Early Perturbations in Red Blood Cells in Response to Murine Malarial Parasite Infection: Proof-of-Concept 1H NMR Metabolomic Study.

BACKGROUND: The major focus of metabolomics research has been confined to the readily available biofluids-urine and blood serum. However, red blood cells (RBCs) are also readily available, and may be a source of a wealth of information on vertebrates. However, the comprehensive metabolomic characterization of RBCs is minimal although they exhibit perturbations in various physiological states. RBCs act as the host of malarial parasites during the symptomatic stage. Thus, understanding the changes in RBC metabolism during infection is crucial for a better understanding of disease progression. METHODS: The metabolome of normal RBCs obtained from Swiss mice was investigated using 1H NMR spectroscopy. Several 1 and 2-dimensional 1H NMR experiments were employed for this purpose. The information from this study was used to investigate the changes in the RBC metabolome during the early stage of infection (~1% infected RBCs) by Plasmodium bergheii ANKA. RESULTS: We identified over 40 metabolites in RBCs. Several of these metabolites were quantitated using 1H NMR spectroscopy. The results indicate changes in the choline/membrane components and other metabolites during the early stage of malaria. CONCLUSIONS: The paper reports the comprehensive characterization of the metabolome of mouse RBCs. Changes during the early stage of malarial infection suggest significant metabolic alteration, even at low parasite content (~1%). GENERAL SIGNIFICANCE: This study should be of use in maximizing the amount of information available from metabolomic experiments on the cellular components of blood. The technique can be directly applied to real-time investigation of infectious diseases that target RBCs.

Early Perturbations in Glucose Utilization in Malaria-Infected Murine Erythrocytes, Liver and Brain Observed by Metabolomics.

Investigation of glucose utilization during an infection is central to the study of energy metabolism. The heavy utilization of glucose by the malaria parasite, and the consequences of this process, have been investigated extensively. However, host glucose utilization during early infection has not been explored to date. In a first attempt, this article investigates the changes in the host glucose utilization in Balb/c mice infected with Plasmodium berghei ANKA using 13C-labeled glucose infusion followed by NMR spectroscopy. The results suggested significant alterations of liver, brain and red blood cell (RBC) glucose utilization during early infection when the parasitemia was <1%. At the pathway level, we observed a decrease in the shunt metabolite 2,3-bisphosphoglycerate in the RBCs. Glycolysis and pathways associated with it, along with fatty acid unsaturation, were altered in the liver. Significant changes were observed in the central carbon metabolic pathways in the brain. These results have implications in understanding the host physiology during early infection and pave the way for detailed flux analysis of the proposed perturbed pathways.

Metabolomic changes in vertebrate host during malaria disease progression.

Metabolomics refers to top-down systems biological analysis of metabolites in biological specimens. Phenotypic proximity of metabolites makes them interesting candidates for studying biomarkers of environmental stressors such as parasitic infections. Moreover, the host-parasite interaction directly impinges upon metabolic pathways since the parasite uses the host metabolite pool as a biosynthetic resource. Malarial infection, although not recognized as a classic metabolic disorder, often leads to severe metabolic changes such as hypoglycemia and lactic acidosis. Thus, metabolomic analysis of the infection has become an invaluable tool for promoting a better understanding of the host-parasite interaction and for the development of novel therapeutics. In this review, we summarize the current knowledge obtained from metabolomic studies of malarial infection in rodent models and human patients. Metabolomic analysis of experimental rodent malaria has provided significant insights into the mechanisms of disease progression including utilization of host resources by the parasite, sexual dimorphism in metabolic phenotypes, and cellular changes in host metabolism. Moreover, these studies also provide proof of concept for prediction of cerebral malaria. On the other hand, metabolite analysis of patient biofluids generates extensive data that could be of use in identifying biomarkers of infection severity and in monitoring disease progression. Through the use of metabolomic datasets one hopes to assess crucial infection-specific issues such as clinical severity, drug resistance, therapeutic targets, and biomarkers. Also discussed are nascent or newly emerging areas of metabolomics such as pre-erythrocytic stages of the infection and the host immune response. This review is organized in four broad sections-methodologies for metabolomic analysis, rodent infection models, studies of human clinical specimens, and potential of immunometabolomics. Data summarized in this review should serve as a springboard for novel hypothesis testing and lead to a better understanding of malarial infection and parasite biology.

Host metabolic responses to Plasmodium falciparum infections evaluated by 1H NMR metabolomics.

The human malarial parasite Plasmodium falciparum causes the most severe forms of malarial infections, which include cerebral malaria and various organ dysfunctions amongst adults in India. So far no dependable clinical descriptor is available that can distinguish cerebral malaria from other symptomatically similar diseases such as sepsis and encephalitis. This study aims at evaluating the differential metabolic features of plasma samples from P. falciparum patients with varying severities, and patients suffering from symptomatically similar diseases. 1H Nuclear Magnetic Resonance (NMR) based metabolic profiling of the plasma of the infected individuals and the control population was performed. The differences in the plasma profiles were evaluated through multivariate statistical analyses. The results suggest malaria-specific elevation of plasma lipoproteins. Such an increase was absent in control populations. In addition, cerebral malaria patients exhibited a decrease in plasma glycoproteins; such a reduction was not observed in malarial patients without cerebral symptoms. The data presented here indicates that the metabolism and/or transport of the plasma lipids is specifically perturbed by malarial infections. The differential perturbation of the plasma glycoprotein levels in cerebral malaria patients may have important implications in the diagnosis of cerebral malaria.

Early prediction of cerebral malaria by (1)H NMR based metabolomics.

BACKGROUND: Cerebral malaria (CM) is a life-threatening disease, caused mainly by Plasmodium falciparum in humans. In adults only 1-2% of P. falciparum-infected hosts transit to the cerebral form of the disease while most exhibit non-cerebral malaria (NCM). The perturbed metabolic pathways of CM and NCM have been reported. Early marker(s) of CM is(are) not known and by the time a patient exhibits the pathological symptoms of CM, the disease has progressed. Murine CM, like the human disease, is difficult to assign to specific animals at early stage and hence the challenge to treat CM at pre-clinical stage of the disease. This is the first report of prediction of CM in mice using a novel strategy based on (1)H nuclear magnetic resonance (NMR)-based metabolomics. METHODS: Mice were infected with malarial parasites, and serum was collected from all the animals (CM/NCM) before CM symptoms were apparent. The assignment of mice as NCM/CM at an early time point is based on their symptoms at days 8-9 post-infection (pi). The serum samples were subjected to (1)H NMR-based metabolomics. (1)H NMR spectra of the serum samples, collected at various time points (pi) in multiple sets of experiments, were subjected to multivariate analyses. RESULTS: The results from orthogonal partial least square discriminant analyses (OPLS-DA) suggest that the animals with CM start to diverge out in metabolic profile and were distinct on day 4 pi, although by physical observation they were indistinguishable from the NCM. The metabolites that appeared to contribute to this distinction were serum lipids and lipoproteins, and 14-19% enhancement was observed in mice afflicted with CM. A cut-off of 14% change of total lipoproteins in serum predicts 54-71% CM in different experiments at day 4 pi. CONCLUSION: This study clearly demonstrates the possibility of differentiating and identifying animals with CM at an early, pre-clinical stage. The strategy, based on metabolite profile of serum, tested with different batches of animals in both the sex and across different times of the year, is found to be robust. This is the first such study of pre-clinical prognosis of CM.

Metabolic perturbations of kidney and spleen in murine cerebral malaria: (1)H NMR-based metabolomic study.

A significant fraction of global population is under the threat of malaria. Majority of annual death is due to the more complicated form of the infection i.e. the cerebral form, also known as Cerebral Malaria (CM). Host parasite interaction is known to cause a cascade of events in various tissues like brain, liver, kidney, and spleen. We have employed (1)H NMR based metabolomics to understand the specific perturbations of various tissues in CM. In our previous paper we have delineated the differences between CM vis-a-vis non-cerebral malaria (NCM) mice in serum, liver and brain. In this paper we focus on their differences of metabolic profile in kidney and spleen as kidney dysfunction and splenomegaly are known to be associated to neurological outcome of the disease. Moreover we have also looked into how the biological compartments (kidney, spleen and serum) interact with each other. The various metabolites involved in such interactions and their correlational aspects across the compartments have been studied in CM, NCM and control mice. The idea was to find out the specific pathways that are altered in CM mice. Our results demonstrate that both the kidney as well as spleen metabolism are differentially perturbed in CM with respect to NCM. The results point out that glutamate levels are decreased in CM mice with respect to NCM mice both in case of spleen and kidney while creatine, myo-inositol and betaine levels are increased in kidney of CM mice with respect to NCM mice. From the analysis of Multiway Principal Component Analysis (MPCA) we see that lipid metabolism and TCA cycle is altered in kidney and spleen.

1H NMR metabonomics indicates continued metabolic changes and sexual dimorphism post-parasite clearance in self-limiting murine malaria model.

Malaria, a mosquito-borne disease caused by Plasmodium spp. is considered to be a global threat, specifically for the developing countries. In human subjects considerable information exists regarding post-malarial physiology. However, most murine malarial models are lethal, and most studies deal with acute phases occurring as disease progresses. Much less is known regarding physiological status post-parasite clearance. We have assessed the physiological changes at the organ levels using (1)H NMR based metabonomics in a non lethal self-clearing murine malarial model of P. chabaudi parasites and Balb/C, far beyond the parasite clearance point. The results showed distinct metabolic states between uninfected and infected mice at the peak parasitemia, as well as three weeks post-parasite clearance. Our data also suggests that the response at the peak infection as well as recovery exhibited distinct sexual dimorphism. Specifically, we observed accumulation of acetylcholine in the brain metabolic profile of both the sexes. This might have important implication in understanding the pathophysiology of the post malarial neurological syndromes. In addition, the female liver showed high levels of glucose, dimethylglycine, methylacetoacetate and histidine after three weeks post-parasite clearance, while the males showed accumulation of branched chain amino acids, lysine, glutamine and bile acids.

Metabolic fingerprints of serum, brain, and liver are distinct for mice with cerebral and noncerebral malaria: a ¹H NMR spectroscopy-based metabonomic study.

Cerebral malaria (CM) is a life-threatening disease in humans caused by Plasmodium falciparum, leading to high mortality. Plasmodium berghei ANKA (PbA) infection in C57Bl/6 mice induces pathologic symptoms similar to that in human CM. However, experimental CM incidence in mice is variable, and there are no known metabolic correlates/fingerprints for the animals that develop CM. Here, we have used (1)H NMR-based metabonomics to investigate the metabolic changes in the mice with CM with respect to the mice that have noncerebral malaria (NCM) of the same batchmates with identical genetic backgrounds and infected simultaneously. The metabolic profile of the infected mice (both CM and NCM) was separately compared with the metabolite profile of uninfected control mice of same genetic background. The objective of this study was to search for metabolic changes/fingerprints of CM and identify the pathways that might be differentially altered in mice that succumbed to CM. The results show that brain, liver, and sera exhibit unique metabolic fingerprints for CM over NCM mice. Some of the major fingerprints are increased level of triglycerides, VLDL-cholesterol in sera of CM mice, and decreased levels of glutamine in the sera concomitant with increased levels of glutamine in the brain of the mice with CM. Moreover, glycerophosphocholine is decreased in both the brain and the liver of animals with CM, and myo-inositol and histamine are increased in the liver of CM mice. The metabolic fingerprints in brain, sera, and liver of mice with CM point toward perturbation in the ammonia detoxification pathway and perturbation in lipid and choline metabolism in CM specifically. The study helps us to understand the severity of CM over NCM and in unrevealing the specific metabolic pathways that are compromised in CM.

Host responses in malaria disease evaluated through nuclear magnetic resonance-based metabonomics.

Malaria is a widespread disease caused by several species of Plasmodium. The parameters that render the hosts susceptible to severe disease complications are not completely understood. Nuclear magnetic resonance (NMR)­based studies offer a convenient platform to investigate the disease process in a noninvasive, nondestructive, and unbiased manner.NMR-based metabonomics allows a systems biological view of the global changes in host metabolism due to the parasite infection. Parasite-infected host red blood cells influence the neighboring uninfected host red blood cells metabolically. In the murine model of malaria, a sexually dimorphic host response is observed upon parasitic infection. Also the animals that are prone to cerebral malaria have different metabolic status vis-a-vis the ones that do not. Early prediction of susceptibility to cerebral malaria may be explored using such metabonomic methods.

Global host metabolic response to Plasmodium vivax infection: a 1H NMR based urinary metabonomic study.

BACKGROUND: Plasmodium vivax is responsible for the majority of malarial infection in the Indian subcontinent. This species of the parasite is generally believed to cause a relatively benign form of the disease. However, recent reports from different parts of the world indicate that vivax malaria can also have severe manifestation. Host response to the parasite invasion is thought to be an important factor in determining the severity of manifestation. In this paper, attempt was made to determine the host metabolic response associated with P. vivax infection by means of NMR spectroscopy-based metabonomic techniques in an attempt to better understand the disease pathology. METHODS: NMR spectroscopy of urine samples from P. vivax-infected patients, healthy individuals and non-malarial fever patients were carried out followed by multivariate statistical analysis. Two data analysis techniques were employed, namely, Principal Component Analysis [PCA] and Orthogonal Projection to Latent Structure Discriminant Analysis [OPLS-DA]. Several NMR signals from the urinary metabolites were further selected for univariate comparison among the classes. RESULTS: The urine metabolic profiles of P. vivax-infected patients were distinct from those of healthy individuals as well as of non-malarial fever patients. A highly predictive model was constructed from urine profile of malarial and non-malarial fever patients. Several metabolites were found to be varying significantly across these cohorts. Urinary ornithine seems to have the potential to be used as biomarkers of vivax malaria. An increasing trend in pipecolic acid was also observed. The results suggest impairment in the functioning of liver as well as impairment in urea cycle. CONCLUSIONS: The results open up a possibility of non-invasive analysis and diagnosis of P. vivax using urine metabolic profile. Distinct variations in certain metabolites were recorded, and amongst these, ornithine may have the potential of being used as biomarker of malaria. Pipecolic acid also showed increasing trend in the malaria patient compared to the other groups.

Multivariate modelling with (1)H NMR of pleural effusion in murine cerebral malaria.

BACKGROUND: Cerebral malaria is a clinical manifestation of Plasmodium falciparum infection. Although brain damage is the predominant pathophysiological complication of cerebral malaria (CM), respiratory distress, acute lung injury, hydrothorax/pleural effusion are also observed in several cases. Immunological parameters have been assessed in pleural fluid in murine models; however there are no reports of characterization of metabolites present in pleural effusion. METHODS: 1H NMR of the sera and the pleural effusion of cerebral malaria infected mice were analyzed using principal component analysis, orthogonal partial least square analysis, multiway principal component analysis, and multivariate curve resolution. RESULTS: It has been observed that there was 100% occurrence of pleural effusion (PE) in the mice affected with CM, as opposed to those are non-cerebral and succumbing to hyperparasitaemia (NCM/HP). An analysis of 1H NMR and SDS-PAGE profile of PE and serum samples of each of the CM mice exhibited a similar profile in terms of constituents. Multivariate analysis on these two classes of biofluids was performed and significant differences were detected in concentrations of metabolites. Glucose, creatine and glutamine contents were high in the PE and lipids being high in the sera. Multivariate curve resolution between sera and pleural effusion showed that changes in PE co-varied with that of serum in CM mice. The increase of glucose in PE is negatively correlated to the glucose in serum in CM as obtained from the result of multiway principal component analysis. CONCLUSIONS: This study reports for the first time, the characterization of metabolites in pleural effusion formed during murine cerebral malaria. The study indicates that the origin of PE metabolites in murine CM may be the serum. The loss of the components like glucose, glutamine and creatine into the PE may worsen the situation of patients, in conjunction with the enhanced glycolysis, glutaminolysis and increased activity of creatine phophokinase which are already reported characteristic pathophysiological features of malaria.

Liver Metabolic Alterations and Changes in Host Intercompartmental Metabolic Correlation during Progression of Malaria.

(1)H NMR-based metabonomics was used to investigate the multimodal response of mice to malarial parasite infection by P. berghei ANKA. Liver metabolism was followed by NMR spectroscopy through the course of the disease in both male and female mice. Our results showed alterations in the level of several metabolites as a result of the infection. Metabolites like kynurenic acid, alanine, carnitine, and ß-alanine showed significant alteration in the liver, suggesting altered kynurenic acid, glucose, fatty acid and amino acid pathways. Distinct sexual dimorphism was also observed in the global analysis of the liver metabolic profiles. Multiway principal component analysis (MPCA) was carried out on the liver, brain, and serum metabolic profile in order to explore the correlation of liver and brain metabolic profile to the metabolite profile of serum. Changes in such correlation profile also indicated distinct sexual dimorphism at the early stage of the disease. Indications are that the females are able to regulate their metabolism in the liver in such a way to maintain homeostasis in the blood. In males, however, choline in liver showed anticorrelation to choline content of serum indicating a higher phospholipid degradation process. The brain-serum correlation profile showed an altered energy metabolism in both the sexes. The differential organellar responses during disease progression have implications in malaria management.

Alterations in urine, serum and brain metabolomic profiles exhibit sexual dimorphism during malaria disease progression.

BACKGROUND: Metabolic changes in the host in response to Plasmodium infection play a crucial role in the pathogenesis of malaria. Alterations in metabolism of male and female mice infected with Plasmodium berghei ANKA are reported here. METHODS: 1H NMR spectra of urine, sera and brain extracts of these mice were analysed over disease progression using Principle Component Analysis and Orthogonal Partial Least Square Discriminant Analysis. RESULTS: Analyses of overall changes in urinary profiles during disease progression demonstrate that females show a significant early post-infection shift in metabolism as compared to males. In contrast, serum profiles of female mice remain unaltered in the early infection stages; whereas that of the male mice changed. Brain metabolite profiles do not show global changes in the early stages of infection in either sex. By the late stages urine, serum and brain profiles of both sexes are severely affected. Analyses of individual metabolites show significant increase in lactate, alanine and lysine, kynurenic acid and quinolinic acid in sera of both males and females at this stage. Early changes in female urine are marked by an increase of ureidopropionate, lowering of carnitine and transient enhancement of asparagine and dimethylglycine. Several metabolites when analysed individually in sera and brain reveal significant changes in their levels in the early phase of infection mainly in female mice. Asparagine and dimethylglycine levels decrease and quinolinic acid increases early in sera of infected females. In brain extracts of females, an early rise in levels is also observed for lactate, alanine and glycerol, kynurenic acid, ureidopropionate and 2-hydroxy-2-methylbutyrate. CONCLUSIONS: These results suggest that P. berghei infection leads to impairment of glycolysis, lipid metabolism, metabolism of tryptophan and degradation of uracil. Characterization of early changes along these pathways may be crucial for prognosis and better disease management. Additionally, the distinct sexual dimorphism exhibited in these responses has a bearing on the understanding of the pathophysiology of malaria.

Kinetics of salt-dependent unfolding of [2Fe-2S] ferredoxin of Halobacterium salinarum.

The [2Fe-2S] ferredoxin from the extreme haloarchaeon Halobacterium salinarum is stable in high (>1.5 M) salt concentration. At low salt concentration the protein exhibits partial unfolding. The kinetics of unfolding was studied in low salt and in presence of urea in order to investigate the role of salt ions on the stability of the protein. The urea dependent unfolding, monitored by fluorescence of the tryptophan residues and circular dichroism, suggests that the native protein is stable at neutral pH, is destabilized in both acidic and alkaline environment, and involves the formation of kinetic intermediate(s). In contrast, the unfolding kinetics in low salt exhibits enhanced rate of unfolding with increase in pH value and is a two state process without the formation of intermediate. The unfolding at neutral pH is salt concentration dependent and occurs in two stages. The first stage, involves an initial fast phase (indicative of the formation of a hydrophobic collapsed state) followed by a relatively slow phase, and is dependent on the type of cation and anion. The second stage is considerably slower, proceeds with an increase in fluorescence intensity and is largely independent of the nature of salt. Our results thus show that the native form of the haloarchaeal ferredoxin (in high salt concentration) unfolds in low salt concentration through an apparently hydrophobic collapsed form, which leads to a kinetic intermediate. This intermediate then unfolds further to the low salt form of the protein.

Glycolysis in Plasmodium falciparum results in modulation of host enzyme activities.

BACKGROUND & OBJECTIVES: Plasmodium falciparum, the causative agent of the most serious form of malaria, infects about 5-10% of the world human population per year. It is well established that the erythrocytic stages of the malaria parasite rely mainly on glycolysis for their energy supply. In the present study, the glucose utilisation of erythrocyte population with parasitaemia levels similar to that of malaria patients was measured. The results allowed us to assess the effect of the parasites on the glucose utilisation of the vast majority of uninfected erythrocytes. METHODS: Using [2-13C]glucose and nuclear magnetic resonance (NMR) technique, the glucose utilisation in normal red blood cell (RBC) and P. falciparum infected red blood cell (IRBC) populations was measured. The IRBC population consisted of > 96% RBC and < 4% of parasite infected red blood cells (PRBC). The glycolytic enzymes were assayed to assess the effect of infected red cells on the enzymatic activities of uninfected ones. RESULTS: The rate of glucose utilisation by IRBC was considerably higher than that of RBC. Upon addition of 25% v/v conditioned culture medium (CM) of IRBC, RBCs exhibited a significant decrease in glucose utilisation. The CM could directly inhibit the activities of RBC glycolytic enzymes-phosphofructokinase (PFK) and pyruvate kinase (PK), without interfering with the activity of the pentose phosphate pathway enzyme-glucose-6-phosphate dehydrogenase (G-6-PD). INTERPRETATION & CONCLUSION: The present study showed that the clinical level of P. falciparum infected RBCs (< 4% parasitaemia) significantly enhance the glycolytic flux as well as down-regulate the glucose utilisation rate in the majority of uninfected RBC population. The mechanism of inhibition seems to be direct inhibition of the regulatory glycolytic enzymes-PFK and PK.

Malaria parasite-infected erythrocytes inhibit glucose utilization in uninfected red cells.

The erythrocytic stages of the malaria parasite depend on anaerobic glycolysis for energy. Using [2-13C]glucose and nuclear magnetic resonance, the glucose utilization rate and 2,3-diphosphoglycerate (2,3-DPG) level produced in normal RBCs and Plasmodium falciparum infected red blood cell populations (IRBCs, with <4% parasite infected red cells), were measured. The glucose flux in IRBCs was several-folds greater, was proportional to parasitemia, and maximal at trophozoite stage. The 2,3-DPG levels were disproportionately lower in IRBCs, indicating a downregulation of 2,3-DPG flux in non-parasitized RBCs. This may be due to lowered pH leading to selective differential inhibition of the regulatory glycolytic enzyme phosphofructokinase. This downregulation of the glucose utilization rate in the majority (>96%) of uninfected RBCs in an IRBC population may have physiological implications in malaria patients.