Gutierrez, Miguel Angel - The minor pilin TcpB is located at the tip of the toxin co-regulated pilus of Vibrio cholerae and i...

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Term: 
Summer 2017
Degree: 
M.Sc.
Degree type: 
Thesis
Department: 
Department of Molecular Biology and Biochemistry
Faculty: 
Science
Senior supervisor: 
Lisa Craig
Thesis title: 
The minor pilin TcpB is located at the tip of the toxin co-regulated pilus of Vibrio cholerae and is the receptor for the filamentous phage CTXφ
Given Names: 
Miguel Angel
Surname: 
Gutierrez
Abstract: 
Type IV pili are polymers of the major pilin subunit found on the surfaces of many Gram negative bacteria. They act like grappling hooks, undergoing cycles of polymerization, adhesion, and retraction, to mediate a diverse array of functions, including twitching motility, DNA uptake and adhesion. T4P possess several minor pilins, which are homologous to the major pilin but are produced in much lower quantities. Minor pilins prime Type IV pili assembly and have been proposed to localize to the tip of the pilus, but this has not been shown definitively. The Vibrio cholerae toxin co-regulated pilus (TCP) is a T4P that mediates microcolony formation, which is critical for the development of the gastrointestinal disease cholera. TCP is the primary receptor for the filamentous cholera toxin phage CTXφ, which binds to the pilus via its tip-associated protein, pIII. TCP possess a single minor pilin, TcpB, which initiates pilus assembly as well as retraction. We hypothesized that TcpB is located at the tip of the pilus and forms the binding site for CTXφ pIII. Here I use direct and competition ELISA to show that recombinantly expressed soluble TcpB and pIII interact. I show that CTXφ phage infection of V. cholerae is reduced 90 % in the presence of soluble TcpB or anti-TcpB antibody. Furthermore, gold-labeled anti-TcpB antibody binds to the tip of purified TCP, providing the first direct localization of a minor pilin to the tip of a T4P. Finally, I show that phage uptake is reduced 98 % in a retraction-deficient V. cholerae strain, demonstrating the role of pilus retraction in this process. My results define a two-step mechanism for CTXφ infection of V. cholerae, which involves (i) binding of CTX via its tip-associated pIII protein to its receptor, TcpB, at the tip of the pilus, and (ii) retraction of the pilus, which pulls CTXφ into the bacterial periplasm as if it were an extension of the pilus.
Keywords: 
Type IV pili; Vibrio cholerae; Toxin co-regulated pili; Cholera toxin bacteriophage; Minor pilin
Total pages: 
103