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Structural characterization of bacterial type III secretion system components Yip, Calvin K.


The virulence-associated type III secretion system (T3SS) mediates the direct translocation of bacterial proteins known as effectors into the cytoplasm of eukaryotic cells, a process essential to the pathogenesis of many Gram negative pathogens. In this thesis, the molecular architecture of the T3SS was investigated through the biochemical and structural characterization of four representative components: EspA, EscJ, and EscC from enteropathogenic Escherichia coli (EPEC), and PrgH from Salmonella typhimurium. EspA is a component of the EPEC T3SS that assembles into extracellular filaments believed to be the molecular conduit for protein translocation. Results from biochemical analysis showed that EspA alone is sufficient to form filamentous structures and that an intact C-terminal coiled coil segment is required for oligomerization. CesA, the EspA-specific chaperone, was found to trap EspA in a monomeric state. Crystallographic analysis of the heterodimeric CesA-EspA complex at 2.8Å revealed that EspA contains two long α-helices, which are engaged in extensive coiled coil interactions with CesA. EPEC EscJ is a member of the highly conserved YscJ family of proteins believed to form the inner membrane ring substructure of the T3SS. Sucrose gradient experiments on EPEC membranes showed that EscJ localizes to the inner membrane. The crystal structure of EscJ, refined to 1.8Å, revealed repetitive and extensive intersubunit packing in the crystal lattice, which allowed the construction of a 24-subunit ring model. Data from stoichiometric and surface mapping analyses of Salmonella typhimurium PrgK validated this model, which possesses surface features indicative of a role as an assembly platform. Salmonella typhimurium PrgH and its distant orthologues represent the second major component of the inner membrane ring complex. Detergent extraction experiments confirmed that PrgH is a membrane protein. A putative transmembrane segment appears to target this protein to the membrane as the predicted cytoplasmic and periplasmic regions of PrgH could exist as soluble, independent folding domains. The crystal structure of PrgH(170-362), the core periplasmic domain, refined to 2.3Å resolution, showed that it possess two EscJ-like domains, which may be involved in intersubunit interactions. The outer membrane channel elaborated by the secretin (YscC) family of proteins represents the second major ring complex in the T3SS. Limited proteolysis and protein expression studies of EscC, the EPEC T3SS secretin, confirmed the modular nature predicted for this family. Surface electrostatic analysis of the crystal structure of the N-terminal domain of EscC, EscC(22-174), determined to 2.2Å, revealed two unique charge patches. Mutagenesis and complementation experiments demonstrated that one of these patches is required for proper function of EscC.

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