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Published in final edited form as: Curr Opin Microbiol. 2015 May 17;26:41–47. doi: 10.1016/j.mib.2015.04.006

Oxygen Sensing by Protozoans: How They Catch Their Breath

Christopher M West 1, Ira J Blader 2
PMCID: PMC4623824  NIHMSID: NIHMS730323  PMID: 25988702

Abstract

Cells must know the local levels of available oxygen and either alter their activities or relocate to more favorable environments. Prolyl 4-hydroxylases are emerging as universal cellular oxygen sensors. In animals, these oxygen sensors respond to decreased oxygen availability by up-regulating hypoxia-inducible transcription factors. In protists, the prolyl 4-hydroxylases appear to activate E3-SCF ubiquitin ligase complexes potentially to turn over their proteomes. Intracellular parasites respond to decreased oxygen by utilizing both types of oxygen-sensing pathways. Since parasites are exposed to diverse oxygen tensions during their life cycle, oxygen sensing is likely a critical process and this review will discuss how these oxygen-sensing mechanisms contribute to the behavior of these unicellular eukaryotes.

O2 sensing is important for cellular life in an aerobic atmosphere

As an atmospheric gas that dissolves in aqueous biological solvents, O2 is a thermodynamically favorable acceptor for electrons and protons in mitochondrial respiration and other metabolic pathways. Its consumption by aerobic metabolism, combined with its limited solubility and slow diffusion in water, leads to concentration gradients of O2 within cells, tissues, and the local environments [1–4]. Cells have evolved multiple autonomous mechanisms to rapidly reorganize their metabolism to cope with O2 depletion [5,6]. These range from the rapid formation of short-lived reactive oxygen species (ROS) to longer-term changes in a cell's transcript and protein repertoire. Together these alterations impact the cell's metabolism and other activities such as motility to enable a cell to migrate to areas of higher O2 availability. As an example, when the soil amoeba Dictyostelium is exposed to low O2 it migrates toward higher O2 levels where it can continue its developmental program and differentiate into a fruiting body [7].

Cells express several types of O2 sensors. One class directly binds O2 effecting a conformational change that modifies the protein's function [8]. A second class includes proteins such as oxidases and non-heme dioxygenases that utilize O2 as a substrate for the biochemical reactions that they catalyze. The non-heme dioxygenases have an Fe center that coordinates and splits O2, distributing one of the O-atoms to the target substrate [9], and the other to α-ketoglutarate (αKG or 2-OG), which decomposes into succinate and CO2 (Fig. 1A). These dioxygenases include a variety of enzymes such as demethylases and hydroxylases that can modify several types of amino acids and nucleic acids. This review will focus primarily on non-heme dioxygenases and in particular prolyl 4-hydroxylases (P4Hs) since they are the best characterized O2 sensors in protozoans and animals and have potential roles in other eukaryotes and bacteria [10].

Fig. 1.

Fig. 1

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The Skp1 modification pathway in Dictyostelium and Toxoplasma. (A) Summary of the reaction catalyzed by the P4H class of non-heme dioxygenases, in which a protein-linked Pro residues is subject to hydroxylation at its 4-position by an O-atom derived from O2 (O-O). The other O-atom is transferred to α-KG, which decomposes into CO2 and succinate. (B) The hydroxyl group generated by the action of the P4H PhyA on Skp1 provides the site for glycosylation by five glycosyltransferase activities encoded by 3 genes in Dictyostelium. (C) Toxoplasma also assembles a pentasaccharide with the same Hex-Hex-dHex-Hex-HexNAc– composition (unpublished data). Tg-PhyA mediates Skp1 hydroxylation and is required for glycosylation, which has been confirmed by mass spectrometry (in preparation). gnt1 and pgtA are conserved in Toxoplasma, and likely modify Toxoplasma Skp1 as they do in Dictyostelium. However, the physical order of the N- and C-terminal catalytic domains of Dd-PgtA, which mediate the addition of β-Gal and α-Fuc, respectively, is reversed in Toxoplasma.

Transcription-dependent O2-sensing in animals

Transcriptome rewiring is a key mechanism by which animals (and many fungi) respond to decreased O2-availability and PHDs (prolyl hydroxylase domain proteins) are the O2 sensing P4Hs most important in regulating these O2-regulated transcription factors. They function by hydroxylating two conserved prolines in the α subunit of the heterodimeric hypoxia inducible factor (HIF) transcription factor, resulting in the formation of (4S,2R)-hydroxyproline, (Hyp) [6,9,11]. Prolyl hydroxylated HIFα is recognized by and targeted for proteasome-dependent degradation by the VHL ubiquitin ligase complex. Thus, HIFα is rapidly degraded under O2 replete conditions. However, the PHDs have low affinity for O2 and when O2 becomes limiting, as can occur in tissue environments, HIFα accumulates, translocates to the nucleus, binds HIFβ (also called ARNT), and activates target gene expression. Many of these genes, such as glycolytic enzymes and vascular endothelial growth factor, are important to coordinate a cell's response to decreased O2 availability. HIFα is regulated by another non-heme dioxygenase, Factor-inhibiting-HIF (FIH), which hydroxylates an asparagine in the protein's C-terminal transcriptional coactivator domain [12,13]. This O2-dependent process diminishes transactivation activity, which reinforces the action of PHDs in regulating HIF1α activity. Besides HIF1α, PHDs regulate other proteins including PKM2 [14] and the TRPA1 [15] ion channels. Thus, hydroxylation of prolines and other amino acid is emerging as a widespread post-translational modification and we expect that these lists will continue to grow.

O2-sensing in amoebae

While HIFα is restricted to animals, P4Hs are evolutionarily conserved and found not only in all animals [16] but fungi, amoebazoa, and, evidently, prokaryotes [10]. Thus P4Hs appear to predate the emergence of HIFα in animals, raising the question of what their targets are in other organisms and whether they are involved in O2-sensing. The discovery of a Dictyostelium P4H (DdPhyA), which hydroxylates Pro143 in the Skp1 subunit of the SCF class of E3-Ub ligases, raised the possibility that this P4H enzyme is an O2 sensor [17] (Fig. 1B).

Dictyostelium resides in the soil where O2 availability is governed by the rate of infusion from the atmosphere, and depletion by microbial and root metabolism. The amoebae use bacteria as a nutrient source and under starvation the amoeba differentiate into dormant spores. This involves a developmental program resulting in cell aggregation to form a polarized multicellular slug that migrates to the soil surface and transforms into a fruiting body consisting of dormant spores that survive until activated by a nutrient rich environment. O2 is one sensory modality used by Dictyostelium to assess whether they are above ground at a site appropriate for fruiting body formation. Culmination therefore occurs in an O2 dependent manner and the O2 set point for culmination, which is well above that required for metabolism, can be raised or lowered by forced under- or over-expression of the DdPhyA [18*]. Since purified DdPhyA has a Km for O2 above 21%, this suggests that O2 control of culmination is mediated via PhyA activity. This mechanism is supported by pulse-labeling studies with 35S-Met, that showed that Skp1 hydroxylation lags behind synthesis at low, but not normal, O2 levels [19*]. When Dictyostelium is cultured under standard submerged conditions, development requires high atmospheric levels of O2 (70–100%) by a mechanism that depends on PhyA and Skp1 hydroxylation [20]. The data suggest that O2 is limiting for hydroxylation of newly synthesized Skp1, which might have a privileged role in forming regulatory SCF complexes composed of nascent FBPs.

Not only is Skp1 a substrate for PhyA it is a functional mediator of PhyA activity in cells. Skp1 under-expression has the same effect on the O2-setpoint for culmination as PhyA over-expression, suggesting that hydroxylation reduces Skp1 activity [21*]. Conversely, Skp1 overexpression raises the O2-setpoint similar to disrupting phyA. As expected, the activity of Skp1 is suppressed by forced overexpression of PhyA, and is not additive with genetic depletion of phyA [21*]. Paradoxically, Skp1 with a point mutation at the position of Pro143 exhibits less activity, though this might reflect separate effects on protein folding and function. Over-expression of Skp1 also inhibits sporulation, a subsequent developmental step that occurs as prespore cells rise to the top of the fruiting body. In contrast to culmination, inhibition depends on its Skp1 hydroxylation. Thus in addition to being the major if not only functional mediator of PhyA action, Skp1 contributes multiple functions in development that vary in their dependence on its hydroxylation.

Skp1 prolyl hydroxylation renders it a substrate for Gnt1 [22], the first of three glycosyltransferases (GTs) that assemble a linear pentasaccharide on Hyp143 (Fig. 1B). This raises the question of whether hydroxylation alone controls Skp1 function, or simply enables subsequent glycosylation. Both answers are correct. Genetic disruption of gnt1 results in an O2 requirement for culmination that lies between that of phyA-KO and w/t cells, and also yields morphologically defective fruiting bodies at any O2 level [22]. A KO of the second GT (pgtA), which allows for only a single sugar (GlcNAc) to be added, has an O2 requirement that is very similar to that of gnt1-KO cells, but rescues the defective fruiting body morphology. Remarkably, KO of the third GT (AgtA), allowing for the trisaccharide to be assembled, reverts the O2-dependence almost back to that of phyA-KO cells and has unexpectedly delayed early development [23]. Accumulating evidence suggests that AgtA has a second function as a Skp1 binding protein, via its WD40 repeat domain, that dampens its function regardless of its glycosylation status [24]. Consistent with this, the high O2-requirement of agtA-KO cells is ameliorated by genetic reduction of Skp1 levels [21*]. Thus glycosylation and the three GTs modulate the signal generated by prolyl hydroxylation of Skp1.

The primary function of Skp1 as part of the E3-SCF ubiquitin complex is to bind the F-box domain of F-box proteins (FBP) (Fig. 2). FBPs are heterogeneous multidomain proteins that serve as receptors for substrates to be poly-ubiquitinated and subsequently degraded [25]. Dictyostelium is predicted to have 55 FBPs and the mechanism(s) controlling binding specificity to Skp1 is complex but important since E3SCFUb-ligases likely control the fates of hundreds if not thousands of cellular proteins. Co-immunoprecipitation studies in Dictyostelium suggest that two distinct FBPs (FbxD and FbxA) preferentially associate with fully glycosylated Skp1 compared to unmodified Skp1 [19*]. In addition, FbxD binds better to the trisaccharide form of Skp1 (in agtA KO strains) than unmodified Skp1 although not as well as it does to fully modified Skp1. While, structural data indicate that addition of GlcNAc promotes increased α-helical content in Skp1 [26], further glycosylation has little on Skp1 structure suggesting that the peripheral sugars of the pentasaccharide regulate FBP binding through different mechanisms. Taken together these data indicate that O2 regulated Skp1 hydroxylation/glycosylation has various novel effects on Skp1 and serves to complement other SCF regulatory mechanisms that include the neddylation of cullin-1 which controls the exchange activity of Cand1 at the Cullin-1/Skp1 interaction site [27,28]. Further studies are needed to identify target substrates of Dictyostelium FBPs to determine how changes to the cell's proteome mediate O2-dependent culmination and development. In addition, we have identified putative Skp1-modification genes in other disease-causing amoebazoa, such as Acanthamoeba, suggesting that O2-sensing may contribute to virulence in ocular keratitis.

Fig. 2.

Fig. 2

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Skp1 hydroxylation and glycosylation likely regulate the SCF complex, an E3 ubiquitin ligase that marks target proteins with K48-linked polyubiquitin chains that are recognized by the proteasome for degradation. Polyubiquitination involves successive transfers of Ub from Ub-E2 donors that cycle on, off, and on again after recharging from Ub-E1. The process is controlled by, in some cases, substrate priming such as phosphorylation (P=PO4) as indicated. Ubiquitination is also regulated by neddylation of Cul1, which is under the control of the COP9 signalosome and other factors and promotes flexible tethering of Rbx1 and its associated Ub-E2 to allow access to the Ub target site (K=Lys) on the substrate. Neddylation also excludes Cand1 (not shown), which allows docking of FBP/Skp1 complexes to Cul1 (dashed interface). Skp1 modifications, which occur near its C-terminus, are proposed to promote interactions of FBPs with Skp1 (dashed interface), providing novel SCF-specific regulation. Modified from [42].

O2-sensing in Toxoplasma

Toxoplasma gondii is an unrelated protozoan that can infect almost all known mammals. It is passed between hosts by digestion of either infectious oocysts shed in feline (the parasite's definitive host) fecal material or tissue cysts found in undercooked meat from other infected mammals [29]. The acidic environment of the stomach releases parasites from oocysts or tissue cysts and once these parasites reach the anaerobic environment of the gut they infect intestinal epithelial cells, transform into replicative tachyzoites, and use recruited inflammatory monocytes and dendritic cells to disseminate via a Trojan Horse mechanism to diverse tissues throughout the body. In tissues, tachyzoites can develop into tissue cysts that are impervious to either anti-parasitic drugs or the resulting immune response.

Thus, like Dictyostelium, Toxoplasma is exposed to diverse O2 tensions during its life cycle and how the parasite senses and responds to changing O2 levels has been an open area of investigation. A clue about how Toxoplasma senses O2 came from the discovery that homologues of the Dictyostelium Skp1 modification pathway genes are conserved in Toxoplasma [30] (Fig. 1C). Toxoplasma PhyA (TgPhyA) is 40% similar in sequence to DdPhyA and can complement DdphyA-KO mutants in O2-dependent culmination [31*]. Furthermore, TgSkp1, which contains the equivalent of Pro143, is glycosylated in a TgPhyA-dependent fashion, and purified TgPhyA is capable of hydroxylating TgSkp1 in vitro [31*]. Genetic disruption of the TgphyA gene reveals that while TgPhyA is not essential its loss does reduce parasite growth at atmospheric O2 and this growth defect is intensified at 0.5% O2, a low but physiologically relevant O2 tension. Slow growth suggests that, in the absence of an O2 sensor, the parasite is unable to accommodate to the stress of low O2. Based on studies in Dictyostelium, these results suggest that Skp1 prolyl hydroxylation and/or glycosylation allows Toxoplasma to adapt to decreased O2 availability by altering the parasite's proteome.

Biochemical analysis of TgPhyA activity revealed an unexpectedly high affinity for O2 [31*], which was in contrast to DdPhyA and the PHDs that have apparent low affinities for O2. Thus, only low O2 levels, like those encountered in the gut, would be expected to impact TgPhyA activity. Nevertheless, TgPhyA can function in place of DdPhyA in O2-dependent culmination [31*] suggesting that TgPhyA is regulated by both O2 (at very low levels) and αKG, as its Km for αKG is similar to that of DdPhyA [31*]. Since αKG is generated by the O2-dependent TCA cycle, it is possible that TgPhyA senses O2 indirectly via changes in αKG. Whether DdPhyA (and the metazoan PHDs) are similarly regulated awaits metabolomic profiling of Toxoplasma and Dictyostelium grown at various O2 tensions.

Other protists and pathogens may employ the Skp1 mechanism to sense O2

Genome analyses indicate that PhyA, Gnt1, and the target Pro residue in Skp1 co-exist in representatives of all 6 major protist phyla, but are evidently absent from fungi, animals, and higher plants (Fig. 3). Their broad but sporadic presence in diverse protozoans suggest that this is an ancient pathway that was retained during evolution via secondary gene loss. A striking example of this conservation is that the Skp1/PhyA/Gnt1 pathway, which was originally discovered in Dictyostelium and is present in other amoebazoae (e.g., Acanthamoeba), is conserved in Toxoplasma and closely related coccidians (e.g. Neospora) as well as in distantly related oomycetes (e.g. Phytophthora). But, the pathway is absent in other amoebazoae (e.g., Entamoeba spp.) and apicomplexans including other coccidia (Eimeria) and hemosporidia (Plasmodium). This is significant because many of the organisms with the Skp1 modification pathway are human and agricultural pathogens and this pathway represents a novel drug target since their hosts do not express it.

Fig. 3.

Fig. 3

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Phylogenetic distribution of Skp1 modification genes. Organisms predicted to modify Skp1, based on possession of phyA-like and gnt1-like genes, and a Skp1 with the equivalent of Pro143 in Dictyostelium Skp1, are encircled. Those in violet can cause human or plant disease. Phylogenetic tree of eukaryotic evolution is from [43].

Other O2-sensing mechanisms in fungal and protist pathogens

The secondary loss of Skp1 modification genes may have occurred in organisms that live anaerobically, only experience constant O2 levels, or acquired other O2 sensing mechanisms. We identified in apicomplexan, including those lacking PhyA such as Plasmodium, a second related P4H we have named PhyB (unpublished data). Further studies are needed to identify PhyB target(s) and determine how PhyB senses O2.

Trypanosomatids, such as the important human parasites Trypanosoma cruzi, T. brucei, and Leishmania spp., and other diversely related protists (e.g., euglenoids) lack genetic evidence for PhyA-related P4Hs. Thus, other O2-sensing proteins are likely to be utilized by these organisms and O2 and α-ketoglutarate utilizing dioxygenases are likely candidates. These organisms have a novel nuclear DNA modification called base J that affects transcription initiation and termination [32,33]. Base J is formed by the action of a DNA-thymine hydroxylase (JBP1/2), which is a non-heme containing O2 and α-ketoglutarate utilizing dioxygenase, and the resulting 5-hydroxymethyluracil (hmU) is capped with a β-glucose. Thus, the base J pathway parallels the Skp1 modification pathway in which a hydroxylated target serves as a glycosyltransferase substrate. Base J synthesis is reduced in Trypanosoma cruzi cells exposed to hypoxia suggesting that the JBP1/2 are O2 sensors in trypanosomes [34*].

Protozoans may utilize other types of O2 sensors. For example, non-heme O2- and αKG-dependent dioxygenases that may function as O2 sensors include asparagine, histidine and arginine hydroxylases [35] and Jumonji-domain containing proteins that epigenetically regulate gene expression by promoting Lys methylation and histone hydroxylation [9,36,37]. Finally other proteins may sense changes in O2 availability due to O2's ability to directly induce structural changes to those proteins that it binds. As an example, O2 binding to a Fe-heme prosthetic group in an adenylate cyclase of L. major stimulates cAMP formation [38*].

Intracellular parasites utilize both parasite and host O2 sensing

As a parasite traverses through the body, oxygen sensors such as PhyA and JBP1/2 are likely important for parasite survival and virulence. An infection is, however, a complex environment consisting of multiple types of cells (e.g. parasites, resident tissue cells, and recruited leukocytes) and each must properly function in order for the host and/or parasite to survive and thrive. Toxoplasma and other obligate intracellular parasites must ensure that both it and its host cell are in a metabolic state permissive for parasite growth. The first clue that host oxygen sensing plays a role in establishing this state was the finding that Toxoplasma activates host HIF1α [39]. Surprisingly, this was not simply a consequence of establishing a hypoxic microenvironment due to parasite oxygen consumption. Rather, Toxoplasma specifically activates HIF-1 by a mechanism that involves activin receptor signaling and inactivation of host PHD2 expression and activity [40]. Most significantly, parasite activation of HIF-1 is required for parasite growth under physiological O2 tensions and does so, in part, by increasing host hexokinase-2 expression (unpublished results).

Other intracellular parasites such as Leishmania and Theileria activate HIF-1 in the infected host cell although it isn't clear whether this is needed for parasite growth or supports a host defense pathway. In macrophages, Leishmania activates HIF-1 through a distinct mechanism involving transcriptional upregulation of HIF-1α and depletion of intracellular Fe to cause reduced PHD activity [41]. This mechanism is reminiscent to how HIF-1 is activated to promote resistance to pathogenic bacteria [42] suggesting that HIF-1 acts to promote resistance to Leishmania.

Summary and implications for the future

O2-sensing is important for protists to grow in the varied environments that they encounter. We are beginning to see that multiple mechanisms have evolved to sense and respond to changes in O2-availability, and even to perhaps manipulate the O2-consumption of host cells for their benefit. We highlighted here recent progress in the elucidation of biochemical mechanisms involving a homolog of the animal PHD O2-sensor whose function is not to regulate the stability of a single transcriptional factor but rather the entire proteome itself. The restriction of this pathway to unicellular parasites and other protists implies that rapid protein turnover in response to altered O2-levels may be more adaptive for single cells than the slower-acting transcriptional mechanism utilized by animals. Clearly much remains to be done to confirm this speculative model, to determine if it is the evolutionary precursor of the animal O2-sensing mechanism, and to assess its utility as a drug target.

Acknowledgments

Research from the authors' labs summarized in this article was supported by grants from the NIH (R01AI069986, R01GM037539, R01GM084383), the Oklahoma Center for the Advancement of Science and Technology, and the Mizutani Foundation for Glycoscience.

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