Date of Award
Doctor of Philosophy (PhD)
Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. These translocated proteins possess a diverse array of biochemical activities, reprogramming eukaryotic cell biochemistry to serve the requirements of the pathogen. Examples of translocated effector proteins hijacking essential host functions such as cytoskeleton assembly, vesicular transport, and apoptosis are numerous (Galan and Wolf-Watz, 2006). However, until recently, less progress had been made with bacteria that also possess virulence mechanisms that target the host cell cycle. Studies have demonstrated the capabilities of pathogenic bacteria in modulating host-cell vital processes with a growing family of bacterial toxins and effectors that interfere with the eukaryotic cell cycle (Nougayrede et al., 2005; Oswald et al., 2005). The EPEC and EHEC T3SS effector Cycle inhibiting factor (Cif) is able to block host eukaryotic cell cycle progression. We present here the crystal structure of Cif. It is a divergent member of a superfamily of enzymes sharing a common Cys-His-Asp/Asn catalytic triad, which includes cysteine proteases and acetyltransferases. Mutation of these conserved active site residues abolishes the ability of Cif to block cell cycle progression in different models. We demonstrate that Cif possesses auto-acetylation activity that is dependent on a wild-type catalytic triad, whereas irreversible inhibitors of cysteine protease activity do not affect the cytopathic phenotype. Finally, we identify the cell cycle regulatory protein Nedd8 as an interaction partner for Cif. Cif binds Nedd8 in vitro through both the catalytic domain and the N-terminal domain, the latter of which we demonstrate to be absolutely required for Cif activity in the host cell.
Hsu, Yun-Yuan, "Structural Analysis of Cycle Inhibiting Factor from Pathogenic Escherichia Coli" (2008). Student Theses and Dissertations. 197.