Metabolic alterations in the erythrocyte during blood-stage development of the malaria parasite

Abstract

Background: Human blood cells (erythrocytes) serve as hosts for the malaria parasite Plasmodium falciparum during its 48-h intraerythrocytic developmental cycle (IDC). Established in vitro protocols allow for the study of host-parasite interactions during this phase and, in particular, high-resolution metabolomics can provide a window into host-parasite interactions that support parasite development.

Methods: Uninfected and parasite-infected erythrocyte cultures were maintained at 2% haematocrit for the duration of the IDC, while parasitaemia was maintained at 7% in the infected cultures. The parasite-infected cultures were synchronized to obtain stage-dependent information of parasite development during the IDC. Samples were collected in quadruplicate at six time points from the uninfected and parasite-infected cultures and global metabolomics was used to analyse cell fractions of these cultures.

Results: In uninfected and parasite-infected cultures during the IDC, 501 intracellular metabolites, including 223 lipid metabolites, were successfully quantified. Of these, 19 distinct metabolites were present only in the parasite-infected culture, 10 of which increased to twofold in abundance during the IDC. This work quantified approximately five times the metabolites measured in previous studies of similar research scope, which allowed for more detailed analyses. Enrichment in lipid metabolism pathways exhibited a time-dependent association with different classes of lipids during the IDC. Specifically, enrichment occurred in sphingolipids at the earlier stages, and subsequently in lysophospholipid and phospholipid metabolites at the intermediate and end stages of the IDC, respectively. In addition, there was an accumulation of 18-, 20-, and 22-carbon polyunsaturated fatty acids, which produce eicosanoids and promote gametocytogenesis in infected erythrocyte cultures.

Conclusions: The current study revealed a number of heretofore unidentified metabolic components of the host-parasite system, which the parasite may exploit in a time-dependent manner to grow over the course of its development in the blood stage. Notably, the analyses identified components, such as precursors of immunomodulatory molecules, stage-dependent lipid dynamics, and metabolites, unique to parasite-infected cultures. These conclusions are reinforced by the metabolic alterations that were characterized during the IDC, which were in close agreement with those known from previous studies of blood-stage infection.

Keywords: Blood-stage infection; Host–parasite metabolism; Lysophosphatidylglycerol; Metabolome; Plasmodium falciparum; Polyunsaturated fatty acids.

Fig. 1

Comparison of metabolite coverage across this study and studies by Babbitt et al. [8] and Olszewski et al. [4]. a Venn diagram showing overlap of metabolites between the three studies. Relative to the other two studies, which quantified comparable numbers of metabolites during the intraerythrocytic developmental cycle (IDC), this study quantified roughly five times more metabolites. b Metabolites detected in the three studies (N_metabolites) mapped onto five major metabolic pathways. In contrast to the previous studies, which quantified ~ 100 metabolites during the IDC, this study quantified over 200 lipid metabolites and more than 100 amino acid metabolites. “Other” denotes metabolites that do not belong to the five major metabolic pathways

Fig. 2

Global metabolomics of uninfected (uRBC) and parasite-infected erythrocyte (iRBC) cultures. a Heatmap of metabolite abundances in uRBC and iRBC at 0, 8, 16, 24, 32, and 40 h. Each of the 501 rows represents a distinct metabolite. There are four replicates for each time point. Orange indicates an abundance level of a metabolite greater than the median value, which is computed across uRBCs and iRBCs, whereas blue indicates an abundance level lower than the median. b Principal component analysis of metabolomic data from uRBCs (black) and iRBCs (red). The uRBC and iRBC data separated along the first (PC1), second (PC2), and third (PC3) principal components, with the maximum separation occurring between the ellipses labelled ‘16–40 h’ and ‘16–32 h,’ respectively. The uRBC data formed two clusters: 0–8 h and 16–40 h. Ellipses are drawn only to visually highlight uRBC and iRBC data that were clustered together; they do not reflect the confidence intervals of the clusters. The ellipses labelled ‘16–32 h’ and ‘16–40 h’ contain 12 and 16 data points, respectively, although they are not discernible because of overlap among some of the data points. The percentage of the total data variance explained by each principal component is shown in parentheses along each axis. c Average variance (σ²) of metabolite abundance at a given time point within replicates. First, the variance within replicates is computed for the abundance of a given metabolite and then the average across all metabolites is computed for each time point. The average variance is shown in black for uRBCs and in red for iRBCs. The dotted horizontal line shows the mean of the average variance, which is ~ 4%. d The average fold change ($\overline{\text{FC}}$) in metabolite abundance between different time points. The fold change in the kth metabolite at time point ‘j’ against time point ‘i’ is computed as ${\text{m}}_{\text{k}}^{\text{j}}/{\text{m}}_{\text{k}}^{\text{i}}$, where i and j are each set to 0, 8, 16, 24, 32, or 40 h. Hence, each element ij indicates the average metabolite fold change computed using the dataset at time points i and j, where N denotes the total number of metabolites. Compared to the average metabolite fold changes in uRBCs, those in iRBCs are more pronounced at all sampled time points. The results are shown on a log₂ scale. e Hierarchical clustering analysis (HCA) of the metabolomic data in a after averaging the metabolite abundances among the replicates. The colour scheme and scale are as shown in a. Metabolites were clustered based on the Euclidean distance similarity of their temporal profiles. HCA identified five distinct clusters, which are shown in distinct colours with a corresponding number. Generally, within each cluster, metabolites that were downregulated in uRBCs were upregulated in iRBCs and vice versa

Fig. 3

Global and temporal changes in metabolite abundance during the IDC. a Temporal fold-change values in significantly altered metabolites. Here, any metabolite that changed (i.e., increased or decreased) twofold or more in abundance was considered as significantly altered. The time-specific fold change was computed as ${\text{m}}_{\text{iRBC}}/{\text{m}}_{\text{uRBC}}$, where m represents the metabolite abundance at 0, 8, 16, 24, 32, or 40 h, and uRBC and iRBC denote uRBC and iRBC cultures, respectively. Fold-change values greater than or equal to two are shown in black, and those smaller than two are shown in grey. b The number of metabolites in a that changed by twofold or more at the indicated time points. The number increased monotonically with time, suggesting that pronounced metabolic changes occur during the later stages of the IDC. c Fold change in the average abundance of metabolites from Clusters 1–5 in Fig. 2e. The fold change in average abundance (FC_IDC) was computed as ${\overline{\text{m}}}_{\text{iRBC}}/{\overline{\text{m}}}_{\text{uRBC}}$, where $\overline{\text{m}}$ represents the average abundance of a metabolite ‘m’ averaged across all time points. Twofold changes in average abundance are shown in red (Cluster 1), magenta (Cluster 2), green (Cluster 4), or cyan (Cluster 5). Fold changes of less than two are shown in grey. Although Cluster 3 showed some temporal changes in metabolites (Fig. 2e), these disappeared when the FC_IDC was computed (hence, all markers are grey). IDC intraerythrocytic developmental cycle, iRBC parasite-infected erythrocyte, uRBC uninfected erythrocyte

Fig. 4

Fold enrichment in human metabolic pathways of parasite-infected erythrocytes at 0–8 h, 16–24 h, and 32–40 h. MetaboAnalyst [10], which takes human metabolome database identifiers as input, was used to compute fold enrichment. Of the pathways in the small molecule pathway database library [11] of normal human metabolic pathways, only those that contained at least five metabolites were used. Asterisks indicate fold-enrichments with an adjusted criterion of p ≤ 0.01 [44]. BCAA branched-chain amino acid, CoA co-enzyme A, FA fatty acid, PPP pentose phosphate pathway, TCA tricarboxylic acid

Fig. 5

Fold change in abundance of lipid and fatty acid metabolites during the intraerythrocytic developmental cycle (IDC). a Lipid metabolites were classified according to the LIPID MAPS Structure Database [51] into 13 subordinate classes. The figure shows fold changes in lipid classes that contain two or more metabolites. The fold changes were greatest for diacylglycerol (DG) and glycerophosphoglycerol (GPG) metabolites. b Fold change in abundance of fatty acids based on different carbon-chain lengths. The fold change was greatest for the 5-carbon fatty acids (~ 1.8-fold in iRBC cultures relative to uRBC cultures), followed by a number of 18-carbon, 20-carbon, and 22-carbon polyunsaturated fatty acids (~ 1.5-fold). The ${\overline{\text{FC}}}_{\text{IDC}}$ was computed as the average ${\text{FC}}_{\text{IDC}}$ (described in Fig. 3c) when a metabolite class contained more than one metabolite. Each error bar shows the standard deviation of the ${\text{FC}}_{\text{IDC}}$ of metabolites present in a metabolite class. Cer ceramide, DG diacylglycerol, FA fatty acid amide, PA glycerophosphate, GPC glycerophosphocholine, GPE glycerophosphoethanolamine, GPG glycerophosphoglycerol, GPI glycerophosphoinositol, GPS glycerophosphoserine, SM phosphosphingolipid, ST sterol

Fig. 6

Normalized abundance of important metabolites of glucose, phospholipid, and pyrimidine metabolism in uninfected (uRBC) and parasite-infected erythrocyte (iRBC) cultures. a Abundance of glucose, phosphoenolpyruvate (PEP), and lactate during the intraerythrocytic developmental cycle (IDC). Glucose decreased in iRBC cultures, whereas it was stable in uRBC cultures. The increase in lactate was commensurate with glucose consumption, indicating active parasite metabolism. b Abundance of phosphocholine (PCho), phosphoethanolamine (PEth), and lyso phosphatidylcholine (PtdCho) 16:0 during the IDC. PCho and PEth are precursors of PtdCho and phosphatidylethanolamine, respectively, which account for ~ 75% to 85% of parasite phospholipids [49]. In addition to PEth, the parasite also utilizes lyso PtdCho to synthesize PtdCho [69], which also decreased over time in iRBC cultures. c Parasites synthesize N-carbamoyl-l-aspartate (NCD) in the first step, dihydroorotate in the second step, and orotate in the third step of de novo pyrimidine synthesis [27]. These metabolites increased in the iRBC cultures, consistent with the synthesis of parasite DNA [70]

Fig. 7

Metabolite and metabolic pathway concordance between this study and studies by Olszewski et al. [4] and Babbitt et al. [8]. a Temporal profiles of metabolite abundance quantified during the intraerythrocytic developmental cycle in the three studies. Metabolite abundances were normalized by their value at t = 0 h (grey vertical bar) to allow comparison across studies. Metabolites are grouped by metabolite class. b Spearman’s ρ computed for metabolites quantified in all three studies (N = 41) and within each metabolite class. The correlation for the lipid class, which contained only two metabolites, was not computed. c Spearman’s ρ computed for all metabolites at 8, 16, 24, 32, and 40 h. In comparisons with both studies, the correlation was near zero at the 8-h time point, i.e., when parasite metabolism is least active [73]. The dotted line shows the average (~ 0.3) of the correlations at each time point for both studies. ADP adenosine diphosphate, AMP adenosine monophosphate, CMP cytidine monophosphate, DHAP dihydroxyacetone phosphate, $\widehat{\text{FC}}$ metabolite abundance normalized with respect to t = 0 h, GMP guanosine monophosphate, IMP inosine monophosphate, NAD⁺ nicotinamide adenine dinucleotide (oxidized), PEP phosphoenolpyruvate, UDP uridine diphosphate, UMP uridine monophosphate

Fig. 8

Distribution of significantly altered metabolites among major metabolic pathways at 0–8 h, 16–24 h, and 32–40 h. Significantly altered metabolites (p ≤ 0.05; q < 0.10) were identified by performing a two-way analysis of variance on the metabolomic data from the cell fractions of uninfected and infected cultures at the indicated time points. At the earliest time points (0–8 h) most of the significantly altered metabolites belonged to the lipid class, but at later time points (16–24 h and 32–40 h) both amino acid and lipid class metabolites were equally perturbed, commensurate with the stage-dependent development of parasite metabolism. The pathway labelled “Cofactors” corresponds to metabolites that belong to cofactor and vitamin metabolism. The pathway labelled “Other” includes metabolites that do not belong to any of the major pathways