The long range goal of the proposed work is to define the molecular components and mechanisms mediatingVibrio cholerae colonization and virulence protein secretion to the point where there is sufficient knowledgeto intelligently incorporate this information into improved cholera vaccine strategies and antimicrobialtherapies designed to inhibit these events. Most of the proposal involves analysis of the molecularmechanisms by which toxin coregulated pilus (TCP) is formed and mediates intestinal colonization. Somesteps in the process by which TCP and other type 4 pili are built are linked and/or related to the process oftoxin and other virulence determinant secretion by type II secretion systems. Thus further understanding ofthe mechanisms of type 4 pilus biogenesis should lead to the characterization of potential antimicrobialtargets involved in multiple virulence pathways. We will examine the aspects of pilus biogenesis in detail.These experiments will be facilitated by our currently available collections of tcp genetic constructs andimmunoreagents. Regarding the mechanism of TCP function, we will utilize the technique of field emissionscanning electron microscopy in combination with specifially engineered tcpA missense mutations and atcpB deletion mutant to address the biophysical mechanisms that lead to pilus supertwist formation,promoting the bacterial associations that represent the basis of microcolony formation in the intestine.Further characterization of TcpF and its role in colonization will be undertaken. This analysis is aided by therecent solution of the crytsal structure of TcpF and the mapping of a functional domain of the protein withinthis structure. Additional steps in the epithelial interaction with V. cholerae will be defined using carbohydratebinding analyses. Taken together, the results of the proposed studies will provide detailed informationregarding the mechanisms of V. cholerae colonization from the levels of defining the components,understanding how they are elaborated to the cell surface and beyond.
(See Instructions):Diarrheal diseases caused by enteric infectious bacteria continue to cause significant morbidity and mortalityworld-wide, including within the US. Despite years of sophisticated research on V. cholerae, the causativeagent of cholera, there is still no effective vaccine. The type 4 pilus, toxin coregulated pilus (TCP), of V.cholerae is the major colonization factor and represents a paradigm for type 4 pilus biogenesis and functionthroughout the realm of enteric bacteria. The proposed studies are relevant to the prevention and cure ofcholera as well as a number of other enteric diseases caused by gram-negative enteric bacterial infectionsworld-wide, which addresses major goals of the NIH mission.