PROJECT SUMMARY
The Gram-negative outer membrane is an effective permeability barrier against numerous chemical
assaults, including antibiotics. The intrinsic barrier capacity of the outer membrane is at least in part due
to its asymmetric nature; the inner leaflet consists of phospholipids while the outer leaflet is composed of
lipopolysaccharide (LPS). The ability of the outer membrane to serve as a permeability barrier relies on
strong lateral interactions between neighboring LPS molecules, which are disrupted by the presence of
phospholipids in the outer leaflet. In addition, the biosynthetic pathways responsible for LPS and
phospholipid production utilize a shared pool of biosynthetic precursors, making it critical that the flux
through both pathways is balanced to allow equitable access to the precursor pool. Several outer
membrane homeostasis factors that confine phospholipids to the inner leaflet or balance LPS and
phospholipid biosynthesis have been well characterized in enterobacteria such as Escherichia coli, but
little is known about outer membrane homeostasis in the human pathogen Pseudomonas aeruginosa.
Further, the increasing rates of multidrug resistant P. aeruginosa highlight the need for a deeper
understanding of the factors required to maintain the barrier capacity of its outer membrane. The
proposed research aims to characterize outer membrane homeostasis in P. aeruginosa. In Aim 1, we will
investigate how flux through the LPS biosynthetic pathway is controlled to fit the needs of the cell. Our
preliminary data suggest that LpxC, which catalyzes the second and committed step in the LPS
biosynthetic pathway, is regulated by a novel mechanism in P. aeruginosa, which will be genetically and
biochemically characterized during the fellowship period. In Aim 2, we will identify and characterize novel
factors required for outer membrane homeostasis in P. aeruginosa, including those responsible for the
maintenance of outer membrane asymmetry as well as those involved in regulating LPS and phospholipid
biosynthesis. Overall, this work will reveal fundamental mechanisms important for drug-resistance in P.
aeruginosa with the potential to guide future antibiotic development. The proposed research has been
designed to build on the applicant's pre-existing skillset while providing ample training opportunities in
new techniques. The work will be completed in a collaborative environment fostered by the sponsor's lab
and the Harvard Medical School Department of Microbiology. In addition, the applicant will regularly
attend scientific conferences and also participate in career development workshops that cover topics
such as scientific communication and lab management. Thus, the applicant will receive comprehensive
training in the skills needed for a successful career as an independent researcher.