Campylobacter jejuni is the leading bacterial cause of human gastroenteritis in the United States. Increasing resistance of Campylobacter to clinical antibiotics raises an urgent need for novel strategies to prevent and control infections in humans and animal reservoirs, which necessitates a better understanding of Campylobacter pathogenesis. We hypothesize that multidrug efflux pump CmeABC and ferric enterobactin (FeEnt) iron acquisition systems, which play a critical role in Campylobacter pathogenesis, are novel targets for developing effective measures against Campylobacter. To test this, the molecular, antigenic, functional, and protective characteristics of two outer membrane proteins, CmeC (an essential component of CmeABC drug efflux pump) and CfrA (a FeEnt receptor), were examined. Both CmeC and CfrA are highly conserved and widely produced in C. jejuni strains. Anti-CmeC and Anti-CfrA antibodies inhibited the function of CmeABC efflux pump and CfrA, resulting enhanced susceptibility to bile salts and reduced utilization of FeEnt of C. jejuni, respectively. Immunoblotting analysis also indicated that CfrA is expressed and immunogenic in vivo. Amino acid substitution mutagenesis demonstrated that a highly conserved basic amino acid R327 in CfrA plays a critical role in FeEnt acquisition. The purified recombinant CmeC and a Salmonella live vaccine expressing the protective epitope of CfrA were evaluated as subunit vaccines against Campylobacter infection in the chicken model. CmeC vaccination elicited immune response but failed to reduce C. jejuni colonization in the intestine. However, Salmonella-vectored vaccine conferred significant protection against C. jejuni challenge. To further elucidate the role of iron acquisition in the pathogenesis of Campylobacter, whole genome sequence of a unique C. jejuni strain was determined using a 454 GS FLX sequencer with Titanium series reagents. Comparative genomics analysis led to the identification of a novel Campylobacter Enterobactin Esterase (Cee) that is essential in the CfrB-dependent FeEnt utilization pathway. Extensive genetic manipulation revealed molecular pathways and mechanistic features of the two orchestrated FeEnt acquisition systems in Campylobacter. This project provides critical information about the feasibility of targeting CmeC and CfrA for immune protection against Campylobacter colonization in the intestine, and increases our understanding of the critical role of FeEnt acquisition in the pathophysiology of Campylobacter.

This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel. In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.