|The type III secretion system (T3SS) is a macromolecular structure assembled by many Gram-negative bacteria in order to invade target host cells. A functional T3SS contains a syringe-like structural component known as the needle apparatus, which works in concert with an export apparatus that recognizes the cargo and an ATPase complex that energizes the transport of bacterial effector proteins. Effectors transported directly into the host cell cytoplasm modulate host cellular functions such as cytoskeletal dynamics and cellular signaling in order to enable the pathogens to invade, survive, and multiply within the host environment. Gram-negative bacteria harboring the T3SS include Salmonella, Shigella, enteropathogenic E. coli, Yersinia, Burkholderia, Pseudomonas, as well as Chlamydia. These organisms are responsible for infectious diseases in humans and pose a threat to human health worldwide. Inactivation of the T3SS, by knocking out structural or functional proteins, renders pathogens incapable of causing infection. Salmonella and Shigella are responsible for millions of cases of food-borne diarrhea annually throughout the world. In addition, large-scale food recalls due to frequent outbreaks of food poisoning has a negative impact on the food industry in the United States. No preventive vaccines are available against Salmonella and Shigella. Study of the T3SS thus has a scope for the development of strategies to combat these pathogens. The T3SS among different bacterial species share common features but also show unique structural and functional characteristics. Therefore, the T3SS provides a suitable target for the development of specific anti-infectives. The needle apparatus of the T3SS consists of a base followed by an extracellular needle. The needle is attached to a tip complex and a translocon. The tip complex serves as a platform for the assembly of the translocon that punctures a translocation pore within the host cell membrane. The tip complex is assembled from several copies of a hydrophilic tip protein and the translocon is assembled from two hydrophobic translocon proteins. This dissertation describes structural and functional studies, as well as characterization of the protein-protein interactions that are important in the assembly of the tip complex and the translocon of Salmonella and Shigella. In Salmonella, the tip complex is formed by the tip protein SipD. A translocon made up of the translocon proteins SipB and SipC is attached on the tip complex. A combination of X-ray crystallography, Nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, as well as functional assays were applied to determine the structure of the Salmonella tip protein SipD and show that the C-terminus is crucial for the function and an antiparallel β-sheet is important for the tertiary structure of SipD. The function of the T3SS can be regulated by small molecules such as bile salts. The T3SS of Salmonella is down regulated by bile salts. The tip protein SipD directly binds to the bile salt deoxycholate using an unknown mechanism. The crystal structures of SipD bound to bile salts reported herein show that the interaction between bile salts and SipD is predominantly hydrophobic. Further, deoxycholate induced the degradation of the Salmonella translocon protein SipB. These observations have led to the hypothesis that deoxycholate might interfere with the interaction between SipD and SipB leading to a down regulation of the T3SS in Salmonella. The structure of the translocon and how it is attached to the tip complex is not clear. Preliminary structural characterization of a folded, hydrophilic domain at the N-terminus of SipB was undertaken to locate two fragments of SipB within residues 82-240 and 82-226, which produced well-dispersed 2D-NMR spectra. Paramagnetic relaxation enhancement (PRE) was employed to define the interaction between SipD and the N-terminal hydrophilic domain of SipB. A region within residues Asp207-Asn283 of SipB bound to a mixed α/β region of SipD. PRE was also used to study how the Shigella tip protein IpaD binds to its needle protein MxiH in order to assemble the tip complex in Shigella. MxiH was shown to bind to the lower portion of a coiled coil region in IpaD. Secretion through the T3SS is enabled by extensive cross-talk within its components. The assembly of the needle apparatus requires polymerization of multiple copies of several different proteins. Regulation of secretion is most probably an outcome of conformational changes relayed in sequence through the needle apparatus. Work described in this dissertation shows that weak protein-protein interactions are a common theme in the assembly of the needle apparatus. Further, T3SS proteins contain discreet functional domains. For example, the coiled coil of the tip protein allows the assembly of the tip complex while the mixed α/β region attaches to the translocon. The structure of theT3SS proteins varies depending upon the role it plays. For example, the extracellular needle is assembled from a small polar protein but the translocon proteins contain both hydrophilic and hydrophobic domains. Nevertheless, further studies are required to fully appreciate certain aspects of the T3SS such as how large proteins are transported through the needle, how the T3SS switches between active and inactive states of secretion, how substrate specificity is controlled, and how effector secretion is energized. Complete understanding of theT3SS requires the determination of high resolution structures of the needle apparatus, direct binding studies analyzing how isolated components of the needle apparatus interact with each other and behave in vitro, computational modeling of larger substructures, and functional assays to test the physiological implications of these in vitro studies.