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 hostparasite 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.

Inter-study and time-dependent variability of metabolite abundance in cultured red blood cells

“…O-positive human RBCs were obtained from healthy blood donors as part of Johns Hopkins University’s phlebotomy protocol (Institutional Review Board protocol number: NA_00019050). Previously described methods were used to deplete white blood cells from the collected blood [ 9 ], and RBCs were maintained in a culture medium that was originally designed to maintain continuous cultures of P. falciparum [ 9 ]. Quadruplicate samples were collected at 0, 8, 16, 24, 32, and 40 h after transfer into fresh culture medium.…”

“…These datasets were included in this analyses because the experiments performed in these studies use methods that are identical to this study and have only minor differences in their culture medium, making these datasets suitable for studying inter-study variations in RBC metabolism. As a perturbation standard, metabolomic data were also analysed from iRBC cultures that were maintained under normal conditions during blood-stage growth [ 9 ]. To ensure the robustness of the analyses, only metabolites with greater than 1000 raw counts at all sampled time points and across all the replicates of uRBC and iRBC cultures were included.…”

HIV-1 Impact on Malaria Transmission: A Complex and Relevant Global Health Concern

“…Foundational and novel studies have identified various factors that correlate with an increase in gametocytogenesis. These include, but are not limited to, host immunity Graves et al, 1988;Motard et al, 1995;Buckling et al, 1999), regulation of the key parasite genes GDV1 and AP2-G (Day et al, 1993;Eksi et al, 2012;Brancucci et al, 2014;Coleman et al, 2014;Kafsack et al, 2014;Filarsky et al, 2018;Josling et al, 2020), host lipid and biomolecule biosynthesis and metabolism (Gulati et al, 2015;Brancucci et al, 2017;Tanaka et al, 2019;Tewari et al, 2020), fluctuations in host hormone production (Trager and Gill, 1992;Lingnau et al, 1993), and chemotherapy (Puta and Manyando, 1997;Robert et al, 2000;Tjitra et al, 2002;Hamel et al, 2005;Elbasit et al, 2006;Schneider et al, 2006). Each of these triggers may be altered in the context of a co-morbidity such as HIV-1.…”

Insights into malaria pathogenesis gained from host metabolomics

“…In the process, Plasmodium produces various by-products including pipecolic acid, a catabolite of lysine ( Fig 1E). Pipecolic acid is detected in in vitro P. falciparum cultures, murine malaria models, and humans with P. falciparum, but not in uninfected RBC cultures or in humans without malaria infection [43][44][45]. Other metabolites potentially generated by the parasite include VOCs pinene and limonene, which may derive from Plasmodium's isoprenoid biosynthetic pathways and are detected in P. falciparum cultures and breath of humans with falciparum malaria [46].…”