ELUCIDATION OF THE NEEDLE-TIP AND TIP-TRANSLOCON INTERACTIONS OF THE SALMONELLA SPI-1 TYPE III SECRETION SYSTEM AND IDENTIFICATION OF SMALL MOLECULE BINDERS OF THE TIP AND TRANSLOCON PROTEINS

Abstract

The type III secretion system (T3SS) is required by many pathogenic Gram-negative bacteria for the initiation and maintenance of infections within eukaryotic host cells. T3SS harboring bacteria include the causative agents of food poisoning/typhoid fever (Salmonella Typhimurium/Typhi), dysentery (Shigella flexneri/dysenteriae), nosocomial pneumonia (Pseudomonas aeruginosa), bubonic plague (Yersinia pestis), melioidosis (Burkholderia pseudomallei), and trachoma (Chlamydia trachomatis). Together, these bacteria are estimated to result in millions of deaths worldwide each year. Therefore, it is of great interest to elucidate the mechanisms of T3SS-mediated virulence utilized by pathogenic Gram-negative bacteria. Salmonella is the focus of this dissertation because it is an excellent model organism for T3SS research due to the ease of genetic manipulation and the availability of biological assays.The T3SS is utilized to inject bacterial virulence factors (also known as effectors) into the host cell cytoplasm, where they manipulate host cell signaling pathways to promote bacterial engulfment, maintenance of infection, and evasion of the host immune system. T3SS effectors are translocated across both bacterial and host cell membranes by the structural component of the T3SS, the needle apparatus. The needle apparatus contains a bacterial membrane embedded base structure, an extracellular needle with a 25A wide channel, a tip complex that regulates secretion and serves as an environmental sensor, and translocon proteins that assemble a pore in the host cell membrane. How the needle, tip and translocon proteins interact with each other to assemble a functional T3SS needle apparatus and coordinate the secretion of T3SS effectors is poorly understood. Because the needle, tip and translocon proteins are essential for the pathogenesis of T3SS harboring bacteria, are exposed to the extracellular environment during infection, and are conserved in structure and function, they are attractive targets for the development of novel virulence based anti-bacterial therapeutics. Hence, the importance of elucidating the structure, function and molecular interactions of the T3SS needle, tip and translocon proteins.This dissertation is focused on two major themes. The first theme is the elucidation of essential protein-protein interactions of the Salmonella T3SS needle apparatus through a combination of solution nuclear magnetic resonance (NMR) and fluorescence spectroscopy. To this end, I used amide (15N) and isoleucine, leucine and valine methyl (ILV 13C-methyl) probes in NMR titrations to map the interaction of T3SS proteins. I additionally labeled T3SS proteins with fluorescent probes to perform fluorescence polarization (FP) and Förster resonance energy transfer (FRET) protein- protein binding assays to complement the NMR studies. Using these methods, the interaction between the Salmonella SP-1 T3SS needle protein PrgI and the tip protein SipD, as well as between SipD and the major translocon protein SipB, are described in detail and validated using bacterial invasion assays. The results of NMR and FP/FRET experiments allowed for the proposal of a model for the needle/tip/translocon protein- protein interaction interface where the proximal end of SipD (the bottom of the coiled- coil) is used for interaction with PrgI, while the distal end of SipD (the top of the coiled- coil and the mixed α/β domain) is the surface used for interaction with SipB. The second theme is focused on the T3SS needle apparatus as an attractive target for the development of inhibitors. A review of the current T3SS inhibitor literature is described. In addition, I identified small molecules binders of the tip and translocon proteins from a surfaceivplasmon resonance (SPR) screen and subsequently validated and mapped the protein- small molecule interactions using titration and saturation transfer different (STD) NMR spectroscopy