Metal nutrients and metallophore-like molecules for a fungal pathogen

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Transition metals including Cu, Mn, Zn and Fe are essential nutrients for all life forms. Microbial pathogens face challenges in acquiring these vital elements, as the host deliberately attempts to starve microbes of their metals through processes collectively known as nutritional immunity. Successful pathogens have evolved elaborate methods to evade nutritional immunity and capture metals from their host; one mechanism involves secretion of ‘metallophores”. Metallophores are small (≤1.5 kDa) molecules that bind extracellular metals with high affinity and deliver the ion to the microbe for uptake. Bacteria secrete various metallophores for Fe, Cu or Zn, although none have been identified for Mn. Pathogenic fungi are thought to only secrete Fe-metallophores and only in certain species. Candida albicans is an opportunistic fungal pathogen believed to not produce metallophores of any kind, although the yeast is dependent on host metals for pathogenesis. Recently, our lab has challenged the dogma of no metallophores for Candida sp and obtained evidence that C. albicans does in fact produce metallophore-like compounds that exhibit strong selectivity for binding Mn2+ or Cu2+. We call these Mn-MBC and Cu-MBC for metal binding complex. The metal coordination sites of Mn-MBC and Cu-MBC are distinct, as are their chromatographic properties, indicating that Mn-MBC and Cu-MBC are different molecules. Interestingly, production of Cu-MBC, but not Mn-MBC, is induced by Fe, indicating a role for Cu- MBC in Fe homeostasis. Currently we do not know the chemical nature of the MBC molecules, their occurrence among diverse fungi, or their roles in metabolism of metals for fungal pathogenesis. Over this two- year research plan, we shall test for MBC production from fungi outside of Candida sp; we will chemically identify C. albicans Mn-MBC and Cu-MBC, and will begin to probe their mechanism of action. Aim 1: To survey MBC across diverse fungi and chemically identify the molecules from C. albicans. Using a size exclusion chromatography/ICP-mass spectrometry/ENDOR spectroscopy approach that we have developed to identify Mn- and Cu-MBC, we will test for MBC production in two non-Candida fungal pathogens. Mn-MBC and Cu-MBC from C. albicans will be chemically identified and composition defined by mass spectrometry. Experiments will engage expert collaborators in ENDOR spectroscopy and mass spectrometry. Aim 2: To gain insight into the fundamental biology of MBC. Our preliminary studies show that both Mn- and Cu- MBC can serve as donors of their respective metals for uptake by C. albicans in culture. We shall test the pathway of metal uptake that uses the MBCs and examine metalloenzyme targets for metal delivery by MBC. Time permitting, we shall test the role of Mn- and Cu-MBC in pathogenesis using appropriate C. albicans mutants and a murine model of candidiasis where the fungal pathogen faces metal starvation stress. Overall, successful completion of this 2-year program will reveal the first non-Fe metallophores for the fungal kingdom and new mechanisms by which fungi can capture metal nutrients that are essential for pathogenesis.