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

Area of Science:

• Mycology
• Antimicrobial drug discovery
• Molecular biology

Background:

• VL-2397 is an antifungal cyclic hexapeptide structurally similar to ferrichrome-type siderophores.
• Its activity is influenced by metal chelation, with iron reducing its antifungal potency compared to aluminum.
• Understanding the uptake mechanism and iron's role is crucial for optimizing VL-2397's therapeutic potential.

Purpose of the Study:

• To elucidate the mechanism of VL-2397 uptake in *Aspergillus fumigatus*.
• To investigate the role of the ferrichrome-type siderophore importer, Sit1, in VL-2397 susceptibility.
• To determine if VL-2397's antifungal activity is dependent on aluminum importation and how iron affects its efficacy.

Main Methods:

• Gene inactivation and expression studies of the *sit1* gene in *Aspergillus fumigigatus*.
• Utilizing xylose-inducible promoters to control *sit1* expression and uncouple it from iron repression.
• Employing fluorescent protein tagging to visualize Sit1 localization and assessing VL-2397 susceptibility under various conditions.
• Comparing VL-2397 activity across different fungal species (*A. fumigatus*, *Candida glabrata*, *Saccharomyces cerevisiae*) and assessing cross-species complementation.

Main Results:

• Inactivation of Sit1 confers resistance to VL-2397 in *A. fumigatus*, indicating Sit1-mediated uptake is essential.
• Sit1 localizes to the plasma membrane and its expression under xylose induction leads to VL-2397 susceptibility.
• VL-2397 retains antifungal activity independent of aluminum importation, and iron-chelated VL-2397 efficacy is reduced due to *sit1* downregulation by iron.
• *Saccharomyces cerevisiae* is intrinsically resistant, but gains susceptibility upon expression of *sit1* or its homologues, suggesting a shared intracellular target.

Conclusions:

• Sit1-mediated transport is essential for VL-2397's antifungal activity in *Aspergillus fumigatus*.
• VL-2397's efficacy is independent of cellular aluminum import but is modulated by iron homeostasis through regulation of Sit1 expression.
• The findings suggest a conserved intracellular target for VL-2397 across *Aspergillus*, *Candida*, and *Saccharomyces* species, with uptake being the primary determinant of sensitivity.