Date of Award
Master of Science
Microbiology and Immunology
Dr. David Heinrichs
The frequent human pathogen Staphylococcus aureus requires a sufficient source of iron to proliferate and, thus, to sustain an infection of a mammalian host. However, the free iron concentration in the host is insufficient for bacterial growth without use of specialized uptake mechanisms. To overcome this, S. aureus utilizes multiple systems to scavenge host iron. One such strategy that S. aureus employs is the use of siderophores: secreted, small-molecular-weight, high-affinity iron chelators. S. aureus synthesizes and secretes two anionic a-hydroxycarboxylate siderophores, staphyloferrin A (SA) and staphyloferrin B (SB). Fe-SA and Fe-SB complexes are transported into the cell through the highly specific ABC-type transporters HtsABC and
SirABC, respectively. The crystal structures of HtsA, the SA receptor, have been solved for both SA bound and unbound forms. Structural data indicate that HtsA has a unique positively charged binding pocket and many charged residues form contacts with the anionic siderophore including R86, R104, R126, K203, H209, and Y239. Their contribution to productive binding and transport is currently unknown. To investigate the role of each residue found to interact with SA, site-directed mutagenesis was used to substitute each interacting residue with either alanine or an amino acid with more conserved properties. Fluorescence titrations of SA, produced and purified in vitro, with wild-type (WT) or mutant rHtsA were used to determine the effect that each substitution had on the binding affinity of HtsA for SA. Growth curves and disk- diffusion assays were completed on strains expressing mutated or WT HtsA to determine the biological significance of each mutation under SA-dependant growth conditions. These studies have confirmed significant roles in SA binding and transport for HtsA residues R104, R126, and H209, strengthening the conclusions drawn from the HtsA crystal structure and providing new insight into the mechanism of SA-dependant iron uptake in S. aureus.
Cooper, John D.E., "CHARACTERIZATION OF HtsA, THE FERRIC-STAPHYLOFERRIN A-BINDING PROTEIN IN STAPHYLOCOCCUS AUREUS" (2011). Digitized Theses. 3282.