IDENTIFICATION AND CHARACTERIZATION OF VIRULENCE DETERMI

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The purpose of this project is to identify and characterize virulence determinants in Corynebacterium diphtheriae in order to understand how this important pathogen causes disease. Expression of diphtheria toxin, the primary virulence determinant of C. diphtheriae, is regulated by iron, and it is likely that additional virulence factors are coordinately regulated with that of the toxin. The ability to acquire iron during an infection is essential for many bacterial pathogens to cause disease. It was shown in earlier studies from this laboratory that the hmuO gene is required for the utilization of iron from heme and hemoglobin in C. diphtheriae. Recent studies have focused on; 1) understanding the molecular mechanisms of hmuO gene regulation, 2) purification and characterization of the activity of the HmuO protein and 3) the characterization of factors involved the transport of heme in C. diphtheriae. Transcription from the hmuO promoter has been shown to be under dual regulation in which expression is positively regulated by a heme source, such as heme or hemoglobin, and negatively regulated by the diphtheria toxin repressor protein (DtxR) and iron. Studies in this laboratory have shown that heme-dependent activation of the hmuO promoter occurs through a two component signal transduction system that requires the chrA and chrS genes, which encode a response regulator and sensor kinase, respectively. The ChrS protein is proposed to detect heme in the extracellular environment and transmit this signal via a phosphotransfer mechanism to the ChrA protein, which then activates transcription of the hmuO gene. An ABC (ATP-binding cassette) type transport system was shown to be required for the utilization of heme and hemoglobin as iron sources in C. diphtheriae. The three proteins identified in this system are homologous to proteins involved in heme transport from gram-negative bacteria. The HmuT protein, which is proposed to be the cell surface receptor for heme, was shown in vitro to bind to heme agarose, and it was shown to be a lipoprotein. These findings suggest that HmuT has the capacity to bind heme and further suggests that this putative receptor is anchored to the cell surface by a lipid moiety at its N-terminus.