Protozoa are known to engage in symbiotic or parasitic relations with their mammalian hosts. Given the optimal concentration of dissolved oxygen in a tissue is dependent on the localization, function, and vascularization, parasites often exhibit specific oxygenic preferences. Considering the steep, dual oxygenic gradients that exist throughout the gut, enteric parasites must be well adapted to the environment to establish an infection and proceed through their life cycle. As a result, most protozoans that colonize the GI tract are accustomed to low oxygen concentrations and may require microaerophilic conditions to survive and thrive. Alternatively, tissue and blood protozoa which are responsible for some of the most prevalent human parasitic infections exhibit a more diverse array of oxygenic preferences depending on where in the body they reside. Numerous protozoa have demonstrated the ability to modulate or exploit the activity of the host Hypoxia-Inducible Factor (HIF) during the course of infection, making the role of HIF an important consideration in the pathophysiology of protozoan parasitic infections.   

In the GI tract, hypoxia occurs upon low blood oxygen concentration or as a result of poor blood flow. Blood flow is important when it comes to gastrointestinal hypoxia as the amount of blood is highly dependent on meal consumption [1,29,30]. Several structural and biological factors can futher determine the oxygen levels in the GI tract, such as the region of the intestine, the mucosal layer, and the gut microbiota [1,4,9,29,30,31,32,33,34]. There are two oxygen gradients in the gut: (i) a longitudinal oxygen gradient, decreasing from the small intestine to the colon, and (ii) a radial gradient, with oxygen levels decreasing from the submucosa to the lumen [1,4,31]. The small intestinal radial gradient ranges from <10 mmHg in the lumen up to 59 mmHg, while the colonic radial gradient ranges from as low as 3 mmHg and 11 mmHg in the lumen (sigmoid and ascending colon, respectively) to as high as 42–71 mmHg in the muscularis externa [4,31]. Intestinal epithelial cells (IECs) are therefore reliant on HIF to promote cellular survival and adaptation when oxygen fluctuations subject them to a state of hypoxia [35]. Furthermore, inflammation in the GI tract leads to an influx of immune cells, resulting in a heightened hypoxic state due to depleted oxygen availability and altered cellular metabolism [36]. Colorectal cancer severity and metastasis have both been linked to increased tissue hypoxia. HIF activates numerous genes involved in tumor proliferation and angiogenesis [37,38,39,40]. For example, HIF-1α can also promote activation of ataxia-telangiectasia mutated kinase to protect colorectal cancer cells against apoptosis, an important mechanism by which tumors utilize hypoxia for enhanced survival [41]. Antibodies against HIF target gene products such as VEGF can further increase the duration of survival in patients when used in combination with other chemotherapeutic drugs [42].

IECs can also utilize the hypoxic response for protection against various system perturbations. For instance, Clostridium difficile toxins increase transcription and subsequent translation of HIF-1α in a dose-dependent manner, suggesting that IEC HIF-1α is protective against C. difficile-induced injury and inflammation [43]. Clinical symptoms of murine TNBS-colitis are exasperated when HIF-1α is decreased and, conversely, overexpression of HIF-1α can be protective [44]. Furthermore, hypoxia is implicated in various inflammatory conditions wherein cellular responses to hypoxia can promote the resolution of inflammation [36,45].

Entamoeba histolytica, the causative agent of amebic dysentery or amebiasis, is a leading cause of diarrheal illness in humans, with 50 million symptomatic infections per year [46]. After ingestion, cysts undergo excystation into trophozoites, which travel to the colon where they colonize and invade the epithelial tissue [46]. During this process, trophozoites traverse a steep gradient from the oxygen-poor colonic lumen to the oxygenated lamina propria [47]. E. histolytica has developed a complex machinery against nitric oxide (NO) and reactive oxygen species (ROS) released by neutrophils and macrophages to prevent macromolecular damage during the tissue invasion process, which subsequently maintains a strict state of intracellular hypoxia [48,49,50,51]. Oxygen is one of the main host stressors that influences E. histolytica’s survival and pathogenicity. E. histolytica can upregulate its antioxidant enzyme machinery, which includes iron superoxide dismutase, peroxiredoxin, and thioredoxin to evade host immune defenses [49,52,53,54].

Alterations to HIF-1α expression and hypoxic-related signaling have been described during amebiasis. E. histolytica is associated with hepatic hypoxic responses as HIF-1α and HIF-dependent genes Vegfa, Icam1, and Il6ra are upregulated in a murine model of E. histolytica infection [55]. Interestingly, HIF-1α is required for adequate secretion of IL-6, indirectly modulating the anti-E. histolytica Th17 immune response [55]. Human intestinal xenografts transplanted into SCID mice exhibit upregulated expression of numerous genes associated with hypoxic responses upon E. histolytica infection, including those coding for HIF1α, metallothioneins (MT1G, MT1H, MT1P, MT2A, MT3), members of the c-Jun and c-Fos family (JUN, JUNB, JUND, FOS), growth arrest and DNA damage inducible 3 (DDIT3), alpha crystallin (CRYAB), immediate early response 3 (IER3), and heat shock protein 70 (HSP70) [56]. Furthermore, intestinal biopsies from humans infected with E. histolytica show elevated HIF activation, as well as enrichment for the response to hypoxia as a biological process [57]. Considering E. histolytica is an anaerobe, this finding suggests that the parasite may modulate its microenvironment to make the conditions more favorable, triggering an HIF-dependent cellular response.

The intestinal protozoan parasite Giardia duodenalis (syn. G. intestinalis, G. lamblia) causes giardiasis, one of the leading causes of diarrheal disease worldwide with over 200 million symptomatic cases annually in humans [73,74]. Clinical manifestations of giardiasis include acute presentations of diarrhea and abdominal pain, as well as post-infectious disorders such as post-infectious irritable bowel syndrome (PI-IBS), chronic fatigue, and failure to thrive in children [75]. After ingestion via the fecal–oral route, cysts undergo excystation and trophozoites colonize the upper small intestine by attaching to the surface of enterocytes [76,77]. Giardia is classified as a microaerophilic parasite, lacking mitochondria and enzymes for the electron transport chain (ETC) and tricarboxylic acid (TCA) cycle [9,78,79]. Instead, Giardia has a fermentative style of metabolism, utilizing glycolysis and substrate-level phosphorylation to generate energy while also possessing oxygen-scavenging enzymes such as flavodiiron proteins (FDPs), peroxiredoxin, flavohemoglobin, superoxide reductase, NADH peroxidase, and NADH oxidase to combat luminal ROS [80,81,82,83,84]. The ability to process oxygen also depends on Giardia’s life stage, wherein Giardia trophozoite uptake of oxygen is stimulated via exogenous glucose while cyst oxygen uptake is triggered by ethanol [85].

G. duodenalis has demonstrated the ability to activate a hypoxic-like response in cells. Transcriptomic analysis on human duodenal organoid-derived monolayers infected with G. duodenalis strain WB clone 6 exhibit an enrichment of hypoxia response-related genes (i.e., Molecular Signatures Database) after 24 h of infection [86]. Similar observations have been made using Caco-2 epithelial cells, where HIF-induced genes including NOS2, ANKRD37, GADD45A, ITF, MIR210HG, and SLC2A3 are also upregulated in Giardia-exposed cells, suggesting that IECs respond to increased oxidative stress upon infection [22,58,59,87]. Furthermore, hypoxia-inducible gene 2 (HIG2), a peptide inhibitor of adipose triglyceride lipase encoded by a target gene of HIF-1 named HILPDA (Hypoxia-Inducible Lipid Droplet-Associated), is upregulated early on in infection, indicating that HIF activation may occur quickly upon cellular interaction with Giardia [58,88] (Table 1). However, little is known about the mechanisms and roles of HIF activation upon exposure to Giardia.

Cryptosporidium species such as Cryptosporidium hominis and Cryptosporidium parvum are leading causes of diarrheal illness [89]. Cryptosporidium oocysts excyst upon ingestion into four sporozoites, which subsequently invade IECs and progress through their life cycle intracellularly [90]. As Cryptosporidium migrates from the gut lumen to the IECs, sporozoites are exposed to fluctuating but predominantly oxygen-poor environments [90]. Cryptosporidium can carry out both aerobic and anaerobic metabolic pathways, encoding a pyruvate:NADP+ oxidoreductase and an alternative oxidase, respectively [91,92]. This suggests that Cryptosporidium can not only tolerate but also make use of the variable oxygenic conditions of the gut [92]. Interestingly, the oocysts are present in higher abundances in water samples with lower dissolved oxygen, implying the oocyst may be more sensitive to oxygen than the invasive merozoites [93].

In a neonatal rat model of cryptosporidiosis, cardiomyocytes have hyperexpression of HIF-1α; however, this finding is more suggestive of a link between gastroenteritis and cardiovascular disease than a direct induction of hypoxia by C. parvum [60]. In the GI tract, the fecal HIF-1 signaling-associated metabolite oxoglutaric acid is increased in C. muris-infected mice, suggesting that mice can enact a hypoxic response upon Cryptosporidium infection [94]. Finally, in HCT-08 colonic monolayers infected with C. parvum, heat shock protein 70 (HSP70), which possesses a hypoxia response element, is also upregulated 24 h post infection [61]. The full biological significance of these observations requires further investigation.

During states of inflammation and hypoxia, epithelial barrier function can be reduced as a result of the dysregulation of various tight junction proteins, including claudin-1 [95,96,97]. To combat this, the hypoxic response activates genes such as ATG9A and CLDN1 to promote tight junction biogenesis and barrier integrity [98,99]. Importantly, enteric protozoa can also directly disrupt tight junctions and increase barrier permeability via the adherence of the active form of the parasite to the epithelial layer. For example, E. histolytica can both alter expression of and degrade zonulin-1, claudin-2, and occludin, a result of secreted cysteine protease A5 [100,101]. Similarly, G. duodenalis secretes cysteine proteases that can disrupt the claudin-1, claudin-4, and occludin arrangement in IECs [102,103]. Under normoxic or anaerobic conditions, G. duodenalis infection is associated with the disruption of epithelial junctional complexes (EJCs), which results in epithelial barrier dysfunction [104,105]. Cryptosporidium spp. can also modulate the organization and expression of various tight junction proteins, including occludin, E-cadherin, and claudin-4; however, these changes are suggested to be mediated via the induction of protein degradation pathways rather than by the secretion of cysteine proteases by the protozoa [65]. Given the evidence of the modulation of both barrier function and HIF activation associated enteric protozoa, the role of hypoxia in modulation of IEC tight junctional complexes requires further investigation.

In the gut, protozoan infections have also been associated with microbiota dysbiosis and biofilm disruption, both of which may lead to the liberation of invasive pathobionts [106,107,108,109,110,111]. Interestingly, microbiota-derived products, including short-chain fatty acids (SCFAs) such as butyrate, play a role in the stabilization of HIF, which in turn improves epithelial barrier functions [112]. In an enteroid model of hypoxia, pre-treatment or concurrent treatment with different ratios of SCFA cocktails such as acetate, propionate, and butyrate led to increased transepithelial electrical resistance, as well as increased expression of key gut barrier and metabolism genes [113]. Thus, more research is warranted to characterize changes in EJCs during protozoan infections under hypoxic conditions, as well as the role of protozoa-associated dysbiotic microbiota and derived products in the loss of barrier functions.

The optimal concentration of dissolved oxygen varies depending on the localization, function, and vascularization of the tissue. Although the state of physiologic hypoxia is pertinent to the gastrointestinal tract, many tissues can become hypoxic and must respond accordingly. For example, the brain must be sufficiently oxygenized (~35 mmHg) to carry out proper functions [114]. Under conditions of cerebral hypoxia due to ischemia or injury, the brain increases metabolic consumption of oxygen [114,115,116]. In the lungs, human influenza A virus infection can lead to localized hypoxia and alveolar cell death, resulting in oxygen depletion below the preferred range of 100–160 mmHg [115,117]. Alternatively, the skin functions optimally with a much lower concentration of oxygen that increases from ~8 to 35 mmHg across the superficial to the subpapillary plexus, respectively, while perturbations to these concentrations can impair keratinocyte proliferation and junction integrity [115,118]. Hence, given the various oxygenic environments sustained by each tissue, fluctuations in oxygen concentration can subject virtually any bodily tissue to hypoxia and incur damage in the absence of an appropriate cellular response.

Tissue and blood protozoan parasites are responsible for some of the most prevalent human parasitic infections, including malaria, Chagas disease, leishmaniasis, and toxoplasmosis [119]. Unlike enteric protozoa, which must be adapted to the fluctuating low levels of oxygen in the gut, tissue and blood protozoa exhibit a more diverse array of oxygenic preferences depending on where in the body they reside, and numerous investigations have aimed to characterize the hypoxic signatures of the host. Indeed, numerous tissue and blood protozoa either require or upregulate expression of HIF to enact a successful infection.

Leishmania spp. are a group of flagellated protozoa that are responsible for three different forms of leishmaniasis: visceral, cutaneous, and mucocutaneous [120]. The promastigote stage of the parasite replicates extracellularly within a sandfly (approximately 90 sandfly species can transmit Leishmania), which can then deliver the protozoan to the mammalian host during a blood meal [120,121]. In the mammalian host, obligate intracellular amastigotes will be phagocytosed by macrophages or dendritic cells, allowing them to travel to distant sites in the body [122]. A study assessing parasitic growth under anoxic conditions noted a loss of motility and increased secretion of lactate in promastigotes, illustrating their poor adaptability to anoxic conditions; hence, they are classified as aerobes [123]. Promastigotes require a high concentration of both extracellular and intracellular oxygen to produce superoxide, enabling the parasite to carry out aerobic metabolism and utilize oxygen as the final electron acceptor in the electron transport chain [124]. Additionally, all Leishmania spp. possess trypanothione reductase to combat oxidative stress, allowing them to survive within a macrophage [125].

Although Leishmania spp. are aerobic, they induce a hypoxic environment within the macrophage, increasing the expression of macrophage HIF-1α and subsequent expression of HIF-target gene micro-RNA-210 (miRNA210) [62,63,64,65]. Increases in miRNA210 enhance L. donovani survival as silencing of both HIF-1α and miRNA210 in macrophages reduces parasitic burden and infectivity [65]. L. donovani also decreases the cellular iron pool, a necessary cofactor for PHDs, preventing the hydroxylation of HIF-1α [126,127]. Drugs capable of targeting HIF-1α, such as resveratrol and echinomycin, decrease L. amazonesis survival in macrophages [128]. Recent studies have also demonstrated that HIF-dependent upregulation of Vegfa results in lymphangiogenesis, a critical step in the resolution of cutaneous lesions during L. major infection [66,129,130]. The host cellular hypoxic response may also have leishmanicidal effects. Exposure of macrophages and dendritic cells to hypoxia during infection improves control of parasitic burden [131,132,133]. Mice deficient in HIF-1α in myeloid cell compartments have a more severe disease outcome and a higher parasitic burden, suggesting that HIF-1α expressed by myeloid cells contributes to the innate immune response against L. major [134]. Conversely, other reports suggest that HIF-1α activation has no impact on the macrophage phagocytosis of Leishmania, nor on control of parasitic burden [66]. HIF-1α expression is downregulated in Leishmania-infected hamsters in a species-dependent fashion, suggesting that different Leishmania spp. may elicit different hypoxic responses [135]. Finally, hyper-virulent Leishmania strains reduced HIF-1α expression, whereas hypo-virulent strains increased HIF-1α expression, indicating different strains within the same species can also elicit different hypoxic responses that may contribute to or mitigate virulence [136].

Toxoplasma gondii is an obligate intracellular protozoan parasite with a global prevalence between 25% and 30% [137,138]. While T. gondii is generally considered to be an aerobe, it must make adaptations to the environmental conditions of the definitive and intermediate hosts [137]. Indeed, T. gondii experiences numerous oxygenic fluctuations during its life cycle. It first colonizes the feline intestinal tract to replicate oocysts, and after sporulation and ingestion by a human, the infective tachyzoite can travel through the bloodstream to numerous bodily locations, including the central nervous system and skeletal muscles [137]. In the small intestine of felines, the merozoite form exhibits a unique gene expression profile compared to the other life stages of the parasite through the increased transcriptomic and proteomic activity of metabolic pathways, a possible adaptation to the low oxygen of the gut and increased growth requirements [139]. Interestingly, T. gondii encodes a prolyl-hydroxylase (TgPhyA) which hydroxylates S-phase kinase-associated protein 1, a protein that can indirectly sense oxygen levels and hence may assist with survival in various tissues [140,141]. T. gondii also possesses a mitochondrion that can carry out aerobic metabolism via an electron transport chain, as well as a necessary assortment of antioxidant enzymes to protect against self- and host-derived ROS [142,143,144].

In the intermediate host, T. gondii is associated with HIF-1α activation. In murine embryonic fibroblasts, luciferase assays indicate the upregulation of HIF-1α transcription upon T. gondii infection [67]. Similarly, increased HIF-1α transcription is observed in human foreskin fibroblasts (HFFs) infected with T. gondii, which correlates with increased stable HIF-1α protein [67]. This accumulation of stable protein can be attributed to T. gondii’s ability to inhibit PHD2 via the stimulation of activin-like kinase 4 (ALK4) and subsequent activation of Rho GTPase and JNK MAP kinase pathways, abrogating the hydroxylation of HIF-1α [69,145,146,147]. Increasing HIF-1α production is of great importance to T. gondii’s fitness as it requires HIF-1α for survival and efficient replication under physiological oxygen concentrations (~3% oxygen) [67]. Moreover, T. gondii Cathepsin C1 can upregulate HIF-1α signaling to increase the production of EPO in HEK and HFF cells, a known HIF-dependent gene target [68]. Additionally, the expression of glycolytic enzymes is increased upon T. gondii infection in an HIF-dependent manner [70,148,149]. Specifically, HK2 is delocalized from the mitochondrial membrane to the cell cytoplasm, making it more accessible to T. gondii [70,148,149]. The characterization of HIF activation in response to T. gondii, an aerobe, provides an example of a hypoxic response in normoxic conditions, suggesting that T. gondii may utilize host response to enhance its own fitness and virulence.

Plasmodium spp. are the causative agents of malaria, a life-threatening vector-borne disease that affects nearly 250 million individuals yearly [150]. Plasmodium begins as a sporozoite in the insect vector, and the parasite undergoes schizogony in the liver of the infected vertebrate upon a blood meal [151]. The schizonts then become merozoites, which can infect red blood cells and subsequently digest host hemoglobin [152,153]. During the asexual stage in the human liver, sporozoites act as microaerophiles as the physiologic oxygen concentration is low [115,154,155]. Some reports suggest that sporozoite mitochondrial oxygen consumption and oxidative phosphorylation is reduced during the liver stage due to sufficient glucose availability [156,157]. However, a switch to dependence on oxidative phosphorylation is observed during the sexual life stages as it is exposed to mosquito salivary glands, which may reach up to 21% oxygen, and the capillaries of the human lung, which can approach 13% oxygen [158,159].

Numerous studies have investigated the role of HIF in Plasmodium spp. infections. Pharmaceutical-induced activation of HIF-1α results in increased survival of P. berghei sporozoites in hepatocytes without altering the growth of the exo-erythrocytic stage of the parasite [160]. Mice infected with P. berghei have increased tissue levels of HIF-1α and VEGF protein, driving angiogenesis as a compensatory mechanism to increased oxygenation in hypoxic infected tissues [71]. Furthermore, insufficient expression of HIF-1α by brain cells has been suggested to contribute to the progression of cerebral malaria [161]. However, increased HIF-1α and VEGFA has not been the consensus in malaria patients. For example, while higher levels of HIF-1α mRNA expression have been identified in placental tissues, VEGFA expression was downregulated in malaria patients [162]. Additionally, post-mortem brain tissue from severe cerebral malaria patients presented no changes in HIF-1α expression but increases in VEGFA, indicating changes in VEGFA may be tissue-dependent or HIF-independent [163].

Chagas disease is a vector-borne parasitic infection caused by the parasite Trypanosoma cruzi. T. cruzi colonizes the gut of triatomine insects to generate infectious trypomastigotes which can then be transferred into the mammalian bloodstream during a blood meal [164]. T. cruzi can invade an assortment of nucleated cells but demonstrates an affinity for cardiac and skeletal muscle tissue, and therefore the amastigote is an obligate intracellular parasite [165]. From the insect vector’s gut to cardiac tissues, T. cruzi is exposed to diverse oxygenic states and hence exhibits variable metabolic behaviors that capitalize on fluctuating microenvironmental conditions [166]. In the low-oxygen environment of the triatomine gut, T. cruzi favors an anaerobic mode of metabolism by increasing glycolytic flux to sustain morphological changes [167,168,169]. In the vertebrate host’s bloodstream, trypomastigotes exhibit a preference for oxidative phosphorylation by increasing the activity of the ETC, utilizing oxygen from the blood to sustain an aerobic mode of metabolism [168,170]. T. cruzi can combat oxidative stress by expressing antioxidant enzymes such as peroxiredoxins and trypanothione synthetase [171]. Interestingly, it has been postulated that the presence of ROS influences the proliferation of epimastigotes, suggesting the parasite utilizes environmental oxygen cues to regulate its life cycle [172].

Few reports have characterized the hypoxic signature of T. cruzi infection. Leukocytes from the cardiac tissue of human patients with late-stage trypanosomiasis exhibit increased expression of HIF-1α in conjunction with increased ATP catabolism and parasitic burden [173]. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) also show significant enrichment for hypoxia-related genes upon infection with T. cruzi, including HIF-1α, VEGFB, HK2, SLC2A3, ENO1, and LDHA [72]. Importantly, reduced expression or inhibition of the glucose transporter GLUT4 in cardiomyocytes lowered parasitic uptake, suggesting T. cruzi can utilize GLUT4 to enter cardiomyocytes [72]. GLUT4 is a passive glucose transporter that is known to have increased action during hypoxia in an HIF-dependent manner, and T. cruzi may hence benefit from the hypoxic response to cellular invasion [174]. However, another report indicates that T. cruzi strain Tulahuen may downregulate HIF-1α in myoblasts, while no alterations to HIF-1α were observed in other strains, suggesting an isolate-dependent phenomenon [175]. Interestingly, T. brucei, the etiologic agent of African trypanomiasis, hydroxylates HIF-1α via the secretion of indolepyruvate, resulting in reduced macrophage glycolysis [176]. This functions as an immune evasion mechanism and prevents macrophages from shifting their metabolism to favor glycolysis, an example of an indirect way in which a parasite can mitigate hypoxic signaling [176]. This observation also supports the hypothesis that HIF-1α can play a role in host immune evasion against protozoan parasites. However, expression of HIF-1α and target genes vegfa, glut1, and il1b is elevated in the median eminence and hypothalamus of both Rag1^−/− and wild-type mice infected with T. brucei brucei (Tbb), suggesting different tissues and cell types may alternatively regulate HIF in the presence of the parasite [177].