Hypoxia and Gene Expression in Eukaryotic Microbes Geraldine Butler School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; email: [email protected]Annu. Rev. Microbiol. 2013. 67:291–312 First published online as a Review in Advance on June 26, 2013 The Annual Review of Microbiology is online at micro.annualreviews.org This article’s doi: 10.1146/annurev-micro-092412-155658 Copyright c 2013 by Annual Reviews. All rights reserved Keywords SREBP, fungi, evolution, biofilm, UPC2, ROX1 Abstract The response of eukaryotic microbes to low-oxygen (hypoxic) conditions is strongly regulated at the level of transcription. Comparative analysis shows that some of the transcriptional regulators (such as the sterol reg- ulatory element-binding proteins, or SREBPs) are of ancient origin and probably regulate sterol synthesis in most eukaryotic microbes. However, in some fungi SREBPs have been replaced by a zinc-finger transcription factor (Upc2). Nuclear localization of fungal SREBPs is determined by regulated proteolysis, either by site-specific proteases or by an E3 ligase complex and the proteasome. The exact mechanisms of oxygen sensing are not fully char- acterized but involve responding to low levels of heme and/or sterols and possibly to levels of nitric oxide and reactive oxygen species. Changes in central carbon metabolism (glycolysis and respiration) are a core hypoxic response in some, but not all, fungal species. Adaptation to hypoxia is an important virulence characteristic of pathogenic fungi. 291 Annu. Rev. Microbiol. 2013.67:291-312. Downloaded from www.annualreviews.org by University of Leeds on 09/16/13. For personal use only.
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MI67CH14-Butler ARI 5 August 2013 17:3
Hypoxia and Gene Expressionin Eukaryotic MicrobesGeraldine ButlerSchool of Biomolecular and Biomedical Science, Conway Institute, University College Dublin,Belfield, Dublin 4, Ireland; email: [email protected]
Annu. Rev. Microbiol. 2013. 67:291–312
First published online as a Review in Advance onJune 26, 2013
The Annual Review of Microbiology is online atmicro.annualreviews.org
This article’s doi:10.1146/annurev-micro-092412-155658
The response of eukaryotic microbes to low-oxygen (hypoxic) conditionsis strongly regulated at the level of transcription. Comparative analysisshows that some of the transcriptional regulators (such as the sterol reg-ulatory element-binding proteins, or SREBPs) are of ancient origin andprobably regulate sterol synthesis in most eukaryotic microbes. However, insome fungi SREBPs have been replaced by a zinc-finger transcription factor(Upc2). Nuclear localization of fungal SREBPs is determined by regulatedproteolysis, either by site-specific proteases or by an E3 ligase complex andthe proteasome. The exact mechanisms of oxygen sensing are not fully char-acterized but involve responding to low levels of heme and/or sterols andpossibly to levels of nitric oxide and reactive oxygen species. Changes incentral carbon metabolism (glycolysis and respiration) are a core hypoxicresponse in some, but not all, fungal species. Adaptation to hypoxia is animportant virulence characteristic of pathogenic fungi.
The level of oxygen in the atmosphere was very low until the evolution of photosynthetic or-ganisms (cyanobacteria), approximately 2.5–3 billion years ago (117). Oxygen concentrations in-creased to significant levels about 1.5 billion years ago, which coincided with the appearanceof the first eukaryotes. These organisms could generate energy by oxidative phosphorylationin internal organelles, the mitochondria. The first fungi probably appeared at the same time(51, 52). Oxygen levels continued to fluctuate, particularly during the expansion of the metazoans(past 500 million years). Oxygen is a toxic molecule; it is therefore not surprising that organismsevolved sophisticated mechanisms for responding to altered levels (130).
Many eukaryotes are also exposed to vastly different levels of oxygen in environmental niches.Some eukaryotic microbes are obligate anaerobes, such as the flagellated Neocallimastigales (re-lated to chytrids; 55), which were first isolated from highly anaerobic sheep rumen (42, 133).Other facultative anaerobes are found in stagnant waters (42). However, even obligate aer-obes and facultative aerobes can be exposed to long-lasting or transient hypoxic conditions.For example, many fungi grow in soil where oxygen levels are low. Others (such as Aspergillusfumigatus) colonize compost heaps where the concentration of O2 approaches 1.5% (136). Fun-gal pathogens are also exposed to variations in oxygen concentrations in the mammalian host(35).
Most organisms can sense oxygen concentrations and respond rapidly to changes. The tran-scriptional response of many fungal species during the early stages of adaptation to low-oxygenconditions has been well characterized (9, 11, 35, 46, 62, 75, 77, 119, 127). Genes that are differen-tially expressed in response to oxygen levels can be divided into two general groups: aerobic genesthat are expressed in normoxic conditions, and hypoxic genes that are expressed when oxygenis low or completely absent (104). A core transcriptional response is shared by most eukaryotes,including increased expression of genes encoding enzymes in oxygen-dependent pathways, such
aSignal of oxygen levels, where known.bSpecies with experimental evidence are listed. Orthologs in other species are likely to have similar functions.cMajor target pathways are listed. Not all target genes in all species have been identified.dTarget genes that may be restricted to one species or a group of species.Abbreviations: NO, nitric oxide; SREBP, sterol-regulatory element binding protein.
as synthesis of sterols, lipids, and heme (14, 35) (Table 1). Other changes seen in some but notall species include an increase in glycolysis and a decrease in respiration, as well as changes in cellwall gene expression (5, 46, 75, 77, 127) (Table 1).
How oxygen is sensed by fungi is not completely understood. Sterol biosynthesis is an oxygen-dependent process, and in several species lowered sterols are apparently used to sense oxygenlevels (24, 27, 29, 62, 134). Similarly, heme (which contains an iron group that binds oxygen)can act as a secondary signal for oxygen, best characterized in Saccharomyces cerevisiae (11, 29,145).
In this review I examine how the transcriptional response of eukaryotic microbes to hypoxicconditions is regulated, concentrating predominantly on the fungi, which are the best character-ized. I also investigate the evolution of core regulatory molecules—some ancient and some veryrecent (summarized in Table 1).
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CONSERVATION AND DISTRIBUTION OF THE MAJORHYPOXIC REGULATORS
Hypoxia-Inducible Factor
Hypoxia-inducible factor (HIF) is probably the best-studied regulator of the oxygen responsein mammals (45). HIFs are dimeric transcription factors, consisting of a regulated subunit (oneof multiple HIFα components) and a constitutively expressed subunit [from multiple HIFβ orthe aryl hydrocarbon receptor nuclear translocator (ARNT) components]. When O2 levels arehigh, HIFα is modified by proline hydroxylases and subsequently degraded via the ubiquitin–proteasome pathway. As oxygen levels drop, hydroxylase activity is inhibited; HIFα enters thenucleus, dimerizes with HIFβ, and regulates the expression of hundreds of genes. Expression ofglucose transporters, glycolytic enzymes, and lactate dehydrogenase A is induced, which increasesglycolysis (129). The efficiency of mitochondrial respiration is increased (117). Genes associatedwith erythropoiesis, angiogenesis, and vasodilation are also regulated (47).
HIF subunits are well conserved throughout the Metazoa, from mammals to arthropods tocnidaria (50). The function is also well conserved; for example, HIFα is also regulated by hydrox-ylation in worms and flies (4, 34). However, HIF orthologs in fungi have never been reported. Todetermine the origin of HIF proteins, we took advantage of recent genome-sequencing projectsaimed at characterizing the evolution of multicellular eukaryotes (112). As suggested previously(88), there are no obvious HIFα orthologs outside the Metazoa (Figure 1). Surprisingly however,there are two HIFβ-like proteins in the genome of Capsaspora owczarzaki, a symbiotic amoeba(54). The proteins have basic helix-loop-helix (bHLH) and PAS (Per/Arnt/Sim) domains, whichcan act as sensors for oxygen, light, and redox potential (128). C. owczarzaki is a member of theFilasterea, a sister group to the choanoflagellates and the Metazoa (Figure 1). We could notidentify other HIFβ orthologs in the choanoflagellates or in older diverging groups, includingFungi; however, very little sequence information is available (Figure 1). It is likely that HIFβ
arose in the common ancestor of the Filasterea, the choanoflagellates, and the Metazoa, but theoxygen-dependent HIFα subunits did not appear until later, in the ancestor of the Metazoa. It isalso possible that C. owczarzaki obtained the HIFβ-like proteins by horizontal gene transfer froman unknown source (e.g., 121); this will be resolved as more sequenced genomes become available.It is not clear what role the HIFβ-like proteins play in C. owczarzaki. However, in mammals,HIF-1β is also involved in the response to dioxins and forms dimers with the aryl hydrocarbonreceptor as well as with HIFα (139).
Distribution of SREBP and Upc2
Sterol regulatory element-binding proteins (SREBPs) are an ancient family of regulators that areassociated with the hypoxic response. Mammals have two SREBP genes (SREBP-1 and SREBP-2)that regulate the expression of fatty acid and cholesterol synthesis and cholesterol uptake genes (60).Invertebrates such as Caenorhabditis elegans and Drosophila melanogaster have only one SREBP or-tholog and tend to be cholesterol auxotrophs (101). Some fungi such as A. fumigatus and Schizosac-charomyces pombe also have SREBP orthologs (101). The localization and activation of SREBPsare described in the next section.
Again, interrogation of recent genome sequences (112) provides insights into the evolutionof the SREBP pathway. Like HIF, SREBPs have bHLH domains but are unique in that a tyro-sine residue replaces a well-conserved arginine (71). SREBP homologs are conserved in Metazoa,Choanoflagellata, Filasterea, Ichthyosporea, and Fungi (Figure 1). The SREBP cleavage acti-vating proteins (SCAPs), which interact with SREBPs, are less widely distributed but have been
Figure 1Conservation of hypoxic regulators in eukaryotic microbes. Phylogenetic relations are shown indiagrammatic form and are adapted from Reference 112. Plus signs and minus signs indicate presence andapparent absence of genes, respectively. No sequence information is available from the Nucleariidae. Themost likely timing of gene gains and losses is shown on the left-hand side.
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SCAP: SREBPcleavage activatingprotein
Opisthokonts:members of the cladethat comprisesMetazoa, Fungi, andtheir close unicellularlineages
Saccharomycotina:a subphylum of theAscomycota thatincludes Saccharomycesand Candida clades
Whole genomeduplication (WGD)event: an event thattook place in theancestor ofSaccharomyces cerevisiaeand closely relatedspecies
identified in some choanoflagellates and Ichthyosporea, in ancient fungal lineages (Spizellomycespunctatus and Mortierella verticillata), and throughout fungi (Figure 1). There are several speciesin which SCAP has been lost, including the entire Eurotiomycetes lineage (e.g., A. fumigatus). Inaddition, there are no obvious homologs in the Amoebozoa. The simplest interpretation is thatthe SREBP signaling pathway arose in the ancestor of the opisthokonts, following the divergencefrom the Amoebozoa, and that components were subsequently lost in some lineages.
SREBPs act as hypoxic regulators in fungi, but it is not clear if they also respond to oxygensignals in the Metazoa (101). In the ascomycetes S. pombe and A. fumigatus, SREBP homologscontrol the expression of genes involved in biosynthesis of lipids, ergosterol, and heme (131, 141).SREBP function is also conserved in the basidiomycete Cryptococcus neoformans (24, 27).
Surprisingly, in some ascomycete species (the Saccharomycotina; Figure 1) there has been anevolutionary rewiring of the sterol synthesis pathway. In these species, the role of SREBP in sterolregulation has been replaced by a different transcription factor, Upc2. Upc2 shares no sequencesimilarity with SREBPs and has a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain.Several fungal proteins have the same domain, but Upc2 appears to be unique to, or highly derivedin, the Saccharomycotina (Figure 1). Upc2 regulates the expression of sterol synthesis and otherhypoxic genes in S. cerevisiae, Candida albicans, and Candida parapsilosis (46, 58, 134).
The most likely homologs of SREBPs in the Saccharomycotina are represented by Cph2 (inC. albicans) (14) and Hms1 (in S. cerevisiae); these have bHLH domains with the characteristictyrosine (14). However, the transmembrane domains associated with SREBP function are poorlyconserved, and the proteins are shorter. In addition, Cph2 regulates hyphal growth (80), andHMS1 is a regulator of pseudohyphal growth (89).
One hypothesis is that Upc2 took over the role of major hypoxic regulator of the sterol pathwayin the Saccharomycotina, allowing the SREBP homologs to degenerate and acquire new functionsas regulators of filamentation. It is also possible that the ancestral fungal SREBP had roles in bothhypoxic regulation and in determination of morphology. This is supported by the observation thatSrbA in A. fumigatus is required both for hypoxic regulation and for hyphal branching (141).
The function of Upc2 in S. cerevisiae and closely related yeasts is complicated because therewas a whole genome duplication (WGD) event in a recent ancestor (142). Although the additionalcopies of most genes were subsequently lost, two Upc2 paralogs (Upc2 and Ecm22) were retainedin S. cerevisiae. Both paralogs play roles in regulating the expression of sterol synthesis genes (134),and the promoters of almost one-third of genes whose expression is induced in hypoxic conditionsin S. cerevisiae contain potential Upc2-binding sites (77). Upc2 probably senses lowered sterollevels and also lowered heme levels as a means of sensing oxygen. Treatment with sterol-depletingdrugs has an effect on transcription similar to that of lowering oxygen, and requires Upc2 inS. cerevisiae, C. albicans, and C. parapsilosis (29, 46, 127).
Although Upc2 and SREBPs are not structurally related, there is an intriguing functionalsimilarity. Both Upc2 and Ecm22 are localized to intracellular membranes and may be traffickedto the nucleus following treatment with sterol-depleting drugs (92). This is similar to the changein location observed following processing of SREBP in mammals and several fungi.
SENSING OF OXYGEN LEVELS
SREBP Pathway
Sensing of sterol levels (and therefore, indirectly, of oxygen) by SREBPs is by far the best-studiedand the best-understood hypoxic mechanism in fungal systems (14, 36, 101). In mammals and insome fungi, SREBPs are synthesized as inactive precursors that are inserted into the endoplasmic
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INSIG:insulin-induced gene
HMG-CoA:5-hydroxy-3-methylglutaryl–coenzyme A
reticulum (ER) membrane (Figure 2). The presence of two transmembrane domains ensures thatboth the C terminus and the N terminus containing the DNA-binding domain face the cytoplasm.The SREBPs associate with SCAP in the ER membrane. SCAPs bind sterols, keeping them ininactive conformations and retaining SREBP in the membrane. As sterol levels drop, the N-terminal region of SREBP is cleaved off and enters the nucleus, where it activates the expressionof target genes.
Although the basic mechanism of SREBP activation is conserved between mammals and fungi,there are also substantial differences. Mammalian SREBP/SCAP complexes are retained in the ERmembrane by interaction with INSIGs (insulin-induced genes). Cholesterol binds to SCAP, in-ducing a conformational change that causes SCAP to interact with INSIGs (2), whereas oxysterolsbind directly to INSIG (106). As sterol levels drop, the interaction between SCAP and INSIG isdisrupted, and the SREBP-SCAP complex is transported to the Golgi apparatus via COPII-coatedvesicles (85). SREBP is cleaved in the Golgi apparatus, and the N terminus is released and entersthe nucleus (36). INSIGs also regulate proteolysis of HMG-CoA (5-hydroxy-3-methylglutaryl-coenzyme A; required for the production of mevalonate, a rate-controlling step in cholesterolbiosynthesis) reductases in mammalian cells (120).
Homologs of INSIG are well conserved across the fungal kingdom [except for C. neoformans(14)]. However, they have no apparent role in regulating transport of the SREBP-SCAP complex.In S. pombe, the INSIG ortholog Ins1 regulates sterol synthesis, but only by inhibiting activityof HMG-CoA reductases (21). Ngs1, the S. cerevisiae ortholog of INSIG, controls HMG-CoAreductase through regulation of the protein levels of one isoform encoded by Hmg2. However,Ngs1 stabilizes rather than inhibits Hmg2 activity (20, 40).
Mammalian SREBP is cleaved by two site-directed proteases. The first (site-1 protease, orSP1) cleaves within the loop in the Golgi lumen, and the second (site-2 protease, or SP2) cleaveswithin the transmembrane domain (32, 33). Site-1 proteases belong to the Kexin 2 family; they arewell conserved throughout fungi and have many functions (114). Site-2 proteases are conservedin C. neoformans and in many basidiomycetes but appear to have been lost from the ascomycetes(13, 14, 25). In C. neoformans, deleting the SP-2 ortholog STP1 results in increased sensitivity tohypoxia (25, 27). Stp1 is required for processing the SREBP Sre1; when residues in the catalyticsite are mutated, processed forms of Sre1 fail to accumulate (13). Stp1 is also required for Sre1processing when sterols are lowered by treatment with azole drugs (13).
Processing SREBPs in the ascomycetes is very different from that in the basidiomyceteC. neoformans. A. fumigatus and related species, for example, have no SCAP homolog (Figures 1and 2), whereas S. pombe contains two SREBPs (Sre1 and Sre2). Sre2 is much shorter than Sre1,it does not bind SCAP, and it is constitutively cleaved irrespective of the levels of sterols (62).Stewart et al. (123, 124) showed that the Dsc E3 ligase complex, which transfers ubiquitin froman E2 ubiquitin-conjugating enzyme to a target, is required for processing both Sre1 and Sre2(Figure 2). Sre1 cleavage also requires the E2-conjugating enzyme Ubc4 and components of theproteasome. Stewart et al. (123, 124) suggest that Scp1 transports SREBPs to the Golgi apparatus,where they are ubiquitinated by Ubc4-Dsc and targeted to the proteasome for cleavage. Thismechanism may be a common hypoxic response in many ascomycete species, compensating forthe loss of SP2, and is supported by the recent demonstration that the complex is required alsofor cleavage of SrbA in A. fumigatus (140).
S. pombe Sre1 is regulated independently of sterol levels, by 2-oxogluturate Fe(II) dioxygenase(Ofd1) (14). Ofd1 is a member of the prolyl hydroxylase (PHD) family, of which some membersregulate degradation of HIFα in mammals. When oxygen levels are high, Ofd1 accelerates thedegradation of Sre1N via the proteasome. However, the transcription factor is stabilized in lowoxygen levels (63). Ofd1 acts as an oxygen sensor and an effector via two distinct domains; the
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– O2
Nro1
O2 , sterols
O2 , sterols
? O2 , sterols
C
Sre1N
C
Sre1N
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SrbA
CSre1
Scp1 Scp1SRE
Ofd1
-Sterol synthesis-Lipid synthesis
Stp1 protease?
Aspergillus fumigatus
Schizosaccharomyces pombe
Cryptococcus neoformans
Mammals
CSre1N
Sre1N
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ENDOPLASMICRETICULUM GOLGI APPARATUS NUCLEUS
-Sterol synthesis-Lipid and heme synthesis
-Sterol synthesis-Iron homeostasis
INSIG
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dioxygenase domain in the N terminus, which senses oxygen, and the C-terminal domain, whichaccelerates degradation.
Subsequent analysis showed that the C terminus of Ofd1 interacts with Nro1, a nuclear protein(82). The interaction is regulated by oxygen; when levels are low, Nro1 binds to Ofd1 and inhibitsdegradation of Sre1N. Nro1 also regulates the nuclear localization of Ofd1 (144). Surprisingly,Ofd1 and Nro1 regulate binding of Sre1N to DNA, as well as proteolysis (83). When oxygenlevels are high, Ofd1 binds (via its C-terminal domain) to Sre1N, preventing binding of the tran-scription factor to regulatory regions on DNA. As oxygen levels drop, Nro1 binds the C-terminaldomain of Ofd1, thus releasing Sre1N and allowing transcription of hypoxic genes (83).
Using a sophisticated mathematical model in combination with experimental measurements,Porter et al. (103) suggest that the inhibitory effect of Ofd1 on DNA binding of Sre1N in highoxygen levels is the most important step in hypoxic regulation. Oxygen-dependent degradation ofSre1N may speed up the response, but it is not absolutely necessary. Hypoxic regulation by Sre1Nis a feed-forward mechanism, in which changes in oxygen levels result in compensatory alterationsin metabolism (such as increased sterol synthesis) (103). This mechanism encompasses positivefeedback through self-regulation of Sre1N (64) and negative feedback through increased produc-tion of Ofd1 in low oxygen. Changing the concentration of Nro1, which competes with Ofd1 forbinding to Sre1N, also allows the cell to fine-tune the response to changing oxygen conditions.
The prolyl hydroxylase activity of Ofd1 does not appear to be important for the hypoxicresponse, which is very different from the regulation of HIF stability by PHD proteins (83). How-ever, Ofd1 is generally well conserved in fungal species (14). The S. cerevisiae ortholog (Tpa1) isrequired for translation termination (53, 67) and binds to Nro1 (72). Tpa1 represses the expressionof respiratory genes regulated by Hap1 (53). It is therefore possible that Ofd1 has been recruitedfor hypoxic regulation in several fungal species but acts through different mechanisms.
Heme-Activated Proteins
Heme biosynthesis requires oxygen, and the expression of many biosynthetic genes is increasedwhen oxygen levels are low. Reducing heme levels is utilized by S. cerevisiae (and probably otherfungi) to monitor oxygen levels by way of the transcription factor Hap1 and, in some cases, Upc2
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2The SREBP pathway in fungi and mammals. The SREBP/SCAP complex in mammals is maintained in theendoplasmic reticulum by interacting with INSIGs. A drop in sterol levels causes a change in theconformation of SCAP, release from INSIG, and transport to the Golgi apparatus. The N-terminal region isreleased by cleavage by two site-specific proteases, enters the nucleus, and activates target gene expression.The transport pathway in fungi, and the role of the Golgi apparatus, is not completely understood. TheSREBP Sre1 is membrane bound in a complex with the SCAP Scp1 in Cryptococcus neoformans andSchizosaccharomyces pombe; there is no SCAP in Aspergillus fumigatus, and the mechanism of retention is notknown. Cleavage of the N-terminal region of SREBP (by one Site-1 protease in C. neoformans, and theproteasome in S. pombe and A. fumigatus) is inhibited by oxygen and high sterol levels. The N-terminaltranscription factor binds to the sterol regulatory element (SRE) in the nucleus and activates the expressionof sterol metabolism and other genes. In S. pombe, when oxygen levels are high, Ofd1 binds to Sre1N,inhibiting DNA binding and targeting the transcription factor for degradation. When oxygen levels are low,Ofd1 is sequestered by the nuclear transporter Nro1. The role of Ofd1 in regulating hypoxia in other fungiis not known. Several putative and/or currently uncharacterized steps in the pathways are indicated withquestion marks. The figure is based on Reference 14, with additional information. Abbreviations: INSIG,insulin-induced gene; SCAP, SREBP cleavage activating protein; SREBP, sterol regulatory element-bindingprotein.
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(29). In the absence of heme, Hap1 forms a high-molecular-weight complex that is incapable ofbinding DNA and is gradually reduced to a dimer as heme binds (145). Hap1 directly regulatesthe expression of many genes associated with respiration, such as the cytochrome c protein CYC1and the cytochrome c oxidase COX5A. However, one of the most important roles of Hap1 isto regulate the expression of ROX1, which encodes an HMG domain protein that represses theexpression of a large number of hypoxic genes (11).
In S. cerevisiae, many duplicated genes retained after the WGD event are associated withrespiration. For several of the gene pairs, the expression of one member is induced in responseto high oxygen, and the expression of the other is induced in hypoxic conditions (146). Thesegene pairs include CYC1 (heme activated) and CYC7 (heme repressed), COX5A (heme activated)and COX5B (heme repressed), and the translation initiation factors TIF51A (heme activated) andANB1 (heme repressed). The expression of the gene pairs is regulated by HAP1 and/or ROX1 (57,90, 146).
Despite the evidence from S. cerevisiae, the breadth of heme sensing in hypoxia is not clear.HAP1 is conserved throughout the ascomycetes but has not been characterized in many species.HAP1 from Kluyveromyces lactis, a close relative of S. cerevisiae, can complement the defect ofa hap1 deletion in S. cerevisiae and also regulate the expression of cytochrome c subunits (8).The expression of HAP1 is induced in hypoxia in both species (8). However, there are significantdifferences in the hypoxic responses. Unlike in S. cerevisiae, Hap1 is not required for the expressionof genes involved in cholesterol biosynthesis or oxidative stress in K. lactis (78).
Many of the differences in the hypoxic response between S. cerevisiae and K. lactis are likely tobe related to differences in Rox1 function. An apparent homolog of ROX1 in K. lactis plays no rolein the hypoxic response (37). Similarly, a homolog of ROX1 in C. albicans (called RFG1) does notregulate hypoxic expression but instead controls filamentous growth (66, 69). The repressive roleof Rox1 is probably confined to S. cerevisiae and some very closely related species, and it is unlikelythat Rox1 orthologs act as major fungal regulators of gene expression in hypoxia.
It is likely that heme sensing is important for assessing oxygen concentrations in manyspecies (19). Other heme-activated proteins have been characterized in S. cerevisiae, includingthe Hap2/3/4/5 complex, which binds to a CCAAT box upstream of genes required for growthon nonfermentable carbon sources (100). Hap2, Hap3, and Hap5 are required for DNA bind-ing, and Hap4 is a regulatory subunit (75). Hap2 and Hap3 are functionally interchangeable withthe CP1-A/B subunits in human cells (26). It is not known how heme activates the Hap2/3/4/5complex, but it is unlikely to be through DNA binding (75). The activity of CCAAT-box-bindingproteins in mammals also is modulated by heme treatment (95), suggesting that the involvementof heme in oxygen sensing may represent an ancient conserved mechanism.
Other Pathways
In S. pombe, Ofd2, a 2-OG-Fe(II) dioxygenase, acts on histone H2A, a major component ofchromatin (79). Ofd2 is similar in structure to Ofd1, which regulates activity of the SREBP Sre1.Lando et al. (79) found that the expression of genes involved in ATP synthesis and mitochondrialelectron transport, which are repressed in hypoxia, are further repressed when ofd2 is deleted.Ofd2 is targeted to the promoters of the oxidative phosphorylation genes. It is not clear exactlyhow Ofd2 modifies histone H2A; possibilities include hydroxylation or demethylation. However,it appears that Ofd2 senses oxygen levels and inhibits repression of respiratory genes.
Considerable evidence suggests that mitochondria and the respiratory chain are important forthe hypoxic response in microbial eukaryotes. In S. cerevisiae, about 70% of the genes that aredifferentially expressed in hypoxia require a functioning mitochondrion (104). It is not known
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how the changes in gene expression are regulated. However, yeast mitochondria produce nitricoxide (NO) from nitrite in low-oxygen conditions, through the activity of cytochrome c oxidase(23). NO is required for induction of CYC7, the hypoxic isoform of cytochrome c (23, 76). NOdirectly affects the level of nitration of tyrosine residues and also causes an increase in the overalllevel of protein carbonylation (23).
Mitochondrial NO also plays an important role in the hypoxic response of mammalian cells (23,104). Mitochondrial function, and particularly cytochrome c, is required for stabilization of HIFα
in hypoxic conditions (91). Recent evidence suggests that stabilization requires NO producedby cytochrome c oxidase in the mitochondrion (7). Other mitochondrial reactive oxygen species(ROS) are also likely to be important for hypoxic signaling in mammalian cells and in yeast cells(48, 49).
Because HIFα is not found outside the Metazoa (Figure 1), the role of the mitochondrionin the hypoxic response for mammalian cells cannot be the same as that for fungi. However, itis likely that NO regulates the stability or activity of other proteins required for the response tohypoxia in S. cerevisiae. Deletion of cytochrome c significantly attenuates growth of A. fumigatus inhypoxic conditions, suggesting that the electron transport chain is also important for the hypoxicresponse in this species (43).
The mitochondrion is also important for the hypoxic response of C. neoformans, where mutantssensitive to CoCl2 and to hypoxia are defective in mitochondrial function (65). CoCl2 is commonlyused as a mimic of hypoxia in mammalian cells, where it stabilizes HIFα by regulating the activityof prolyl hydroxylases (116). In C. neoformans and S. pombe, CoCl2 targets the sterol synthesispathway, probably by replacing iron in Erg25 and Erg23 enzymes (84). Some of the C. neoformansmitochondrial mutants produce increased levels of ROS, supporting the hypothesis that ROS maybe used as an oxygen-sensing mechanism in many fungi.
REGULATION OF STEROL, LIPID, AND IRON METABOLISM GENES
Sterols and lipids are essential for membrane fluidity, and levels are therefore carefully controlled.Induction of sterol synthesis gene expression is one of the most conserved responses to hypoxiain fungi and has been reported in every species studied (e.g., 9, 16, 22, 27, 29, 62, 141). Genesrequired for the oxygen-dependent steps are particularly upregulated (9, 127). Regulation isassociated most closely with activation of SREBPs (in basidiomycetes and many ascomycetes)or with members of the Upc2 family, as described above. However, other mechanisms mayalso be used. For example, in A. fumigatus, the expression of genes required for the early stepsin ergosterol synthesis (terpenoid synthesis) is reduced in hypoxia, leading to a decrease insubstrates such as mevalonate. Barker et al. (9) have suggested that there may be a direct linkbetween hypoxic regulation of terpenoid biosynthesis and ergosterol synthesis that bypassesdirect regulation by SREBP, possibly by acting through HMG-CoA reductase (108).
The expression of many other lipid biosynthesis genes is increased in low-oxygen conditions.In S. cerevisiae and K. lactis, hypoxic induction of fatty acid synthesis genes requires the Mga2transcription factor (94, 96). The expression of lipid synthesis may also be directly regulated bySREBP/Upc2 [e.g., in C. neoformans (27) and in S. pombe (62)] or other transcription factors [e.g.,Efg1 in C. albicans (119)]. However, it is likely that other mechanisms, such as a general stressresponse, are also involved (108).
The fungal response to hypoxia overlaps the response to low iron; many genes involved inheme biosynthesis and/or iron acquisition are upregulated in both conditions (9, 15, 25, 27, 119,127, 131). In A. fumigatus, iron acquisition genes are probably regulated directly by the SREBPSrbA (15). However, because the reductive transport system in species such as C. albicans requires
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oxidation of Fe2+, it is hard to separate the direct effects of low oxygen from those of low iron(reviewed in 3). It is possible that iron is used as an oxygen sensor, distinct from its previouslydescribed role in heme-activated proteins, but this remains to be characterized (22).
REGULATION OF GLYCOLYTIC GENE EXPRESSION
Glycolysis is one of the oldest metabolic pathways, and glycolytic enzymes predate the appearanceof oxygen-requiring species by at least 2 billion years (137). In multicellular eukaryotes (includingmammalian cells), the expression of glycolytic enzymes is strongly and coordinately induced inhypoxic conditions, and it has been hypothesized that coregulation of glycolytic genes may dateback to the origins of life (137). However, the regulatory mechanisms are not conserved. Inmammalian cells, glycolytic gene expression is regulated by HIF; in fungi, it is not yet clear if there isa shared core mechanism; and in bacteria, expression is regulated by a two-component redox sensor(represented by ArcBA in Escherichia coli ) and by anaerobic activation of the FNR pathway (137).
Hypoxic induction of glycolytic genes is well characterized in S. cerevisiae (77). Similar changesin expression are observed in other ascomycetes such as C. albicans, C. parapsilosis, Pichia pastoris,S. pombe, and the aquatic Blastocladiella emersonii (5, 10, 22, 46, 111, 119, 127, 131), suggest-ing that changes in central carbon metabolism may be a shared response to hypoxia among theopisthokonts. The expression of glycolytic enzymes is also increased in A. fumigatus, at least atthe protein level (9). There are some exceptions, however, particularly among obligate aerobicspecies. For example, in Trichoderma reesei, glycolytic gene expression is repressed when oxygenlevels are drastically lowered (16), and in C. neoformans, respiratory gene expression is inducedrather than repressed when oxygen levels are low (27).
Even species with a shared hypoxic response may regulate expression in a different manner. InS. cerevisiae, glycolytic gene expression is controlled by two unrelated proteins, Gcr1 and Gcr2,in response to glucose levels (6). Both proteins are well conserved in the Saccharomyces clade andregulate glycolytic gene expression in K. lactis (98). However, Gcr1 and Gcr2 are likely to haveevolved relatively recently as they are not present in sister clades to Saccharomyces, such as theCandida clade.
The expression of glycolytic genes in S. cerevisiae also requires Tye7, which was originallyidentified through genetic interactions with Gcr1 and Gcr2 (99, 113). Tye7 is well conservedthroughout the Saccharomycotina and binds DNA through a bHLH domain similar to that inSREBPs (14). In C. albicans, Tye7 binds promoters of glycolytic genes (5). Tye7 is required forregulation of the glucose-6-phosphate flux and is a determining factor of glycolysis or glycogenmetabolism (5). Importantly, Tye7 is necessary for the hypoxic induction of glycolysis (5, 17).The prevalence of Tye7 as a hypoxic regulator is not clear, because few species have been tested.In C. albicans, for example, Gal4 acts with Tye7 to regulate glycolytic gene expression (5, 93). InS. cerevisiae, however, Gal4 controls galactose metabolic gene expression, not glycolysis (132).
Other potential hypoxic regulators of glycolysis in C. albicans have been identified. Efg1 facil-itates upregulation of glycolytic genes in hypoxia, which is particularly important during biofilmdevelopment (17, 119, 125). In C. albicans cells lacking the transcription factor ACE2, glycolyticgene expression is reduced and respiratory gene expression is increased. Ace2 is required for in-duction of filamentous growth in hypoxic conditions; however, the connection with glycolyticgene expression is unknown (97).
EXPRESSION OF CELL WALL GENES
Growth in hypoxia has dramatic effects on fungal cell walls. In S. cerevisiae, expression of theDAN/TIR family of mannoproteins is highly induced in hypoxic and anoxic conditions (28). The
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Common in FungalExtracellularMembranes(CFEM): a proteinfamily
GPI: glycophos-phatidylinositol
expression of a related family of secreted seripauperin (PAU) genes is also induced (105). InC. albicans, hypoxia affects transcription and protein levels of GPI-anchored proteins and membersof the CFEM (Common in Fungal Extracellular Membranes) family (122, 127), and increasedexpression of cell wall genes is observed in A. fumigatus during hypoxic growth (9).
The regulation of cell wall gene expression is best studied in S. cerevisiae. The expression of theDAN/TIR family requires Upc2 (1) and is blocked by the addition of heme (118). Repression ofPAU family expression is mediated by heme, sterols, and Upc2 (56, 77, 105). Recently, Hickmanet al. (56) showed that Hog1 and other members of the osmotic stress pathway are required forhypoxic induction of the PAU genes. Changes in membrane fluidity (influenced by both steroland heme levels) may activate Hog1 in hypoxia, which then controls the expression of the PAUgenes via Upc2. The hypoxic role of Hog1 is distinct from its role in the stress response.
Hypoxic induction of the CFEM family of cell wall genes in C. albicans is mimicked by low-ering sterol levels with azole drugs, and it requires Upc2 and the transcription factor Bcr1 (127).However, expression of the CFEM family is apparently unaffected by hypoxia in C. parapsilosis(111). Therefore, changes in oxygen levels likely play a general role in modifying fungal cell wallstructure, but with major species-specific effects.
HYPOXIA AND PATHOGENESIS
Fungal pathogens of mammalian hosts are routinely exposed to varying oxygen levels (14, 35,44). Oxygen on the skin is at atmospheric concentrations (21% or 160 mm Hg partial pressure),whereas organs and tissues are generally hypoxic (with levels ranging from 1 to 110 mm Hg;reviewed in 35). Most infection sites are assumed to be hypoxic, even those in the lungs (43, 44).
Biofilms and Hypoxia
Communities of cells forming biofilms on indwelling medical devices are hypoxic environments(17, 111, 115, 125). Rossignol et al. (111) showed that the transcriptional response of biofilmcommunities of C. parapsilosis overlaps the response of this organism to hypoxia, with increasedexpression of lipid, ergosterol, glycolysis, and alcohol metabolism in both conditions. A similarexpression pattern was observed in cells detaching from C. albicans biofilms (115). The availabilityof oxygen does not regulate the detachment process, suggesting that biofilms respond to hypoxicenvironments and are not regulated by them (115). However, subsequent analysis (17, 125) showedthat hypoxic adaptation is important for biofilm development.
Whereas several transcriptional regulators are required for biofilm development in normoxicconditions only, others such as Efg1 and Flo8 also regulate biofilms in hypoxia (125). Efg1 isparticularly involved in the upregulation of glycolytic genes and is required for hypoxic adaptationduring biofilm development. Disrupting other regulators of glycolytic gene expression [such asTYE7 (17)] results in the formation of biofilms with reduced cohesiveness. Increased glycolyticflux is therefore likely to be important for biofilm development in Candida species. It is not clear,however, how conserved this response is in biofilms formed by other fungi (such as Cryptococcus)that do not increase glycolytic gene expression during hypoxia.
Hypoxic Regulators and Virulence
The importance of Efg1 in the hypoxic response extends beyond its role in biofilm formation.Efg1 is a member of the APSES (Asm1, Phd1, Sok2, Efg1, and StuA) family of transcriptionalregulators, which have important roles in regulating morphology in many fungal species (35).
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C. albicans efg1 deletions are more invasive in mice transiently exposed to hypoxic conditions (70).In addition, cell-to-cell variability in EFG1 expression affects colonization of the gastrointestinaltract, and expression is influenced by the host immune system (102). Low oxygen levels in thegastrointestinal tract may influence the activity of EFG1.
In C. albicans, EFG1 is also required for the switch from yeast to hyphal growth when oxygenlevels are low (31); hyphal growth is generally associated with infection (87). Deleting EFG1 resultsin hyperfilamentous growth in hypoxic conditions (31). The transcription factor CZF1 is requiredfor filamentous growth when cells are embedded in agar where oxygen levels are low (18). Theexpression of CZF1 is regulated by Efg1 (135), and filamentation in embedded media appears toresult from modulation of the repressive role of Efg1 by Czf1 (41). Czf1 and Efg1 are componentsof a transcriptional network that regulates white-opaque switching in C. albicans (147). This switchis required for efficient mating in C. albicans, but it is also associated with pathogenesis; white cellsare more virulent in intravenous models of infection, whereas opaque cells are more virulent inskin infections (73, 74). Conversion of white to opaque cells is induced by low oxygen and highcarbon dioxide (61, 107).
Other regulators, such as Rho G protein Rac1 and its guanine exchange factor Dck1, havebeen associated with hyphal growth in hypoxic conditions (59). Hypoxic induction of filamen-tous growth requires the G protein Ras1 and the adenylate cyclase Cdc35 (127), similar to otherfilamentation pathways (81). Deleting UPC2, the major regulator of ergosterol synthesis, also abol-ishes filamentation in hypoxia, whereas strains carrying deletions of the BCR1 transcription factorare hyperfilamentous (127). Changes in the cell wall, such as alterations in sterol levels via Upc2 orcell wall proteins by Bcr1 (30, 39), may therefore be important for the hypoxic response. In addi-tion, it is difficult to separate the effect of reduced oxygen levels from effects of increases in carbondioxide, both of which are likely to occur within tissues. For example, the kinase Sch9 repressesfilamentation in hypoxic conditions but only when carbon dioxide levels are also high (126).
Although fungal pathogens likely share a common transcriptional response to hypoxia withnonpathogenic organisms, the effect of gene deletions on virulence suggests that hypoxic adap-tation is especially important for pathogenesis. For example, deleting components of the signaltransduction pathway that regulate filamentation of C. albicans in response to hypoxia (e.g., RAS1,CDC35, SCH9) and some of the transcription factors (TYE7, ACE2) greatly attenuates virulencein animal models (5, 68, 81, 86, 110). The role of regulators of sterol metabolism in pathogenesisof Candida species has not been investigated, although it is known that at least some sterol genesare required for survival and virulence (12).
The correlation between the hypoxic response and virulence is clearer in other species. Delet-ing components of the SREBP pathway reduces virulence in both C. neoformans and A. fumigatus(13, 24, 27, 140, 141). The Tco1 histidine kinase in C. neoformans is also necessary for both thehypoxic response and for virulence (27). The unfolded protein response, important for hypoxic reg-ulation in mammals (143), might also play a role in fungi. For example, Feng et al. (38) showed thatA. fumigatus strains deleted for the endoplasmic reticulum stress sensor IreA are avirulent and havereduced growth in hypoxic conditions. Finally, Grahl et al. (44) suggest that hypoxia may influencethe host response to infection with fungal pathogens, particularly because HIFα is important forcombating bacterial infection. Interestingly, C. albicans, like many bacterial pathogens, increasesthe stability of HIFα in the presence of oxygen (138).
CONCLUSIONS
Although much is known about the regulation of the hypoxic response in fungi, even more re-mains to be investigated. One fertile area is the identification of the signal that controls the
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expression of glycolytic genes in hypoxia. In Candida species, for example, expression is not in-duced by exposure to sterol-lowering drugs (46, 127), suggesting that oxygen-induced changes incentral carbon metabolism are not sensed through lowered sterol levels. The role of heme proteinsand the potentially important roles of the mitochondria and the respiratory chain remain to befully explored. Other areas likely to be of future interest include the regulation of translation inhypoxia (109) and the nearly unstudied modulation of the host by fungal pathogens in hypoxicconditions.
SUMMARY POINTS
1. HIFα has evolved recently as a regulator of the hypoxic response in multicellular animals,but its partner, HIF-1β, may be conserved in some unicellular organisms (e.g., Capsasporaowczarzaki ), excluding the fungi.
2. The SREBPs are ancient regulators of sterol synthesis that were present in the ancestorof all the opisthokonts.
3. SREBPs are highly degenerate in the Saccharomyces and Candida clades, where their rolein hypoxic regulation has been replaced by the transcription factor Upc2.
4. Fungi sense low oxygen via reduced heme and sterol levels.
5. Fungi have a shared transcriptional response to reduced oxygen, which includes increasedexpression of lipid, sterol, and heme biosynthesis. Changes in central carbon metabolism(e.g., increased glycolysis, reduced respiration) are observed in some species.
6. Reduced sterol levels control cleavage and activation of SREBPs in some fungi, but bydramatically different mechanisms.
7. Adaptation to hypoxic conditions is important for biofilm formation and for virulence inhuman fungal pathogens.
FUTURE ISSUES
1. How important are the respiratory chain and the mitochondrion for regulating the hy-poxic response?
2. How are oxygen levels correlated with the expression of glycolytic genes and changes incentral carbon metabolism?
3. How important is protein hydroxylation for sensing oxygen in fungi?
4. Does hypoxia influence the immune response of the host to fungal pathogens?
DISCLOSURE STATEMENT
The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.
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