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REGENTS OF THE UNIVERSITY OF COLORADO, THE

The envelope of Gram-negative bacteria consists of two membranes separated by the periplasmic compartment that contains the peptidoglycan wall. The inner membrane (IM) is in contact with the cytosol while the outer membrane (OM) contacts the extracellular environment. The OM is a unique structure, essential for Gram-negative bacteria, composed of lipopolysaccharide (LPS), phospholipids and proteins. It is a very selective permeability barrier that allows the bacteria to survive in hostile environments such as the gut, where the OM resistance to bile salts allows enteric bacteria to thrive. The components of the OM are the first to come in contact with a host upon infection and strongly modulate the interaction of symbiotic and pathogenic bacteria with their host. A clear grasp of the OM biogenesis process is essential to understand host-pathogen interactions as well as a fundamental aspect of bacterial physiology. Outer membrane proteins (OMPs) are integral membrane proteins with 2-barrel structures embedded in the OM. Many OMPs are immunogenic and some of them serve as adhesins mediating adhesion and colonization of host tissues. OMPs are synthesized in the cytosol and translocated across the IM by the SEC translocation machinery. However, how these hydrophobic proteins cross the periplasm and insert specifically into the OM is poorly understood. A number of periplasmic proteins and one OMP (Omp85/YaeT) have been implicated in the transport and insertion of OMPs. In this proposal we will establish the mechanisms of OMP transport and assembly focusing on the following proteins: (i) the YaeT complex; a protein complex embedded in the outer membrane containing YaeT and the lipoproteins YfiO, YfgL, SmpA and NlpB, proposed to work as the machine inserting OMPs into the outer membrane; (ii) SurA, a periplasmic chaperone that facilitates OMP folding and insertion; and (iii) the Seventeen Kilodalton Protein (Skp); a trimeric periplasmic chaperone that prevents protein aggregation and is proposed to escort OMPs across the periplasm and assist in their insertion into membranes. A combination of crystallographic, NMR and biochemical approaches will be brought to bare on this crucial mechanistic problem. PUBLIC HEALTH RELEVANCE: Transport and assembly of outer membrane proteins is an essential process in bacteria required for viability. Interfering with this process can be an effective strategy for antibiotic development much like beta-lactams are effective by interfering with cell wall synthesis. This proposal is focused on understanding the mechanisms controlling transport and assembly of outer membrane proteins.

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