Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

David Heinrichs

Abstract

The high affinity iron scavenging glycoprotein transferrin sequesters trace amounts of serum Fe3+ to concentrations below what is required to sustain microbial life. Iron may be liberated from this important innate immune factor after interaction with molecules that chelate or reduce Fe3+. Organisms with cognate transport systems for these iron coordinating molecules can survive in the bloodstream using transferrin iron.

Staphylococcus aureus is an opportunistic bacterial pathogen. S. aureus executes numerous strategies for overcoming the innate immune barrier of iron deprivation in the bloodstream. In addition to specialized mechanisms for hemoglobin iron extraction, S. aureus can proliferate on serum iron, but factors enabling this growth are not described. Production of at least two siderophores (microbial iron chelators) has been documented on numerous occasions, and their contribution to growth on transferrin is documented here. Genomic inactivation of genes involved in production of molecules subsequently termed staphyloferrin A (SA; the sfa locus) and staphyloferrin B (SB; the sbn operon) resulted in a mutant severely incapacitated for growth in serum as well as on rarefied human transferrin as sole iron sources. Transport of staphyloferrins was correlated to adjacently encoded cognate ABC type transporter operons, hts (SA) and sir (SB), using previously constructed transport mutant strains. Mass spectrometry confirmed the molecular structure of SB as being the same as the previously described S. aureus metabolite, staphylobactin.

Alternate siderophores were not detectable for the double biosynthetic mutant. Growth in the presence of transferrin could be rescued by addition of saturating concentrations of iron, and restored by molecules that bind Fe3+ through catechol-iron coordination, including mammalian catecholamine stress hormones. In silico analysis and mutational inactivation confirmed this transport function to be encoded by the sst operon. Biochemical assays revealed that the Sst transporter lipoprotein has a high affinity for ferrated catecholate iron ligands.

Siderophore biosynthesis and transport mutants displayed reduced virulence during systemic mouse infection. Decreased bacterial loads were documented in mouse hearts, an important finding as S. aureus is a leading cause of endocarditis. The data collected in this study show that acquisition of serum iron is an important part of staphylococcal pathogenesis, and suggest that therapeutics targeting the numerous facets of this process may be effective in combating invasive infection.

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