To replicate in mammalian hosts, bacterial pathogens must acquire host iron. Most iron, however, is sequestered in the iron-porphyrin heme which is bound to circulating hemoglobin (Hb). To access this iron pool and enhance replication in blood, it is hypothesized bacteria require specialized protein-based systems to liberate and transport the heme across the cell envelope. Recent studies have demonstrated that heme acquisition in Gram-positive bacteria is mediated by a structural module called the near-iron transporter (NEAT) domain. NEAT proteins are a hallmark of iron- regulated surface determinant (Isd) systems, a network of transport proteins that function to import and catabolize heme. However, analysis of the genomes of sequenced Gram-positive pathogenic bacteria indicates genes encoding NEAT proteins can also be distinct from Isd operons. This raises the question as to the role of these additional NEAT proteins in the heme uptake process. This application seeks to determine the function of two non-Isd NEAT proteins, BslK and BAS0520, in B. anthracis pathogenesis and heme import. Further, the molecular mechanisms by which these NEATs acquire heme from host heme reservoirs and initiate heme transfer into the cell will also be determined. We hypothesize BslK and BAS0520 promote heme extraction from hemoglobin (Hb) and transfer the heme to envelope proteins, thereby enhancing B. anthracis replication during host infection. This hypothesis will be tested with the following specific aims: 1. Determine the contribution of non-Isd NEAT proteins to anthrax pathogenesis and heme import. --- B. anthracis strains lacking one or both genes will be tested for growth on Hb or heme as the sole iron source and their role during infection determined using fully-virulent models of anthrax disease. 2. Determine the mechanism of BslK- and 0520-mediated heme acquisition and identify new genes involved in heme assimilation. --- Novel biophysical methods will be used to determine the mechanism of BslK and BAS0520 function. A transposon mutant library will be screened to uncover new heme transport systems.
This study investigates the mechanism of action of a class of iron-uptake proteins that contribute to the survival and disease-causing ability of certain bacterial species. Knowledge generated from these studies will increase our understanding of iron acquisition in bacterial pathogens, facilitate the development of novel anti-infectives targeting these systems, and generate reagents for the creation of more safe and effective vaccines.
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