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Molecular characterization of the TCP biogenesis apparatus in Vibrio cholerae.

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Call No.	Ph.D. 2004
Author	Bose, Niranjan.
Title	Molecular characterization of the TCP biogenesis apparatus in Vibrio cholerae.
Date	2004
Abstract	Vibrio cholerae is a water-borne, Gram negative enteropathogen that causes the gastrointestinal disorder, cholera, in humans. A critical step in cholera pathogenesis is the attachment and colonization of intestinal cells, and the formation of bacterial microcolonies. Type 4 pili serve as attachment and colonization factors for many Gram negative bacteria, and a type 4 pilus, the toxin coregulated pilus (TCP), serves as a critical colonization factor for V. cholerae. In our efforts to better understand TCP biogenesis, we created in-frame deletions in the tcp genes, and studies revealed that all the tcp genes except tcpF were required for TCP biogenesis. This lead to the identification of TcpF, a secreted colonization factor, which utilizes a Sec dependent mechanism to enter the periplasm and exits the periplasm using the type 4 pilus. We utilized the set of in-frame deletions to further characterize the biogenesis apparatus. We identified a TcpC-TcpQ outer membrane (OM) complex that is required for TCP biogenesis. Furthermore, we determined that TcpC is the secretin (OM pore) for the biogenesis apparatus. The interaction between TcpC and TcpQ was confirmed using biochemical methods, and TcpQ was demonstrated to have a role in the stability and proper localization of TcpC. By using tonB mutants, we determined that TonB mediated proton motive force is not required for type 4 pilus biogenesis and type II secretion systems. Pilus biogenesis and protein secretion were affected in cognate ATPase mutants, suggesting that ATP is the prime source of energy for these systems. Further efforts lead to the identification of a putative TcpQ-TcpS-TcpD periplasmic bridge, which has lead us to believe that this transmembrane bridge could either be serving as an energy transducer or a translocation channel. Finally, heterologous expression studies in Escherichia coli and cross-complementation studies performed using enteropathogenic E. coli components have demonstrated specificity for TCP biogenesis.
Description	xii, 322 leaves : ill. (some col.) 28 cm.
Format	application/pdf
Advisor	Ronald K. Taylor.
Subject	Vibrio cholerae -- Molecular genetics. Pili (Microbiology). Cholera -- Pathogenesis. Proteins -- Secretion.
College	Dartmouth Medical School -- Department of Biochemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.48

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Full Text	Molecular characterization of the TCP biogenesis apparatus in Vibrio cholerae A Thesis submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by Niranjan Bose DARTMOUTH COLLEGE Hanover, New Hampshire October 31, 2004 Examining Committee ________________________ Ronald K. Taylor, Ph.D. (Chair) ________________________ Ambrose Cheung, M.D. ________________________ George A. O’Toole Jr., Ph.D. ________________________ ________________________ Charles K. Barlowe, Ph. D. Matthew R. Chapman, Ph.D. Dean of Graduate Studies

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