Given the evolutionary conservation of sterol biosynthesis in eukaryotes, much can be learned about sterol biosynthesis mechanisms in A. fumigatus through examination of more well studied systems in yeast and mammals among others. While many mechanisms are conserved, it is clear that each organism has evolved unique regulatory mechanisms and this is particularly true for A. fumigatus as discussed below. Importantly, these unique mechanisms present opportunities to develop novel therapeutic strategies to augment existing antifungal drugs or identify new targets and molecules with activity against A. fumigatus.

De novo synthesis and LDL receptor mediated endocytosis are two major pathways through which mammalian cells fulfill their sterol requirement (Brown and Goldstein, 1986; reviewed in Espenshade and Hughes, 2007). In mammals, levels of cholesterol are known to control both of these pathways at the transcriptional level (Brown and Goldstein, 1997). The Sterol Regulatory Element Binding Protein (SREBP) class of transcription factors (TF) bind SRE elements and function as major regulators of sterol levels in mammalian cells (Yokoyama et al., 1993; Brown and Goldstein, 1997). When cells are depleted of sterol, the transcription of HMG-CoA reductase and LDL receptor increase through SREBP binding of sterol regulatory element (SRE) DNA elements in promoter regions of these genes. Three major SREBP’s have been identified in mammalian cells viz., SREBP-1a and SREBP-1c encoded by the same gene, and SREBP-2 (Yokoyama et al., 1993; Hua et al., 1995).

Sterol Regulatory Element Binding Proteins are bHLH TFs synthesized as ER resident proteins with their N and C terminals in the cytosol. The SREBP binding partner, SCAP (Sterol Cleavage Activating Protein), regulates the activation of SREBPs (Rawson et al., 1999). When cellular sterol levels are replete, SCAP binds sterols and consequently promotes binding to INSIG (another ER resident protein) to prevent ER exit of SREBP-SCAP. When cellular sterol levels are reduced, SCAP does not bind INSIG which allows ER exit of SREBP via COPII vesicles (Brown et al., 2002; Yang et al., 2002; Sun et al., 2005). In the Golgi, SREBPs are sequentially proteolytically cleaved by golgi resident site-1 serine protease (S1P) that cuts SREBP in the luminal loop (Duncan et al., 1997) and site-2 zinc metallo protease, (S2P) that cuts in the transmembrane domain (Rawson et al., 1997; Duncan et al., 1998). These proteolytic cleavage events release the N terminal TF for nuclear localization and activation of gene expression (Espenshade, 2006; Espenshade and Hughes, 2007).

In the fission yeast Schizosaccharomyces pombe, seminal studies identified and characterized fungal homologs of SREBP (Sre1), Scap (Scp1), and Insig (Ins1) (Hughes et al., 2005). As in mammalian cells, Sre1 physically interacts with Scp1 and Sre1 activity is essential to maintain cellular sterol levels through transcriptional regulation of target genes including oxygen dependent steps in the ergosterol biosynthesis pathway (e.g., erg11, erg25, erg3, erg5, erg6) (Hughes et al., 2005, 2007a; Todd et al., 2006). A major activation signal for S. pombe Sre1 proteolytic cleavage is hypoxia. Under low oxygen concentrations that induce hypoxia in fission yeast, ergosterol levels are reduced and this in turn induces proteolytic cleavage of Sre1. Thus, in fission yeast ergosterol levels act as an indirect oxygen sensor (Hughes et al., 2005). These data further highlight the mechanistic relationship between sterol biosynthesis and oxygen discussed previously. Cleavage of Sre1 is also stimulated by drugs that inhibit ergosterol biosynthesis including the azoles (Hughes et al., 2005, 2007a). Consequently, in fission yeast, ergosterol regulates Sre1-Scp1 ER-Golgi transport, cleavage and activation (Porter et al., 2010). Like mammalian cells, direct binding of ergosterol to Sre1-Scp1 complex determines the fate of Sre1 cleavage, however, unlike mammalian cells, this is independent of Ins1 binding (Porter et al., 2010). In S. pombe, the INSIG homolog Ins1 does not regulate the SREBP pathway. Rather, Ins1 regulates HMG-CoA-reductase (hmg1) that catalyzes HMG-CoA to mevalonate; the first committed step in sterol synthesis. Unlike in mammalian cells, S. pombe Ins1 regulates the activity and not stability of Hmg1 (Burg et al., 2008).

As in fission yeast, A. fumigatus contains a membrane bound SREBP homolog, SrbA, required for sterol biosynthesis, hypoxia fitness, iron homeostasis, and azole drug tolerance/resistance (Willger et al., 2008; Blatzer et al., 2011; Chung et al., 2014). The sterol profile of ΔsrbA shows a significant decrease in total ergosterol content and accumulation of 4-methyl fecosterol and 4,4 dimethyl fecosterol (Willger et al., 2008; Blosser and Cramer, 2012). This likely stems from direct transcriptional control of cyp51A/B and erg25A/B expression by SrbA in A. fumigatus though DNA binding of an SRE motif in their promoters (Blosser and Cramer, 2012; Chung et al., 2014). Overexpression of cyp51a in the ΔsrbA strain does not restore a WT sterol profile or hypoxia fitness which suggests SrbA regulates multiple steps in the ergosterol biosynthesis pathway (Figure 1) (Blosser and Cramer, 2012). In fact, over-expression of cyp51A in ΔsrbA exacerbates the accumulation of C4-methyl sterols. However, intriguingly, this strain’s voriconazole and fluconazole MICs are restored to WT levels perhaps due to a decrease in toxic 14-α methylated sterol accumulation (Blosser and Cramer, 2012).

While loss of cyp51a or erg25 is dispensable for virulence of A. fumigatus, loss of SrbA severely attenuates A. fumigatus virulence in multiple immune compromised IPA murine models (Mellado et al., 2005; Alcazar-Fuoli et al., 2006; Blosser and Cramer, 2012; Willger et al., 2008, 2012). Supporting a partial role for accumulation of C4 methyl sterols as a mechanism to explain the hypoxia fitness defect of ΔsrbA, generation of an erg25A/B double genetic null mutant was not possible in A. fumigatus (Blosser et al., 2014). It is thus possible that accumulation of 4-methyl sterols in the absence of SrbA contributes in part to its inability to grow in hypoxia and cause invasive disease. It would be intriguing to examine the sterol profile of ΔsrbA in the presence of high iron levels where a small but significant restoration of hypoxia growth occurs (Blatzer et al., 2011). Further studies are needed to fully define SrbA’s role in A. fumigatus hypoxia fitness and virulence.

Intriguingly and worth consideration for therapeutic development, it is clear that significant differences exist in SREBP regulation between mammals, the yeast S. pombe, and the human pathogen A. fumigatus. The difference in SREBP activation mechanisms in A. fumigatus is highlighted by the observation that a homolog of SCAP is absent in the A. fumigatus genome. Moreover, significant levels of the N terminal bHLH portion of SrbA are detectable in normoxia conditions in A. fumigatus suggesting that proteolytic cleavage occurs at some level when sterols levels are presumably high. It is worth noting that under these conditions NGFP::SrbA localizes to the ER/NE rather than nuclei (Willger et al., 2012). It is plausible that post-translational mechanisms control the levels of active SrbA, in a manner similar to S. pombe (Hughes and Espenshade, 2008), however, this hypothesis needs further investigation. These observations raise an important question that remains to be answered in A. fumigatus: how does the A. fumigatus SREBP pathway monitor cellular sterol levels? It is plausible that a SCAP like protein remains to be identified. A protein with sequence similarity to mammalian Insig – InsA (AFUB_064770) is present in the genome but is yet to be characterized in A. fumigatus. Moreover, it is plausible that sterol levels fluctuate with filamentous fungal development in batch culture perhaps explaining the apparent constitutive cleavage of A. fumigatus SrbA in sterol replete conditions. Intriguingly, mRNA levels of SrbA do not change in the presence of voriconazole in at least 2 independent studies (da Silva Ferreira et al., 2006; Krishnan et al., 2013). This is noteworthy as SrbA has been observed to bind its own promoter in response to hypoxia and azole drug treatment (Chung et al., 2014; Gsaller et al., 2016). Thus, it is also plausible that SrbA activity in A. fumigatus is regulated by another signal related to oxygen and antifungal drug responses. Given that genes encoding key enzymes in heme biosynthesis are transcriptionally regulated by SrbA, heme or heme intermediates are a potential possibility through which SrbA activity is regulated that remains to be explored (Davies and Rine, 2006; Chung et al., 2014).

An additional important mechanistic difference in regulation of SREBP activity in S. pombe and A. fumigatus compared to mammalian cells is at the level of proteolytic cleavage. S. pombe and A. fumigatus SREBP pathways lack homologs of S1p and S2p. Sre1 is activated by a different mechanism requiring golgi resident defective in sre1 cleavage (Dsc) proteins which form a complex collectively referred to as the Dsc complex that encode components of an E3 ligase (Stewart et al., 2011). Homologs of S. pombe Dsc1-4 are present in the A. fumigatus genome (dscA-D) and genetic null mutants largely phenocopy the A. fumigatus srbA genetic null mutant including important azole drug and virulence phenotypes (Willger et al., 2012). For example, loss of DscA significantly reduces SrbA N terminus protein levels in normoxia and hypoxia and mRNA levels of known SrbA target genes including cyp51A. Phenotypes of ΔdscA could largely be rescued by ectopic expression of the SrbA N terminus, amino acid residues 1-425. These data link Dsc proteins with regulation of SrbA function in A. fumigatus.

Recently, a homolog of S. pombe Dsc5, another component of the Dsc complex, was characterized (dscE) in A. nidulans and showed to be essential for low oxygen survival in this model organism. A. nidulans dscE has a UAS and UBX domain at the C terminus, similar to S. pombe Dsc5 (Stewart et al., 2012; Bat-Ochir et al., 2016). Interestingly, non-sense mutation in UAS domain of dscE results in loss of function of this key hypoxia regulator indicating a role of the UAS and possibly downstream UBX domain of DscE in hypoxia fitness, similar to the mechanism of Sre1 cleavage in S. pombe (Bat-Ochir et al., 2016). The UBX domain is known to interact with Cdc48, a major regulator of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway (reviewed in Stolz et al., 2011). In S. pombe, the UBX domain of Dsc5 is essential for recruitment of Cdc48 to Dsc2, a component of the Dsc complex; however, this recruitment is intriguingly dispensable for Sre1 cleavage (Stewart et al., 2012). It is worth noting that loss of Cdc48 is indispensable for Sre1 cleavage in S. pombe. Thus, further studies are needed to confirm if Cdc48 is recruited to other components of the Dsc complex apart from Dsc2 in a Dsc5-UBX domain independent manner or if expression and not recruitment is essential for its function in Sre1 cleavage. At this time, the role of the UAS domain of Dsc5/DscE is not completely understood. Future studies will determine if the UAS domain has a role in recruitment of Cdc48 or association of Dsc5 to other components of Dsc complex.

Consequently, the mechanism of Sre1 and SrbA cleavage remains to be fully elucidated. Recently, a rhomboid protease Rbd2 has been suggested to be necessary for cleavage and activation of Sre1 in S. pombe (Kim et al., 2015). Further mechanistic studies are necessary to determine the exact role of Rbd2 and other yet unidentified mechanisms for cleavage and activation of Sre1 in fission yeast. Recently two proteases including an Rbd2 homolog – rbdB and sppA - have been identified in Neurospora crassa and A. fumigatus mediating the cleavage of SrbA (Figure 2) (Bat-Ochir et al., 2016; Dhingra et al., 2016; Vaknin et al., 2016). How SrbA interacts with the Dsc complex, RbdB and SppA is an important area of ongoing research. It is important to note that canonically both rhomboid protease and aspartyl protease cleave the intramembrane domains and/or near the N terminal site of their substrates through regulated intramembrane proteolysis (Lohi et al., 2004; Ha et al., 2013; reviewed in Sun et al., 2016). In A. fumigatus both proteases appear essential for SrbA activation, thus future experiments will reveal if their functions are conserved or if they act in a non-canonical fashion. It is important to note that the DscE3 complex and SppA are golgi and ER resident localized proteins respectively (Stewart et al., 2011; Willger et al., 2012; Bat-Ochir et al., 2016). Thus, it is unclear if the processing of nascent SrbA happens in ER or if it is a dynamic process requiring anterograde and retrograde transport from ER to golgi and vice versa (Figure 2). Additionally, in mammals, SREBP cleavage in the ER lumen by S1P is a prerequisite for S2P activity, thus additional, yet unidentified, proteases may be involved in activation of SrbA (reviewed in Sun et al., 2016). As mammals and A. fumigatus have a distinct mechanism of SREBP activation, molecular dissection of SrbA proteolytic cleavage in A. fumigatus is an important area of future investigation to fully understand sterol biosynthesis and harness this pathway for novel therapeutic development.

Regulation of SREBPs in other fungi is complex and involves additional regulatory layers including post-translational mechanisms that could potentially be unique to filamentous fungi. These mechanisms are high priority targets for investigation in A. fumigatus. For example, a second non-sterol dependent pathway also controls levels of Sre1 in S. pombe. When oxygen concentration is high, a prolyl 4-hydroxylase-like 2-oxoglutarate-Fe(II) dioxygenase Ofd1 negatively regulates Sre1N levels in a proteasome dependent manner (Hughes and Espenshade, 2008). Under low oxygen concentrations another protein, Negative Regulator of Ofd1 – Nro1, binds to the C terminal degradation domain of Ofd1 and prevents Sre1N degradation leading to stability and accumulation of Sre1N in cells (Lee et al., 2009). It is interesting to note that while a putative homolog of Ofd1 is present in the A. fumigatus genome and remains to be characterized, a homolog of Nro1 is not present bringing into question whether this elegant regulatory mechanism is in play for SrbA regulation.

In mammals, SREBP TF levels and function are also controlled by post-translational modifications and this remains a promising area of investigation in A. fumigatus. In mammalian cells, GSK-3 (glycogen-synthase kinase) phosphorylates SREBP1 in response to DNA binding. GSK-3 mediated phosphorylation leads to docking of the ubiquitin ligase FBW7 on phosphorylated residues and subsequent proteasome-mediated degradation of active SREBP TFs (Sundqvist et al., 2005; Punga et al., 2006; Bengoechea-Alonso and Ericsson, 2009). Thus, in A. fumigatus, levels of active SrbA may be regulated through phosphorylation by GSK-3 and/or a different kinase followed by proteasome mediated degradation. This hypothesis awaits testing though is particularly attractive given the plethora of kinase inhibitors available to explore for antifungal development.

Another important area for investigation is the relationship between multiple SREBP genes in A. fumigatus and sterol biosynthesis. Similar to the mammalian SREBP pathway, three SREBPs have been identified in A. fumigatus – SrbA, SrbB, and SrbC (Willger et al., 2008; Chung et al., 2014). A second SREBP, Sre2, is present in S. pombe. Sre2 contains the hallmark tyrosine residue in the bHLH DNA binding domain, and two transmembrane domains however, Sre2 does not bind Scp1 and is not regulated by levels of sterols. Interestingly, Sre2 is constitutively cleaved and requires the Dsc complex for processing (Hughes et al., 2005; Stewart et al., 2011). However, loss of Sre2 does not appear to affect hypoxia fitness in S. pombe. Further studies are needed to determine the role and targets of Sre2 in S. pombe.

In A. fumigatus, SrbB and SrbC both have the canonical tyrosine residue in the bHLH DNA binding domain but lack the predicted transmembrane and C terminal domains of SrbA. SrbB mRNA levels are massively induced in response to hypoxia through an unknown mechanism. SrbA contributes in part to srbB hypoxia mRNA levels through direct binding to the srbB promoter region (Chung et al., 2014). However, loss of SrbA does not completely attenuate srbB mRNA levels. Importantly, loss of SrbB in A. fumigatus attenuates low oxygen fitness and virulence but not tolerance to azole antifungal drugs. Direct target genes of SrbB remain to be elucidated, but initial studies suggest it contributes to regulation of erg25A and hem13 mRNA levels. Importantly, SrbB is a critical regulator of the ethanol fermentation pathway through direct regulation of the alcohol dehydrogenase alcC mRNA levels (Grahl et al., 2011; Chung et al., 2014). SrbC is expressed at low levels in conditions examined to date including low oxygen and its role in sterol biosynthesis and SREBP gene regulation are under investigation (Chung et al., 2014).

An important area of future research is determining how the 3 SREBPs coordinate target gene expression which is expected to yield novel insights into the role of these TFs in A. fumigatus biology and pathogenesis. There at least appears to be co-regulation of the key ergosterol biosynthetic genes erg1 and erg25A by SrbA and SrbB (Chung et al., 2014). In mammals, hetero-dimerization of SREBPs controls regulation of SRBEP target genes (Datta and Osborne, 2005). Whether this co-regulation of sterol biosynthesis genes observed in A. fumigatus involves heterodimer formation between SrbA and SrbB is not known, and direct regulation of SrbB by SrbA and vice-versa cannot be ruled out and is likely. It is interesting to note the presence of a canonical SRE in the promoter region of SrbA and data suggest SrbA auto-regulates its mRNA levels (Chung et al., 2014). Further research will elucidate if binding of different SREBPs to the SRE element in the promoter region of SrbA confer differential protein regulation for activation of SrbA under hypoxic and sterol-level mediated stress. In general, rigorous promoter analysis of critical genes in ergosterol biosynthesis remains an important but understudied area of A. fumigatus biology.

Cytochrome P450 enzymes are heme dependent monooxyge nases and represent an important class of enzymes for normal levels of ergosterol production (and some enzymes in this class are the major targets of the azole class of anti-fungal drugs as previously discussed (reviewed in Nebert and Russell, 2002)). Two major cytochrome P450 heme containing enzymes are present in the ergosterol biosynthesis pathway viz., Erg5 and Erg11 (Cyp51) (Song et al., 2016). Despite their importance in sterol synthesis and removal of toxic sterol intermediates, cytochrome b5 and P450 oxidoreductase are the only known proteins that interact with and control the levels of cytochrome P450 enzymes (Hughes et al., 2007b; reviewed in Pandey and Flück, 2013; reviewed in Schenkman and Jansson, 2003). In fission yeast, the damage response protein Dap1 physically interacts with both Erg5 and Erg11 and is necessary for normal sterol levels (Hughes et al., 2007b). This physical interaction requires heme binding and a stable complex formation between Dap1 and P450 enzymes. Mutant cells lacking Dap1 accumulate sterol intermediates and lower levels of ergosterol (Hughes et al., 2007b).

In A. fumigatus, sequence similarity searches led to the identification and characterization of three Dap proteins, DapA-C with antagonistic functions. A DapA null mutant is hypersensitive to itraconazole, whereas a DapC null mutant is more resistant compared to the wild type strain. This sensitivity may stem from the fact that abnormal levels of sterols accumulate in Dap genetic null mutants. A DapA null mutant accumulates lower ergosterol levels and a subsequent increase in levels of ergosta-5,7,24(28)- trienol and ergosta 5,7 dienol indicating a blockage at Erg5 (cytochrome p450 desaturase) (Figure 1). The current model of Dap protein mediated regulation of sterol biosynthesis suggests DapA is necessary for stability of Erg5 and Cyp51A and loss of DapA leads to degradation of Cyp51B. Heme binding may be critical for DapA mediated stability of Erg5 and Cyp51A but not Cyp51B. Where DapA is necessary for function of P450 enzymes, DapB and DapC have antagonistic role through iron binding. DapB and DapC are predicted to irreversibly bind iron affecting the local iron concentration and thereby altering heme-dependent P450 enzyme function (Song et al., 2016). The presence of multiple Dap proteins with antagonistic functions allows complex regulation between sterol synthesis and iron availability. As Dap proteins control levels of P450 enzymes, targeting DapA might provide a novel therapeutic target to treat azole resistant cases of IA and or potentiate the efficacy of existing triazoles targeting Cyp51 enzymes.

Stress conditions can overwhelm ER capacity to correctly fold proteins that are destined for the membrane and cytosol. Perturbation in lipid synthesis is known to activate the Unfolded Protein Response (UPR) in S. cerevisiae and A. fumigatus (Pineau et al., 2009; Han et al., 2010; reviewed in Volmer and Ron, 2015). The UPR pathway alleviates ER stress by balancing the proteins entering the ER for folding and the rate of ER protein folding capacity (reviewed in Moore and Hollien, 2012). Proteins that fail to fold correctly are degraded by a proteasome-mediated degradation pathway called ER-associated degradation (ERAD) (reviewed in Tsai and Weissman, 2010; reviewed in Wang and Kaufman, 2014). Mutants defective in ERAD constitutively activate the UPR, indicating regulatory crosstalk between these two pathways (Travers et al., 2000). A. fumigatus detects ER stress via the ER stress sensor IreA, which activates HacA – the major regulator of the UPR (Feng et al., 2011). Ergosterol levels are negatively affected in both ireA and hacA null mutants and sterol intermediates accumulate in ΔhrdA, a strain with a deficient ERAD pathway (Feng et al., 2011; Krishnan et al., 2013). This increase in sterol intermediates in ΔhrdA might be the result of HMG CoA reductase turnover as Hrd1 is required for degradation of HMG CoA reductase in yeast (Hampton et al., 1996). Interestingly, ΔhrdA is resistant to voriconazole compared to the WT strain, although mRNA levels of cyp51A and cyp51B are not different in these two strains (Krishnan et al., 2013). While the ERAD pathway appears dispensable for virulence in A. fumigatus, mutants that lack the UPR regulators ireA and hacA show significant virulence attenuation in murine models of IPA (Richie et al., 2009; Feng et al., 2011; Krishnan et al., 2013).

Intriguingly, like the SREBP pathway, the UPR is involved in hypoxia and iron stress responses in A. fumigatus (Feng et al., 2011; reviewed in Grahl et al., 2012; reviewed in Krishnan and Askew, 2014). This raises an interesting question of interplay between the UPR and SREBP activation in A. fumigatus given also that SrbA is an ER resident protein. It is not clear if SREBP activation requires the UPR, as hypoxia is known to activate UPR in tumors (Feldman et al., 2005; reviewed in Romero-Ramirez et al., 2004; Koumenis and Wouters, 2006). In support of this hypothesis, a mechanism by which the UPR is activated in response to lipid stress is different compared to protein misfolding activation, and ireA mediated hypoxia fitness is hacA independent in A. fumigatus (Feng et al., 2011; Lajoie et al., 2012). As UPR mutants ΔhacA and ΔireA have increased sensitivity to azoles and reduced total ergosterol content (Richie et al., 2009; Feng et al., 2011), future research should elucidate whether the role of UPR in mediating azole sensitivity is SREBP dependent or independent. Also, the role of the UPR pathway in azole resistance needs further exploration. This provides a novel genetic opportunity to decipher the link between the UPR, SREBPs and lipid homeostasis and highlights a potential therapeutic opportunity to target the UPR and/or SREBP pathway in A. fumigatus.

Iron is a major cofactor for essential life processes and is indispensable for growth of microbes including A. fumigatus (reviewed in Chung et al., 2012; reviewed in Haas, 2012; reviewed in Kaplan and Kaplan, 2009). Microbes have elegant strategies to acquire iron under iron deplete conditions such as in the tissue microenvironments of the host (Schrettl et al., 2007, 2008). Under these conditions, A. fumigatus relies mainly on siderophore production for iron acquisition (Schrettl et al., 2004). SrbA is required for production of siderophores and fitness in low iron liquid environments (Blatzer et al., 2011). There is a direct correlation between iron availability and sterol levels in A. fumigatus and this is mediated in part by SrbA (Blatzer et al., 2011; Yasmin et al., 2012). Mevalonate is a key metabolic intermediate between ergosterol and siderophore production, and its fate is dependent on iron availability. Under iron replete conditions, mevalonate is preferentially converted to ergosterol, however, in iron deplete conditions mevalonate is converted to the siderophore TAFC. In iron deplete conditions, ergosterol levels are reduced by ∼50% and sterol intermediates accumulate rendering A. fumigatus more sensitive to voriconazole (Yasmin et al., 2012). Consequently, these data suggest that HMG CoA reductase is a potential target for treatment of IA. Cholesterol lowering agents known as statins target HMG CoA reductase (reviewed in Tobert, 2003). Statins have fungicidal activity against A. fumigatus, however at concentrations higher than safe physiological levels used to control cholesterol in humans (Qiao et al., 2007). In plants, bacteria and protozoa, a non-mevalonate pathway (NMP) exists for isoprenoid biosynthesis (reviewed in Hunter, 2007; Odom, 2011). As NMP is not reported in A. fumigatus and homologs of enzymes in NMP are absent in A. fumigatus, modifying current statins or development of new drugs to target mevalonate production at clinically relevant concentrations needs further exploration.

Hsp90 is molecular chaperone that regulates diverse client proteins, many of which are involved in cell signaling (Cowen and Lindquist, 2005). Hsp90 is conserved across eukaryotes and gene deletion strategies reveal it is necessary for survival in A. fumigatus (Lamoth et al., 2014). In S. cerevisiae and C. albicans, Hsp90 function is critical for the emergence of azole resistance. Hsp90 mediated azole resistance is associated with loss of erg3 function, leading to accumulation of ergosta-7,22-dienol in C. albicans. This prevents accumulation of toxic 14-α-methyl-3,6-diol. This allows, alternatively, 14-α methyl fecosterol to incorporate into the membrane and circumvent cyp51 mediated azole activity (Martel et al., 2010; reviewed in Shapiro et al., 2011). Thus, it is plausible that Hsp90 directly or indirectly affects the ergosterol biosynthetic pathway in A. fumigatus. Mutagenesis experiments revealed deacetylation of K27 is necessary for Hsp90 mediated azole resistance in A. fumigatus, however no positive interaction was observed between a lysine deacetylase inhibitor and azole drugs in A. fumigatus (Lamoth et al., 2014). As erg3 associated azole resistance is reported for C. albicans (Kelly et al., 1997) and has not been identified or reported in clinical azole resistance strains of A. fumigatus to date, further research is needed to determine the role of Hsp90 on long-term azole therapies for IA and to completely understand the role of Hsp90 in azole mediated drug resistance.