Date of Award

Spring 1-1-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Marcelo C. Sousa

Second Advisor

Natalie G. Ahn

Third Advisor

Rob Knight

Fourth Advisor

Amy E. Palmer

Fifth Advisor

Thomas T. Perkins

Abstract

Pathogenic bacteria have developed numerous mechanisms to subvert host cell physiology and create unique niches that facilitate colonization. Within γ-proteobacteria, a specialized molecular machine, the Type III Secretion System (T3SS), is responsible for translocating bacterial proteins directly into host cells. These virulence factors, called effectors, rewire various host cell processes for bacterial gain and are the key for a successful infection. Because many effectors share little if any sequence similarity to proteins of known function, biological knockouts, biochemical assays, and structural studies are necessary to elucidate their functions in pathogenesis.

Enterohemmorhagic E. coli (EHEC) encodes its T3SS on a chromosomal pathogenicity island that also contains effectors essential for its unique attaching and effacing phenotype when infecting epithelial cells. However, numerous effectors outside of this pathogenicity island are required to suppress inflammation and the host immune response, and are thus crucial for infection. Identified as a zinc protease of NFκB transcription factors, NleC is one such effector that is essential for EHEC suppression of inflammatory cytokine secretion.

Here, I show that NleC cleaves NFκB transcription factors with high specificity, recognizing residues proximal to the cleavage site in the NFκB DNA-binding loop. However, elements outside of this site are also necessary for recognition by NleC making it an unusual protease with exquisite selectivity. I determined the crystal structure of NleC revealing that despite undetectable sequence similarity to other Zn2+proteases, it is a member of the Zincin zinc protease fold superfamily. Nevertheless, NleC displays distinct structural differences from currently known Zincins that may contribute to its extraordinary selectivity and the unique folding/refolding requirements implicit in translocation. Finally, I propose that NleC recognizes the NFκB DNA-recognition sequences by mimicking DNA in both shape and electrostatic character. This would make NleC the first example of both a DNA-mimicking T3SS effector, and a DNA-mimicking protease, representing a truly unique mechanism for subverting host signaling pathways for the benefit of the pathogen.

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