The current diphtheria epidemic in the former Soviet Union has resulted in a heightened interest in the bacterium Corynebacterium diphtheriae, the causative agent diphtheria. With the exception of diphtheria toxin, no studies have been done to identify virulence determinants in this important bacterial pathogen. Additionally, little is known on how C. diphtheriae invades, colonizes and survives within the human host. The acquisition of iron is an important virulence determinant for many bacterial pathogens, and numerous virulence factors including diphtheria toxin are regulated by iron.
The aim of this project is to identify and characterize putative iron-regulated virulence determinants from C. diphtheriae. A number of approaches will be employed in order to clone these iron-regulated genes; 1) Expression cloning of iron and dtxr (diphtheria toxin repressor) regulated promoters from C. diphtheriae will be done. Genes downstream from the iron-regulated promoters will be sequenced and tested for regulation and expression. Homologous genes from other species will be identified in an attempt to determine the possible function for these iron-regulated gene products; 2) A repressor titration assay using dtxR and a plasmid library carrying DNA fragments from C. diphtheriae will be used as an alternative method to identify iron-regulated promoters and their associated operons. This procedure was originally developed for the E. coli Fur (ferric uptake repressor) protein, and will be modified for use with the DTXR protein; 3) Convalescent serum from diphtheria patients will be that are specifically expressed during infection. These proteins will be purified and a partial amino acid sequence will be determined so that DNA oligo primers could be designed to locate chromosomal genes in C. diphtheria which encode these immunogenic proteins.; 4) Experiments will be done to determine if C. diphtheriae possesses cell surface receptors for transferrin, lactoferrin, heme, and heme associated proteins, and for its ability to utilize these compounds as a mechanism for obtaining iron. The acquisition of iron from these compounds has been shown to be essential for survival and growth for numerous bacterial pathogens, and the membrane receptors for these compounds in other organisms are known to be iron-regulated.
