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Degree Name

Doctor of Philosophy



First Advisor

Michael S. Donnenberg


Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. The type IV pilus (T4P) of EPEC, known as the bundle-forming pilus (BFP), plays an important role in EPEC pathogenesis. T4Ps are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. One essential component of the BFP system is the secretin, BfpB. Secretins are a large family of integral outer membrane proteins found in T4Ps as well as type II and type III secretion systems, and filamentous phages. Details of the secretin structure have been limited to the overall shape, with atomic resolution of only the soluble amino-terminus domains, which impede our understanding of T4P biogenesis. The goals of this project are: 1) determine the structure of BfpB via cryo-electron microscopy; 2) define the amino-terminus domains of BfpB and its interactions with BfpU, an essential periplasmic protein of the system. We present a 7.1 Å resolution cryo-EM structure of BfpB, the first of a type IVb pilus secretin. Internal features suggest that BfpB is composed of 17 subunits with C17 symmetry. Structural and bioinformatic analyses suggests that monomeric BfpB possesses two amino-terminal domains, N0 and N3, which allowed us to successfully purify BfpB N0 and N0+N3 domains for interaction studies. Additionally, we have successfully purified BfpU for structure determination and interaction studies. Also, a random mutagenesis approach was used for further characterization of BfpU. Furthermore, surface plasmon resonance suggests the possibility that BfpB and BfpU interact with affinity in the micromolar range, but this result must be interpreted cautiously in light of similar interactions between BfpU and proteins chosen as negative controls. Results from these studies will not only further our understanding of BFP biogenesis, but also enhance research for understanding other T4Ps and secretion systems that are confirmed virulence factors.


© Janay I. Little

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