Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor(s)

Dr. Martin J. Stillman

Abstract

The antibiotic resistant bacterium Staphylococcus aureus is a significant problem in hospitals and communities worldwide. Survival of the bacterium in the host is reliant on iron scavenging. Staphylococcus aureus has adopted specialized mechanisms for scavenging iron from the host. The cell wall and membrane-associated iron regulated surface determinant (Isd) proteins allow Staphylococcus aureus to scavenge iron from the heme in hemoglobin. There are nine Isd proteins (IsdH, IsdB, IsdA, IsdC, IsdE, IsdD, IsdF, IsdG and IsdI) located at different depths in the cell wall and membrane. Magnetic circular dichroism (MCD) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) have been used to determine the direction, mechanistic details and heme binding ligands of the Isd heme transfer system. Ferric heme extracted from metHb by IsdB and has been demonstrated to transfer in a unidirectional fashion with the heme transferring along the pathway in the sequence (proximal amino acid) IsdB-N2 (Tyr) -> IsdA-N (Tyr) -> IsdC-N (Tyr) -> IsdE (His) or, alternatively, when initiating from IsdH-N3 (Tyr) the transfer sequence is -> IsdA-N (Tyr) -> IsdC-N (Tyr) -> IsdE (His). Heme transfer through the cell wall must occur through IsdC indicating that IsdC acts as the central conduit of the Isd system. MCD and ESI-MS data show that disruption of unidirectional heme transfer occurs with heme analogs and protein mutational studies. Finally kinetic analysis provides rate constants for the major unidirectional reaction steps.


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