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A malaria parasite phospholipase facilitates efficient asexual blood stage egress.

ABSTRACT: Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300). Conditional ablation of LCAT resulted in abnormal egress and a reduced replication rate. Lipidomic profiles of LCAT-null parasites showed drastic changes in several phosphatidylserine and acylphosphatidylglycerol species during egress. We thus show that, in addition to its previously demonstrated role in liver stage merozoite egress, LCAT is required to facilitate efficient egress in asexual blood stage malaria parasites.

SUBMITTER: Ramaprasad A

PROVIDER: S-EPMC10325081 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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A malaria parasite phospholipase facilitates efficient asexual blood stage egress.

Ramaprasad Abhinay A Burda Paul-Christian PC Koussis Konstantinos K Thomas James A JA Pietsch Emma E Calvani Enrica E Howell Steven A SA MacRae James I JI Snijders Ambrosius P AP Gilberger Tim-Wolf TW Blackman Michael J MJ

PLoS pathogens 20230623 6

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the pa ...[more]

PMID: 37352369

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Project description:BACKGROUND: The carbon metabolism of the blood stages of Plasmodium falciparum, comprising rapidly dividing asexual stages and non-dividing gametocytes, is thought to be highly streamlined, with glycolysis providing most of the cellular ATP. However, these parasitic stages express all the enzymes needed for a canonical mitochondrial tricarboxylic acid (TCA) cycle, and it was recently proposed that they may catabolize glutamine via an atypical branched TCA cycle. Whether these stages catabolize glucose in the TCA cycle and what is the functional significance of mitochondrial metabolism remains unresolved. RESULTS: We reassessed the central carbon metabolism of P. falciparum asexual and sexual blood stages, by metabolically labeling each stage with 13C-glucose and 13C-glutamine, and analyzing isotopic enrichment in key pathways using mass spectrometry. In contrast to previous findings, we found that carbon skeletons derived from both glucose and glutamine are catabolized in a canonical oxidative TCA cycle in both the asexual and sexual blood stages. Flux of glucose carbon skeletons into the TCA cycle is low in the asexual blood stages, with glutamine providing most of the carbon skeletons, but increases dramatically in the gametocyte stages. Increased glucose catabolism in the gametocyte TCA cycle was associated with increased glucose uptake, suggesting that the energy requirements of this stage are high. Significantly, whereas chemical inhibition of the TCA cycle had little effect on the growth or viability of asexual stages, inhibition of the gametocyte TCA cycle led to arrested development and death. CONCLUSIONS: Our metabolomics approach has allowed us to revise current models of P. falciparum carbon metabolism. In particular, we found that both asexual and sexual blood stages utilize a conventional TCA cycle to catabolize glucose and glutamine. Gametocyte differentiation is associated with a programmed remodeling of central carbon metabolism that may be required for parasite survival either before or after uptake by the mosquito vector. The increased sensitivity of gametocyte stages to TCA-cycle inhibitors provides a potential target for transmission-blocking drugs.

Project description:The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. Insect cells have supplied the production of numerous vaccine antigens in a fast and cost-effective manner; improving this platform further could prove key to its wider use. In this study, insect (Sf9 and High Five) and human (HEK293) cell hosts as well as process-optimizing strategies (new baculovirus construct designs and a culture temperature shift to hypothermic conditions) were employed to improve the production of the malaria asexual blood-stage vaccine candidate PfRipr5. Protein expression was maximized using High Five cells at CCI of 2 × 106 cell/mL and MOI of 0.1 pfu/cell (production yield = 0.49 mg/ml), with high-purity PfRipr5 binding to a conformational anti-PfRipr monoclonal antibody known to hold GIA activity and parasite PfRipr staining capacity. Further improvements in the PfRipr5 expression were achieved by designing novel expression vector sequences and performing a culture temperature shift to hypothermic culture conditions. Addition of one alanine (A) amino acid residue adjacent to the signal peptide cleavage site and a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag (His6) induced a 2.2-fold increase in the expression of secreted PfRipr5 over using the expression vector with none of these additions. Performing a culture temperature shift from the standard 27-22°C at the time of infection improved the PfRipr5 expression by up to 1.7 fold. Notably, a synergistic effect was attained when combining both strategies, enabling to increase production yield post-purification by 5.2 fold, with similar protein quality (i.e., purity and binding to anti-PfRipr monoclonal antibody). This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost the expression of secreted proteins.

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A malaria parasite phospholipase facilitates efficient asexual blood stage egress.

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A malaria parasite phospholipase facilitates efficient asexual blood stage egress.

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