The long-term goal of this project is to increase the understanding of the physiology and virulence of the human pathogen Streptococcus pyogenes. This investigation is specifically concerned with the molecular mechanisms used by S. pyogenes to obtain iron during infection. Most bacteria require iron, which serves as a catalyst for electron transfer and is an essential component of many important enzymes. The vast majority of iron in the mammalian body is sequestered by high affinity proteins, resulting in an environment that is essentially free of unbound iron, which in turn presents a challenge to invading bacteria that need iron for growth. Heme and heme-compounds are valuable sources of iron for the hemolytic Streptococcus. Yet, the mechanisms used by S. pyogenes and related pathogens to capture and transport heme or iron are not well characterized. Genetic studies done in this laboratory identified two iron-regulated operons named sia (for Streptococcal Iron Acquisition) and sit (for Streptococcal Iron Transport) that are involved in the utilization of hemoglobin. The first specific aim dissects the function of the sia and the sit operons and their role in S. pyogenes physiology. Genetic and biochemical analysis that includes mutant characterization and employs in vivo binding and transport assays will be used. The second specific aim analyzes the operon role in virulence; studies will be conducted in zebrafish and mice infection models. The third specific aim analyzes the structure and function of surface receptors involved in hemoprotein utilization. Substrate recognition and heme or iron capture will be investigated by in vitro binding assays; site-directed mutagenesis and arbitrary PCR mutagenesis will be used to identify functional receptor domains. The interactions between different receptor components will be studied with solid phase binding assays, cross-linking, and immunoprecipitation. Iron acquisition from host hemoproteins is likely to have important implications on the physiology and virulence of S. pyogenes, an obligate human parasite. The proposed characterization of iron acquisition in S. pyogenes will advance our understanding of this important pathogen, and will add to the understanding of the process of iron uptake in other Gram-positive bacteria, many of which are important human pathogens.
