Staphylococcus aureus heme acquisition from hemoglobin

Staphylococcus aureus is a bacterial pathogen of major health concern. Furthermore, the emergence of methicillin-resistant S. aureus (MRSA) strains limit the number of treatment options. Iron is essential for S. aureus survival and growth; however, the majority of iron in the human body is stored as hemoglobin (Hb) in erythrocytes. Hb released from lysed erythrocytes is quickly captured by serum haptoglobin (Hp) and the complex is degraded in the liver where released iron is then recycled. To date, the Iron Surface Determinant (Isd) system is the only heme utilization pathway identified in S. aureus. IsdB is the primary receptor at the cell surface that binds Hb to extract heme. Heme is then transferred to IsdA or IsdC, which then relay it to other Isd proteins for internalization. IsdB contains two NEAT (NEAr Iron Transporter) domains, and both domains and the intervening linker are needed for efficient heme uptake from Hb. In the first part of my study, the molecular mechanism of heme transfer from Hb to IsdB was investigated using site-directed mutagenesis. Residues in the heme-binding pocket were identified by inspection of a crystal structure of the complex of IsdB and Hb. The Y440F/Y444F (YFYF), E354A, M363L and S361A variants were found to be deficient in heme transfer. In particular, spectroscopic analysis of the YFYF mutant mixed with Hb provided evidence of a trapped heme transfer intermediate similar to that observed in the crystal structure of the wild-type protein. In the second part of my study, Hp was found to inhibit heme transfer from Hb to IsdB in a concentration-dependent manner and heme transfer was completely blocked when Hp was added to Hb above the binding stoichiometry determined by structural and solution studies. Moreover, Hb mixed with excess Hp did not support growth of S. aureus on iron-restrictive media. This study gained insight into the heme transfer mechanism between Hb and IsdB and revealed a novel role of Hp in blocking heme uptake by S. aureus.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat