Regulation of Expression of Adherence Factors

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease in infants and small children and in travelers. The first step in establishment of infection is attachment of the pathogen to the host tissue, which is believed to be mediated by pili (and/or thinner, more flexible fibrillar structures). The major long-term goals of this work are to understand the mechanisms by which pili are synthesized ad the mechanisms that regulate their synthesis. A limited number of serologically different pili are synthesized and the mechanisms that regulate their synthesis. A limited number of serologically different pili appear to be prevalent among ETEC strains isolated from human disease. Thse include CS1 and CS2, studies in this project, as well as Cfa/I (which appears to be similar, based on DNA sequence), CS4 and CS5. The genes needed for synthesis of human ETEC pili are completely unrelated to those identified for morphogenesis of the better studied E. coli pili like pap and type 1. CS1 pili appear to be much simpler structures, so it seems possible that a detailed understanding of their morphogenesis is within reach. Aims 1 and 2 of this proposal are directed at approaching this. (Aim 1: Characterize the morphogenesis of CS1 pili by determining the role of each gene product in pilus assembly and Aim 2: Characterize protein interactions in pilus assembly and adherence.) CS1 and CS2 are positively regulated by Rns, which shows homology to AraC, as do global regulators of virulence determinants of many enteric pathogens. A Rns homolog seems to be necessary for expression of many different serological types of ETEC pili, as well as pili of other diarrheagenic E. coli strains. Current information indicates that regulation of expression of and by Rns is unusual, involving DNA regions both upstream and downstream of the regulated promoter. In addition, it may involve a cascade of regulatory factors. Thus, because it may illustrate new principles of control of gene and because it is likely to be important for bacterial pathogenesis, regulation of expression of CS1 pili will be analyzed further. (Aim 3: Determine the mechanism of regulation by rns and Aim 4: Investigate other regulation of CS1.) Pili appear to be good candidates for development of human anti-ETEC vaccines because of their limited serological variability, as well as their surface localization and immunogenicity, and because of the success of pili based vaccines in prevention of diarrheal diseases in domestic animals. This work should facilitate the development of such vaccines.