV production. These data indicate that OprF plays a significant role in anchoring the outer membrane to the peptidoglycan and that, in its absence, more MVs are formed.