Combined inhibition of polyamine biosynthesis and uptake blocksvirulence.

Abstract

Polyamines, small organic polycations, are essential for life, and cells maintain polyamines via synthesis and uptake. Recently, we identified Hol1 as the high-affinity polyamine transporter in. Hol1 is a conserved fungal-specific transporter withhaving twohomologs (orf19.4889 and orf19.2991) and no identifiablehomolog in mammals. Deleting bothhomologs blocked efficient polyamine uptake in, establishing Hol1 as the high-affinity polyamine transporter in. Combined deletion ofandencoding ornithine decarboxylase (ODC), the first enzyme in the polyamine synthesis pathway, resulted in a severe growth defect, confirming the importance of polyamines forgrowth. In addition, cells lackingandfailed to form hyphae when exposed to serum, suggesting a role for polyamines invirulence. In accord with this hypothesis, in the mouse model of disseminated candidiasis, the homozygousmutant, like the WT strain, readily colonized the kidney and all mice died within 2 weeks following intravenous inoculation. In contrast, the homozygousmutant was avirulent with all mice surviving the infection. Consistent with these genetic results, simultaneously treating WT cells with l-α-difluoromethylornithine, an irreversible inhibitor of ODC, to inhibit polyamine biosynthesis and with the polyamine transport inhibitor Trimer44NMe to inhibit uptake substantially impairedgrowth and hyphal differentiation. We conclude that polyamines are critical forvirulence and could be of potential therapeutic interest via combined targeting of polyamine synthesis and the fungal-specific polyamine transporter Hol1.IMPORTANCEFungal infections are a growing concern, and a predominant human opportunistic pathogen is the fungus. Current antifungals commonly target cell wall and cell membrane biosynthesis or integrity; however, resistant strains are emerging. Polyamines are essential small organic cations that cells synthesize and import. We identify polyamines as a possible new target for antifungal therapies. The Hol1 polyamine transporter is unique to fungi and is distinct from mammalian transporters, so it is an intriguing antifungal target. As proof of concept, we show that combined knock-out of Hol1 and a polyamine biosynthesis gene impairsgrowth and hyphal differentiation in culture and virulence in mouse infection assays. Moreover, we identify a polyamine analog that robustly inhibits Hol1 function, providing insights into potential new therapeutics.