Shared host, distinct invaders: metabolomic footprints of plasmodium and babesia in host red cells

Abstract

Purpose of review

Malaria and babesiosis are important transfusion-transmitted diseases, therefore, it is important to report novel insights into the complex interactions the causative parasites share with their common host RBCs. Metabolomics is an important tool that can be used to reveal an in-depth analysis of parasite infections in the context of the host. Similarities and differences in the biochemical fingerprints between malaria and babesia infected RBCs are reviewed with potential reasons for these differences and implications for the host.

Recent findings

Recent results from Babesia-infected RBCs offer an opportunity to develop comparative models of pathogenesis for both infections. Perturbation in the levels of key biomolecules including sugars, amino-acids and lipids, along with redox homeostasis, and heme utilization, are hallmarks of both diseases. Key similarities include enhanced glycolytic rate in both infected RBCs together with lipid scavenging from RBC membranes. Differences relate to hemoglobin breakdown and the use of resultant amino acids for propagation.

Summary

Altered metabolic profiles reflect the unique lifecycles of Plasmodium and Babesia, pointing to how they carve out a niche for successful proliferation. A comprehensive understanding of the metabolic similarities and differences between the two parasites will aid in identifying new biomarkers as well as specific, effective targeted therapies.

INTRODUCTION

Human red blood cells (RBCs) serve as the common host cells for both the malaria parasite Plasmodium and the babesiosis causing parasite Babesia during their intraerythrocytic asexual cycles [1,2]. This phase forms the pathogenic stage of the lifecycle and is responsible for all clinical symptoms of disease, thus forming an important research focus. Both parasites are obligate intracellular parasites whose development is supported through a shared resource environment within the RBC as well as scavenged from the plasma [1,3,4]. This auxotrophic lifestyle requires complex interactions with the RBC and has been studied using high resolution metabolomics, although currently more data is available for Plasmodium than Babesia[5^▪▪]. These studies reveal how these parasites alter host metabolic processes for survival, proliferation as well as transmission to the insect vector. Critically, these altered metabolites can be used as biomarkers and diagnostics as well as provide new insights that can aid in the development of antiparasitic therapies [6^▪,7]. New chemotherapeutic targets are urgently needed for both diseases given the emergence of resistance to multiple drugs [8]. Here, we explore how these parasites alter the metabolomic profile of the host RBC, focusing on both similarities and differences and potential reasons for these characteristics. As most of the metabolomic data is available and relevant for the human malaria and Babesia parasites, Plasmodium falciparum and Babesia divergens, much of the review focuses on these parasites. 

Box 1.

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DIFFERENCES IN ASEXUAL STAGE BIOLOGY AND MORPHOLOGY MAY EXPLAIN DIFFERENCES IN METABOLIC PROFILES

Although both parasites infect human RBCs, there are a number of biological features that differ between them, reflected in changes in the metabolic framework of infected RBCs (Fig. 1).

FIGURE 1.

Model shows human host RBCs infected with Babesia divergens (left) and Plasmodium falciparum (right) with their corresponding lifecycle stages. Overlapping regions highlight the metabolic similarities between the two parasites along with matching color code showing physiological impact of metabolic alterations on host system. Unique features and metabolic alterations are shown in purple for B. divergens and green for P. falciparum. PVM, Hz, Hemozoin; NPP, New Permeability Pathways; PVM, Parasitophorous Vacuolar Membrane.

Genome size

While Plasmodium genomes average 22–30 Mb, with 14 chromosomes [9], Babesia genomes are much smaller, with the smaller babesia species like that of B. microti encoding only four chromosomes with a genome size of 6–8 Mb and larger ones like B. divergens and B. duncani having genome sizes typically around 10–12 Mb [10,11^▪▪]. The smaller genome size of Babesia results in a lack of genes for essential metabolic pathways, reduced metabolic pathways and greater reliance on host metabolism.

Asexual life cycle parameters

Malaria parasites primarily reproduce by schizogony within the RBC while Babesia divide asynchronously via binary fission [12,13]. While a single schizont can produce numerous merozoites, up to 30 in P. falciparum, Babesia spp. typically produce 4–8 merozoites per infected RBC [14]. P. falciparum, the dominant malaria pathogen resides in the RBC for approximately 48 h and P. vivax for 24 h, however Babesia spp. typically have much shorter RBC cycles of 6–8 h [15,16]. (Fig. 1). As a result, the metabolic needs and demands of Plasmodium are higher than Babesia resulting in differences in host cell modification to acquire nutrients as well as differences in infected metabolic profiles.

Parasitophorous vacuolar membrane (PVM) is the membrane that envelops the parasites as they enter the host erythrocyte and is critical to maintain an optimum environment within the RBC. However, while malaria parasites maintain the PVM throughout their intra-erythrocytic life cycle, using it for nutrient uptake, waste removal, and protein export [17], Babesia dismantles it soon after invasion [18,19] (Fig. 1). Therefore, Plasmodium resides and replicates within the PVM in the RBC and breakdown of the membrane is essential for egress and new infections. As Babesia lives free within the RBC cytoplasm, the route for acquiring nutrients is less complex compared to Plasmodium where the PVM serves as a membrane barrier in the flux of both nutrients and wastes [7]. New data also reveal that in malaria, the PVM is not uniform but has specialized domains that facilitate various processes, including membrane transport and lipid exchange [17].

Hemoglobin utilization

As hemoglobin is the most abundant protein in the RBC, it appears the most likely source of nutrition for both parasites. However, current evidence suggests that Babesia spp. lacks direct homologues of hemoglobin-digesting clade A plasmepsins and complete pathways required for direct hemoglobin breakdown [20,21^▪]. Plasmodium, on the other hand, degrades hemoglobin, both to acquire amino acids and to maintain osmotic homeostasis [22,23]. This difference in hemoglobin consumption may be linked to the longer time Plasmodium spends in RBC and its metabolic demands as a more vigorous multiplier requiring a constant stream of in-house nutrients. Additionally, malaria parasites have a cytosome, that bridges the PVM and parasite membranes and serves as a funnel for hemoglobin directly to the parasite's digestive vacuole where further proteolysis occurs [24]. The resultant heme is incorporated and detoxified into hemozoin (Fig. 1). Babesia does not produce hemozoin and this lack may reflect no or sparse degradation of hemoglobin [25,26]. These fundamental differences in heme metabolism are thought to contribute to differences in sensitivity to antimalarial drugs such as artemisinin [21^▪].

Permeability pathways

Plasmodium and Babesia scavenge extracellular nutrients including amino acids, thiamine, purines and lipids from the human serum [27,28]. While endogenous RBC transporters can be used for some nutrients, others need specialized transporters to provide for efficient transport into the RBC and eventually into the parasites. Parasites exploit new permeability pathways (NPPs), which serve as channels for a wide array of molecules including structurally unrelated low molecular mass solutes like sugars, amino acids, vitamins, purines, and ions (Fig. 1). In P. falciparum, the proteins implicated in NPP activity are parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3 [29]. These channels are called surface anion channel (PSACs) in Plasmodium, while in Babesia divergens, these are not anion-selective and utilize fundamentally different but understudied mechanisms [30]. Chemically blocking these channels arrest parasite growth [31], and more recently, studies in Babesia have shown specific susceptibility to drugs such as Fosidomycin which are taken up by infected RBCs through NPPs [31,32].

SIMILARITIES AND DIFFERENCES IN INFECTED RED BLOOD CELL METABOLOMIC PROFILES

Comparative metabolomic profiling of malaria and babesia-infected RBCs reveal their shared role in host nutrient hijacking, but importantly also point to differences in their metabolic needs, resulting in identification of metabolites that can be exploited as disease specific biomarkers as well as therapeutic targets.

Energy

As both Plasmodium and Babesia require to expand their populations in a short time, energy pathways are critical for successful proliferation and represent viable drug targets. Given the glucose-rich environment of the host RBC, both parasites rely heavily on glycolysis for ATP production. As a result, lactic acidosis and hypoglycemia are clinical features observed in both infections [33–35] (Fig. 1). All glycolytic metabolites have been detected during the asexual phase of both parasites [36,37]. Hexose transporters are abundant on RBC membranes and a 50 to 100-fold increase in glucose consumption has been reported for infected RBCs [38,39]. As expected, a large increase in pyruvate was also reported in B. divergens infected RBCs [5^▪▪]. In-silico analysis of both P. falciparum, the B. divergens genome shows that genes encoding the various TCA cycle enzymes are present, and this has been confirmed by metabolomic analyses, showing alteration in key TCA metabolite levels, like isocitrate, fumarate, malate, and α-ketoglutarate between uninfected and infected RBCs. However, other studies in Plasmodium point to a limited carbon flux through the mitochondrial tricarboxylic acid and six of eight TCA cycle enzymes have been found to be dispensable in asexual blood stages [40]. Thus, it is not clear to what extent mitochondrial aerobic respiration is used to fulfill parasite energy needs.

Lipids

In both parasite-infected RBCs, the host RBC's lipid profile is significantly altered, with overall cellular lipid content increasing as well as the composition of specific lipid classes. Parasite lipids are obtained both through de-novo synthesis using host-derived metabolites and by scavenging lipids from the extracellular environment and the RBC membrane [41]. As cholesterol and fatty acids cannot be synthesized de novo by both parasites, they are forced to scavenge them from host RBC (Fig. 1) [42]. Tracking studies have shown that host RBC membrane cholesterol is gradually depleted as the parasite proliferates within the cell and gets incorporated into the parasite plasma membranes [5^▪▪,43]. Another striking observation was that the levels of neutral lipid species diacylglycerol (DAG) and triacylglycerol (TAG) were highly elevated in RBCs from both infections. This was accompanied by a high proportion of lipid droplets which contain these glycerides. It has been hypothesized that lipid droplets are central to lipid and energy homeostasis in these parasites and inhibition of the formation or breakdown of TAG can kill the parasites [5^▪▪,41]. Both parasites can synthesize phosphatidylcholine and phosphatidyl ethanolamine and use it for membrane building [44]. Thus, metabolites involved in their syntheses were also elevated. Interestingly, poly unsaturated fatty acids (PUFA), including arachidonate (20 : 4n6), linoleate (18 : 2n6), docosapentaenoate (22 : 5n3), and docosahexaenoate (22 : 6n3) were also detected in higher amounts in infected RBCs than uninfected RBCs [5^▪▪,38]. These PUFAs are precursors of eicosanoids, which mediate important inflammatory and immunosuppressive roles in the host [38].

Sphingolipids

Sphingosine-1-phosphate (S1P) is a phosphorylated product of sphingosine produced by sphingosine kinase-1 and -2 (SphK-1 and SphK-2) in RBCs. S1P, a pleotropic signaling, is critical for cell proliferation and survival, endothelial cell migration, maintenance of endothelial barrier integrity, and bone marrow trafficking [45,46]. S1P has been implicated in various cellular functions; however, in the context of malaria, the intra-erythrocytic pool of S1P is essential for parasite growth. Recent studies have shown the link between reduced S1P levels and decreased glycolysis, thereby linking lipid and energy metabolism in parasite-infected RBCs [46]. Interestingly, S1P has been shown to play a double-edged sword in malaria pathogenesis, being essential for parasite development, but its low plasma levels are associated with cerebral malaria in mouse models and human studies [47]. More recently, a role for S1P has emerged in epigenetic control of virulence and sexual differentiation in P. falciparum, thereby warranting further investigation [48]. Although limited data are available for sphingosine in Babesia pathogenesis, a recent metabolomics study showed a marked reduction of S1P in infected cells, thereby mirroring the results obtained in Plasmodium [5^▪▪]. However, the implications of S1P on glycolysis and overall host-Babesia metabolism remain unknown.

Lysophospholipids

Are a class of chemically related membrane lipids that regulate a wide range of cellular and physiological functions in mammalian systems including cell growth and death, cell trafficking, and membrane shaping. They are roughly divided into two groups of molecules, namely lysosphyngolipids and lysoglycerophospholipids. During the pathogenic, asexual stage, high rates of phospholipid synthesis are required to support the expansion of parasite membranes and vigorous anabolic activity. Host serum contains two abundant fatty acid sources: free fatty acids and lyso-phosphatidyl-cholines (LPC) and studies with isotope-labeled LPC have demonstrated that LPC-derived fatty acids are readily incorporated into parasite lipids. LPC catabolism also provides choline, an important precursor for phosphatidylcholine biosynthesis [49^▪▪,50]. LPC was also reported to be a critical molecule found in human serum that significantly impacts gametocyte formation [51,52]. Recent metabolomics report of B. divergens has corroborated this shift and identified a significant decrease in this class of lipids in infected cells [5^▪▪].

Amino acids

Both parasites depend on their hosts for amino acids, which are absolutely essential and needed in large quantities to sustain their fast growth. Arginine is an important amino acid, which has been implicated to be clinically important in malaria. Hypoarginemia as a result of severe malaria correlated with decreased nitric oxide production and cerebral malaria [53]. Metabolomics studies have shown that as Plasmodium matures, the extracellular concentration of arginine reduces, with concomitant increase if ornithine and citrulline via the activity of parasite enzyme arginase [36,54]. It has been suggested that the parasite depletes the host arginine pool in order to modulate the activity of the host enzyme nitric oxide synthase (NOS), which is used by the human immune system to generate antimicrobial nitric oxide radicals from arginine. Depletion of the plasma arginine pool may suppress the immune response by removing the substrate for nitric oxide production, and may serve as an important pathway for immune evasion [55]. A very recent study in Mali confirmed that L-arginine metabolites are decreased in cerebral malaria and suggested a putative mechanism impairing cerebral vasodilation [56]. Our recent global metabolomics study demonstrated similar metabolite profiles in culture supernatants of Babesia-infected RBCs, suggesting that both parasites may be using similar immune evasion strategies [5^▪▪].

An important nonproteinogenic amino acid is pipecolic acid, which is the catabolic product of lysine metabolism and can serve as an important biomarker for severe malaria [54,57]. A significant change of 30-fold at 24 h and 60-fold at 40 h was observed in levels of pipecolic acid in culture supernatant of P. falciparum in vitro cultures as well as serum of P. berghei-infected mice (Fig. 1) [54]. In contrast, in B. divergens culture supernatant, pipecolic acid level was unaffected at low parasitemia and was significantly downregulated at higher parasitemia, suggesting that though these parasites are apicomplexans invading the same host cell and evolutionarily close to each other, they seem to have unique metabolic pathways, which can be useful in both disease detection by biomarker analysis and development of new chemotherapeutics.

Redox metabolites

Redox perturbation and loss of host redox homeostasis is a hallmark of several parasitic diseases- and is especially important in malaria and babesiosis due to the limited metabolic capacity and enzyme turnover of critical redox related enzymes in the host RBCs to combat this insult. Infected RBCs face severe ROS flux, owing to parasite metabolism and host ROS produced by immune system in response to infection (Fig. 1). Redox metabolic fluctuations in plasma have been shown to corelate with severity of malaria, emphasizing the role of these changes in disease severity. Several metabolites are responsible for this redox stress, including perturbation of GSH/GSSG [58], increased homocysteine levels [54], RBC lipid peroxidation [59] and free heme [60]. A new report shows that redox protection can be conferred through the RBC GSH biosynthesis pathway, as total RBC glutathione was increased when cultures were supplemented with glutamine, cysteine and glycine [61^▪▪]. Several interesting observations relating to Plasmodium antioxidant biology such as absence of crucial enzymes like catalase, classical form of glutathione peroxidase, and the endoplasmic reticulum (ER) resident enzyme peroxiredoxin IV have been shown recently. Additional evidence for the role of redox stress is evidenced by the parasite's vulnerability in sickle or G6PD-deficient RBC and the use of several antimalarials like artemisinin which challenge the parasite's antioxidant defense system [62–67]. The study of redox in Babesia is in its infancy as only a few studies have investigated redox homeostasis in infected RBCs. A recent study showed that like malaria, Babesia-infected cells had high redox activity and disturbed GSH/GSSG ratio [5^▪▪,68^▪]. Interestingly, artemisinin and plasmodione mechanistically disrupt malarial redox homeostasis, but only mildly inhibit Babesia [69^▪,70]. This observation underscores the importance of further study of redox biology in Babesia to understand these key differences and identify chemotherapeutics that might be better suited to kill this specific parasite.

CONCLUSION

Comparative metabolomics of malaria and babesiosis infections underscore both shared and distinct metabolic strategies employed by these parasites to survive and replicate within RBCs. By identifying molecular pathways underlying both malaria and babesiosis pathogenesis, metabolomics increases our understanding of disease pathology and enables the development of strategies to disrupt these interactions. Metabolic biomarkers are objective indicators and can quantitatively measure the progression of disease. Comparative metabolomics can facilitate the identification of Plasmodium and Babesia species-specific features and differences such as metabolic signatures, mechanisms of drug resistance mechanisms, as well as host immune responses, leading to the development of tailored interventions for both infections.

Acknowledgements

None.

Financial support and sponsorship

This work was supported by the National Institutes of Health (NIH), National Heart, Lung, and Blood Institute (Grant P01 HL149626 [C.A.L.]) and the NIH, National Institute of Allergy and Infectious Diseases (Grants R61AI187095 [C.A.L.] and 1K99AI182422 [D.B.]).

Conflicts of interest

There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

• ▪ of special interest

• ▪▪ of outstanding interest