The EspF protein is translocated into host cells by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). EspF sequences differ between EPEC and EHEC serotypes in terms of the number of SH3-binding polyproline rich repeats and specific residues in these regions as well as residues in the amino domain involved in cellular localization. In this study we have compared the capacity of different espF alleles to inhibit: (i) bacterial phagocytosis by macrophages; (ii) translocation through an M-cell co-culture system; (iii) uptake by and translocation through cultured bovine epithelial cells. The espFO157 allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell co-culture system in comparison to espFO127 and espFO26. In contrast, espFO157 was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonisation site of EHEC O157 in cattle and a site containing M-like cells. As functional differences could not be simply assigned to variation in established interactions of EspF with Sorting Nexin 9 and N-WASP, yeast-2-hybrid screening was used to identify additional host proteins that may interact with EspF. The anaphase promoting complex inhibitor, Mad2L2, was identified from this screen. Mad2L2 was then demonstrated to interact with EspF variants from EHEC O157:H7, O26:H11 and EPEC O127:H6 by Lumier assays. While Mad2L2 has been shown to be targeted by the non homologous Shigella effector protein IpaB to limit epithelial cell turnover, we presume that EspF interactions with this protein may indicate a similar function to promote EPEC and EHEC colonization. The final section of work addressed whether bacterial interactions can actually induce M-cell differentiation on follicle-associated epithelium. The work focused on bovine rectal primary cell cultures interacting with Salmonella enterica serovar Typhimurium. The type III secreted protein, SopB, was required for Salmonella to: III (i) activate parts of epithelial to mesenchymal transition (EMT) pathway; (ii) transform a subset of epithelial cells to a cell type that phenotypically and functionally resembles specialized antigen sampling M cells; (iii) induce RANKL and downstream RelB dependent NFkB signaling. The work suggests that Salmonella may induce this cellular transformation to promote its invasion and colonization of intestinal mucosa.

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacteria responsible for significant morbidity and mortality in young children. EPEC elaborates a type III secretion system (T3SS), which translocates bacterial effector proteins into the host intestinal epithelial cell. To this date, 23 effector proteins are known to be secreted by EPEC. Over the past two decades, traditional studies uncovered the functions of some of these effector proteins. While there was an initial rise in the EPEC effector function discoveries, we now observe a plateau in the identification of host-EPEC interactions. Thus, the aim of my dissertation is to define novel virulence strategies in EPEC pathogenesis, and to demonstrate how traditional reductionist and global systems biology approaches can be utilized in uncovering functions of individual effectors, as well as the complex interplay of effectors in modulating host functions. Specifically, we defined the novel cytoprotective function of a T3SS effector EspZ. We further illustrated the complex interplay of EPEC effectors by defining how EPEC utilizes EspZ and EspF to dynamically regulate the prosurvival epidermal growth factor receptor signaling pathway. Finally, by integrating comparative proteomics and traditional reductionist approaches, we identified a novel function for EspH, and defined the mechanism by which EspH perturbs epithelial cell structure and function.

A three-stage Experimental Spatial Power Flow (ESPF) method which computes an experimentally derived, spatially continuous representation of the structural power in l-D and 2-D structures is presented. The three stages of the ESPF method consist of first using a scanning laser Doppler vibrometer (SLDV) to acquire spatially dense measurements of the structure's dynamic response. Second, the continuous 3-D complex-valued velocity field is solved from the laser data. Third, a spatially continuous model of the power is computed from the velocity-field model obtained in stage 2. The results of the ESPF method were validated by using both simulated and experimental laser data. In the simulated laser data cases, the power injected into a simply supported plate computed analytically, compared to within 1.33% of the power injected as computed by the ESPF method. In the experimental validation, three methods were used to compute the power injected and extracted from a simply supported plate forced with two shakers. The three methods consisted of the ESPF method and two methods of computing the injected power using impedance head type measurements. The injected power results were compared at four different frequencies. These frequencies were 79.0 Hz, 311.0 Hz, 909.0 Hz, and 1100.0 Hz. In all cases, the injected and extracted power results of the three methods compared to within 20% and to within 12% for all cases except the 909.0 Hz case. These results are currently better than other experimental techniques. Advantages of this method are 1) a spatially continuous representation of the power is computed 2) the affects of the actual boundary conditions and near-field effects of the structure are inherently measured by the SLDV 3) the SLDV does not affect the response of the structure by mass loading and is fully portable for in-field testing 4) the method allows for convergence of the power-flow vector field in addition to convergence of the velocity field and 5) the ESPF method is extendable to account for power due to in-plane motion and to account for shells of arbitrary geometry. / Ph. D.