The Siderophore Transporter Sit1 Determines Susceptibility to the Antifungal VL-2397

VL-2397 (previously termed ASP2397) is an antifungal, aluminum-chelating cyclic hexapeptide with a structure analogous to that of ferrichrome-type siderophores, whereby replacement of aluminum by iron was shown to decrease the antifungal activity of this compound. Here, we found that inactivation of an importer for ferrichrome-type siderophores, termed Sit1, renders <i>Aspergillus fumigatus</i> resistant to VL-2397. Moreover, expression of the endogenous <i>sit1</i> gene under the control of a xylose-inducible promoter (to uncouple <i>sit1</i> expression from iron repression) combined with C-terminal tagging with a fluorescent protein demonstrated localization of Sit1 in the plasma membrane and xylose-dependent VL-2397 susceptibility. This underlines that Sit1-mediated uptake is essential for VL-2397 susceptibility. Under xylose-induced <i>sit1</i> expression, VL-2397 also retained antifungal activity after replacing aluminum with iron, which demonstrates that VL-2397 bears antifungal activity independent of cellular aluminum importation. Analysis of <i>sit1</i> expression indicated that the reduced antifungal activity of the iron-chelated VL-2397 is caused by downregulation of <i>sit1</i> expression by the imported iron. Furthermore, we demonstrate that defects in iron homeostatic mechanisms modulate the activity of VL-2397. In contrast to <i>A. fumigatus</i> and <i>Candida glabrata</i>, <i>Saccharomyces cerevisiae</i> displays intrinsic resistance to VL-2397 antifungal activity. However, expression of <i>sit1</i> from <i>A. fumigatus</i>, or its homologue from <i>C. glabrata</i>, resulted in susceptibility to VL-2397, which suggests that the intrinsic resistance of <i>S. cerevisiae</i> is based on lack of uptake and that <i>A. fumigatus</i>, <i>C. glabrata</i>, and <i>S. cerevisiae</i> share an intracellular target for VL-2397.