The importance of secreted siderophores in diagnosis and virulence of invasive fungal infections

Iron acquisition is essential for growth. In pathogenic Mucorales fungi, reductive iron assimilation contributes to virulence whereas in Aspergillus fumigatus, siderophore biosynthesis is essential for pathogenesis. The overall aim of my research was to investigate the synthesis and diagnostic importance of siderophores of these two groups of pathogenic fungi. Biosynthesis of select siderophores has been shown to occur using non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) synthetases. I used crystal structures to compare NIS enzymes involved in the biosynthesis of 8 siderophores. I proposed a new class of Type C’ synthetases, responsible for dimerization/macrocyclization of more complex and substituted intermediates. I also demonstrated that NIS enzymes are predicted to be present in all Domains of life. I identified an NIS homolog in the fungal pathogen, Rhizopus delemar, and confirmed that the enzyme (Rfs) was responsible for synthesis of the polycarboxylate siderophore, rhizoferrin. Rfs kinetic parameters were comparable to bacterial NIS enzymes. Rfs used diaminobutane, ornithine, diaminopentane and diaminopropane as nucleophiles. Site-directed mutagenesis showed that H484 is essential, and that L544 may govern nucleophile specificity. These data provide the first characterization of a fungal NIS enzyme. We identified an uncharacterized siderophore, termed Band 4, in Aspergillus terreus. Band 4 did not restore growth of a sidA mutant of A. fumigatus suggesting that Band 4 is structurally unrelated to A. fumigatus hydroxamate siderophores. Studies of an A. terreus sidA mutant indicated that biosynthesis of Band 4 is not co-regulated with hydroxamate siderophores. An iron-regulated NIS homologue (AtNIS) was identified and recombinant AtNIS was generated. rAtNIS can be used to confirm its involvement in Band 4 biosynthesis. Finally, I investigated whether siderophores could be detected in serum of patients at risk for invasive aspergillosis (IA) caused by A. fumigatus. We used UPLC-MS/MS to quantify the major A. fumigatus siderophore, N,N’,N”-triacetylfusarinine C (TAFC) in serum. In >90 serum samples from at-risk patients, TAFC was diagnostic in 29 samples, and identified in an additional 28 serum samples. TAFC was not detected in healthy serum. Thus, serum siderophore detection may be a useful tool in the diagnosis of IA caused by A. fumigatus.