Streptococcus pyogenes (Group A Streptococcus) is a Gram-positive bacterial pathogen that causes significant superficial and invasive diseases. Iron acquisition is an important component of GAS pathogenesis in the human host. The 10 gene sia operon of GAS is involved in the acquisition of iron via heme or heme-binding proteins and encodes an ABC transporter as well as the large multifunctional receptor Shr. Domain analysis of Shr shows that it contains two copies of the DUF1533 (domain of unknown function) in its N-terminal part and two NEAT (NEAr Transporter) domains. NEAT domains are found in variable copy number in surface proteins of Gram-positive pathogens and are implicated in binding to various ligands. A new recombinant Shr protein was cloned and a purification protocol was developed, improving the yield of the full-length protein. A solid phase binding assay was developed and used to demonstrate Shr binding to hemoglobin. Several truncated Shr proteins were expressed and purified: the N-terminal Domain (NTD) up to but not including the first NEAT domain of Shr, the NTD plus the first NEAT domain (NTD-NEAT1) and the second NEAT domain alone (NEAT2). It was determined that Shr’s NTD mediates hemoglobin binding, demonstrating that a new protein pattern in Shr is involved in hemoglobin binding, and implicating the DUF1533 in this process. It was also determined that NTD-N1 and NEAT2 bind heme while NTD does not. Therefore, both NEAT domains may participate in the capture of heme from the host hemoglobin by Shr.

The bacterium Streptococcus pyogenes requires heme, which is taken up via an ABC transporter. An understanding of this pathway may result in new approaches to antibacterial agents. Both SiaA and NEAT2 (NEAr Transporter 2) are proteins involved in heme binding. One of the axial ligands of SiaA, His 229, was purified to study how mutagenesis affects heme binding. UV-visible studies showed a small band at 420 nm with respect to the protein band at 288 nm which probably indicates that heme was lost easily from this mutant. We have also worked to optimize the yield of Shr-NEAT2 by changing different variables. For each of the batches, the yield of holoNEAT2 was calculated by UV-visible spectroscopy. Increasing oxygen during growth did not improve holoNEAT2 yield. On the other hand, lower temperature, decrease in time after induction, and addition of ALA all increased the protein production.

Group A streptococcus (GAS) is an important pathogen that produces a wide spectrum of suppurative infections and autoimmune sequelae in humans, ranging from less complex pharyngitis, impedigo to more severe manifestations such as necrotizing fasciitis, toxic shock syndrome, rheumatic fever and glomerulonephritis. The worldwide burden of GAS infections and sequelae is considerable, but an immunization program that defends against the hyper-variable GAS is missing. The streptococcal hemoprotein receptor (Shr), is an iron-regulated protein involved in heme acquisition. An unspecified region in the amino terminus of Shr mediates the interactions with hemoglobin and two protein modules named NEAT1 and NEAT2 bind heme. In this study, we analyzed the molecular structure and function of Shr, investigated its antigenic properties and role in GAS disease production. We demonstrated that Shr is a new type of GAS adhesin that contributes to the pathogen interactions with extracellular matrix (ECM) proteins. Shr enabled bacterial adherence to host cells and was important for GAS virulence in vivo. Immunizations with Shr protein by intraperitoneal or intranasal administration conferred resistance to systemic GAS challenge in mice. Shr antiserum allowed bacterial opsonization and defended against GAS diseases in a murine model for passive vaccination. Studies with isolated Shr domains localized ECM-binding to the NEAT domains and showed that most of the protein is exposed on the bacterial surface. In addition, Shr N-terminal region and both of the NEAT modules elicited strong antibody response in rabbits. In conclusion, Shr is a protective antigen that contributes to GAS pathogenesis by facilitating both heme uptake and bacterial adherence. Since Shr is conserved among GAS strains and other pyogenic streptococci, this study demonstrates that Shr may be used to develop a vaccine against GAS strains and related pathogens.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Gram-positive bacteria are characterized by a single lipid bilayer with a thick peptidoglycan layer. This group of organisms contains bacteria commonly associated with human infection, including: Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis and Streptococcus pneumoniae among others. These bacteria have a common system for importing iron in the form of heme, which is acquired by proteins containing heme-binding NEAT (NEAr iron Transporter) domains. The heme acquisition system in S. aureus is termed the Iron Surface Determinant (Isd) system and in L. monocytogenes is termed Heme Binding Protein (Hbp) and Heme/Hemoglobin Uptake Protein (Hup). These proteins work together to obtain heme from hemoglobin and then transport the heme into the cytoplasm via well characterized ABC-transporters. Although there have been clinical trials with antibodies directed against Isd proteins, there are currently no antibiotics targeting iron uptake systems in bacteria in general. Building upon fluorescent approaches for detection of iron uptake in Gram negative organisms, this work develops fluorescent heme acquisition detection in Gram positive organisms. The spectrofluorimetric methodology facilitates the understanding of heme acquisition protein interactions and mechanisms in bacteria. This work could subsequently be used to identify inhibitors of Gram positive bacterial iron uptake systems, and develop a new target for antibiotic action.