*2.3. Iron*

Iron (Fe) is a transition metal belonging to group eight of the periodic table and can exist as ferrous (Fe2+) or ferric (Fe3+) iron [211,212]. As an essential nutrient, Fe is significant for the virulence of fungi that cause disease. In *A. fumigatus* and *F. oxysporum*, survival depends on the ability to sequester iron from the host and a well-functioning homeostatic system to maintain this delicate balance [213,214]. Incapacitating the ability to do so reduces virulence and becomes a growth limiting factor, such as in the use of excessive amounts of Fe to completely overrun homeostatic systems [215–217]. Thus, homeostatic mechanisms are integral.

#### 2.3.1. Iron Transport and Homeostasis

Generally, in *S. cerevisiae*, two iron uptake systems are described, the reductive and nonreductive systems. The reductive system recognizes Fe2+ salts and chelates for uptake through importers, while the nonreductive systems utilizes iron siderophores [218–221].

In the reductive system, high-affinity (aerobic) and low-affinity (anaerobic) transporters are responsible for ferric and ferrous iron transport, respectively [222,223]. For low-affinity uptake, iron must be reduced by ferric reductases Fre1 or Fre2, initially described by Lesuisse et al. in 1987 and later coined Fre1 and Fre2 by Georgatsou and Alexandrakin in 1994 [221,224]. Since then, both metallo-reductases have also been found to reduce both cupric and ferric ions, where *FRE1* expression induces the reduction of Cu2+ when transcription factor Mac1 is bound, and Fe3+ reduction occurs via binding of transcription factor Aft1 [176,181,225]. After Fe3+ reduction, Fe2+ is then ready for uptake by a six domain, transmembrane, metal transporter, Fet4 [54,55]. Fet4 can also import other metals, but is mostly responsible for Fe2+ uptake in iron-restricted cells [223]. In anaerobic conditions, transcription factor Aft1 is required for activation, and in aerobic conditions, expression of *FET4* is repressed by Rox1, which has two binding sites in the *FET4* promoter region [223]. This repression is necessary to prevent the unintended uptake of toxic metals, such as Cd, where it is demonstrated that *rox1*∆ mutants have increased sensitivity to Cd under aerobic conditions [223,226]. A second, less utilized iron transporter in the low-affinity uptake system is Smf1, responsible for the uptake of the Fe2+/H complex [51,52]. This metal transporter is mostly known for the uptake of Cu, Mn, and Cd; however, in a study completed by Cohen et al. in 2000, it was shown that overexpression of *SMF1* also results in significant iron uptake [52,65,227]. High-affinity iron uptake is also part of the reductive system. In low-iron conditions, this system dissociates and reduces ferric iron, via Fre1 and Fre2, from a wide array of Fe3+ substrates such as ferric chelates, salts, and siderophores [218,219]. Fe2+ then transitions through the Fet3/Ftr1 complex [58]. Fet3 is activated by transcription factor Aft1 in iron-deficient conditions and contains four Cu<sup>+</sup> binding domains that must be metalated for activation [53,178,181]. Activated Fet3 goes through an aerobic reaction that oxidizes Fe2+ to Fe3+ for passage to the cytosol via iron permease Ftr1 [58,178]. The final destination and the cell's utilization of Fe3+ is not fully elucidated.

The nonreductive system utilizes siderophores. *S. cerevisiae* is incapable of producing siderophores, but can sequester siderophores produced by other microorganisms via siderophore iron plasma membrane transporters Arn1—Arn4 [16,224]. Arn1 transports ferrichrome into the cell for iron acquisition; however, Arn1 is not always readily available in the plasma membrane because it is localized to endosomes or is routed to vacuoles for degradation when ferrichrome is unavailable [16,220,228]. When ferrichrome is present, Arn1 is routed through the plasma membrane, where ferrichrome adheres to either the low or high-affinity binding site and is transported to the cytosol [16,220,228]. It remains intact in the cytosol and serves as an intracellular Fe3+ storage reservoir until the cell needs iron; in this event, Fe3+ is reduced via metallo-reductases, or released via ferrichrome degradation [16,220,228,229]. Arn2 (also known as Taf1) is the second siderophore transporter in the *ARN* family, responsible for transporting tri-acetyl-fusarinine to the cytosol; it is unclear if Arn2 is located anywhere else aside from the plasma membrane when tri-acetyl-fusarinine is unavailable [220,230,231]. The literature is not very informative on the functions of tri-acetyl-fusarinine, but it does appear to have a similar role to ferrichrome as a store reservoir for ferric iron [230,231]. Arn3 (also known as Sit1) is a transporter for ferrioxamine B and is situated within intracellular vesicles. It appears to have a similar function to Arn1 and can progress to the plasma membrane when ferrioxamine B is available [229,232]. After ferrioxamine B is transported inside the cell, it is stored in the vacuole, likely for subsequent dissociation [232]. The first three mentioned siderophores transported by Arn1–Arn3 belong to the hydroxamate class of siderophores. However, the final transporter Arn4 (also known as Enb1) transports a siderophore of the catecholate class, ferric entero-bactin [220,233]. Unlike the other siderophore transporters, Arn4 remains at the plasma membrane regardless of the presence of its substrate [218]. Philpott and Protchenko suggested the difference in plasma membrane cycling between hydroxamate and catecholate transporters may be due to the possibility that there are toxins that can adhere to the hydroxamate transporters and not the catecholate transporters [218]. In

the act of self-preservation, those transporters remove themselves as a potential source of toxicity [218]. Ferric entero-bactin is not well-studied in *S. cerevisiae*, but based on the function of other siderophores it may be reasonable to conclude that, upon cellular entry, ferric entero-bactin is also used as an Fe3+ storage system.

After Fe uptake, there are many intracellular destinations. Two briefly discussed here are the cytosol and the nucleus [61,62]. In the cytosol, iron–sulfur assembly (CIA) proteins Npb35 (binds two Fe–S clusters), Nar1, Cfd1 (binds one Fe–S cluster), and Cia1 form an iron–sulfur complex [61,62,234]. These complexes transfer Fe–S clusters to various apoproteins for activation [61,62,234]. In the nucleus, CIA proteins deliver Fe–S clusters to various nuclear proteins involved in DNA repair and replication [61,235].

Iron homeostasis in the fission yeast *S. pombe* is also well-studied and has three mechanisms of iron uptake [236]. One involves cell surface ferric reduction, and the other, in contrast to *S. cerevisiae*, involves the production of siderophores to capture extracellular iron and heme [236]. The first iron uptake system described here is through use of siderophore synthesis [237]. Under iron-deficient conditions, Sib2, a catalyst for ferrichrome synthesis, hydroxylates ornithine to N<sup>5</sup> -hydroxyornithine, a newly formed hydroxy-mate group molecule, and then undergoes processing by Sib1 [236,237]. This non-ribosomal peptide synthase yields the desferri-form of ferrichrome [236,237]. Schrettl, Winkelmann, and Haas suggested that the resulting ferrichrome is excreted from the cell to capture extracellular Fe3+ from the surrounding environment [237]. In an iron-dependent response, transcription factor Fep1 activates ferrichrome transporters Str1, Str2, and Str3, and the iron-loaded ferrichrome re-enters the cell (predominately by way of Str1) [59,63]. *S. pombe* is also able to import exogenous iron-loaded ferrioxamine B via Str2 [63]. In addition to the previously mentioned siderophore functions, it had also been suggested that, as in *S. cerevisiae*, imported siderophores also serve as iron storage vesicles [63,237].

The second iron uptake mechanism employed by *S. pombe* is the high-affinity, reductive system that depends on cell surface ferric reductase Frp1. *frp1*<sup>+</sup> shares 27% homology with the *S. cerevisiae* Fe3+/Cu2+ reductase encoding gene, *FRE1*, and reduces extracellular Fe3+ to Fe2+ [238]. Transcription of *frp1*<sup>+</sup> may also have some functional relation to the vacuole/cytoplasmic transporter Abc3 that transports iron from the vacuole to the cytosol in iron-deficient conditions [238,239]. Pouliot et al. found that *abc3*∆ mutants resulted in the activation of *frp1*<sup>+</sup> ; however, a nucleotide-based transcription factor directly linked to *frp1*<sup>+</sup> has not yet been determined and it appears to be solely activated or repressed by the absence or presence of iron, respectively [238,239]. After ferric reduction, Fe2+ enters an oxidase-permease complex, similar to that of the *S. cerevisiae* Fet3/Ftr1 complex, composed of proteins Fio1 and Fip1 [50]. Fio1 is a Fe2+ oxidase that shares 37% homology with the *S. cerevisiae* Fet3, and in an iron deprived environment, oxidizes Fe2+ in preparation for transfer across the plasma membrane via Fip1 [50]. fip1<sup>+</sup> is a ferrous permease having 46% homology with the *S. cerevisiae* Ftr1 [50,236].

Heme is an iron-containing compound and its acquisition and biosynthesis are the finally discussed mechanisms of iron uptake in *S. pombe*. It is notable to state that while *S. cerevisiae* does utilize heme in other processes such as respiration and ergosterol biosynthesis, it has not been determined to be used to acquire iron [240,241]. *S. pombe* imports exogenous heme for iron uptake through Str3 and Shu1 [56,57]. Shu1 is a plasma membrane protein induced during iron deprivation, when heme biosynthesis is not attainable, or if Fep1 is inactivated [56,57]. The second protein involved in heme uptake is Str3, previously mentioned as a part of a ferrichrome transporter family (Str1, Str2, and Str3). Str3 shares the lowest homology (25.1%) with Str1 when compared to Str2 (29%), and its substrate specificity is undetermined [57,59,63]. Iron release and utilization from heme is not yet fully understood in *S. pombe*; however, studies in *C. albicans* (and other fungi) show that heme degradation is catalyzed upon cellular entry via heme oxygenase [56,57,242]. *S. pombe* also biosynthesizes heme and is encoded by *hem1*<sup>+</sup> , *hem2*<sup>+</sup> , *hem3*<sup>+</sup> , *hem12*<sup>+</sup> , *hem13*<sup>+</sup> , *hem14*<sup>+</sup> , *hem15*<sup>+</sup> , and *ups1*<sup>+</sup> [56,57]. In iron-deficient conditions, a cascade of events between the mitochondria and the cytoplasm occurs to synthesize heme for further utilization [56,57,243].

In addition to iron acquisition in *S. pombe*, regulation mechanisms must be in place to prevent over-accumulation. Mercier, Pelletier, and Labbé identified the gene *pcl1*<sup>+</sup> to play a role in vacuolar iron storage [244]. *pcl1*<sup>+</sup> shares homology to *S. cerevisiae* Ccc1, an iron vacuolar transporter, and it has been shown that *pcl1*∆ mutants have increased sensitivity to iron; this together with the study of Mercier, Pelletier, and Labbé suggests that Pcl1 might play a similar role in iron storage in *S. pombe* [63,239]. As mentioned, the final destinations of heme are somewhat unclear, but based on research in other fungi, heme may be degraded, and literature suggested that there may be a group of proteins responsible for transporting those ions to the vacuole for degradation or storage [56,57,245]. Much is known about iron homeostasis in *S. pombe;* however, there are apparent gaps in knowledge of specific processes.

In filamentous fungi, iron homeostasis is less documented. It has been investigated in *U. maydis*, a pathogenic fungus that causes corn smut disease and whose virulence is associated with iron acquisition [60,121]. There are two iron uptake mechanisms, one through hydroxamate siderophores, and the other an oxidase-permease system, similar to *S. pombe* [60,233,246,247]. In the latter, exogenous ferric iron is reduced by a seemingly unknown reductase (possibly Fer9) and then re-oxidized by ferroxidase Fer1 for uptake through the high-affinity ferric iron permease Fer2 [60,121]. In the former, siderophore iron uptake is mediated by siderophore biosynthesis encoding genes *Sdi1* and *Sid2*, and both are negatively regulated by transcription factor Urbs1 [60,121,246,247]. These siderophores play a role in iron acquisition; however, deletion mutants showed they are not necessary for virulence [121].
