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Polyunsaturated fatty acids promote Plasmodium falciparum gametocytogenesis.

ABSTRACT: The molecular triggers of sexual differentiation into gametocytes by blood stage Plasmodium falciparum, the most malignant human malaria parasites, are subject of much investigation for potential transmission-blocking strategies. The parasites are readily grown in vitro with culture media supplemented by the addition of human serum (10%) or by a commercially available substitute (0.5% AlbuMAX). We found better gametocytemia with serum than AlbuMAX, suggesting suboptimal concentrations of some components in the commercial product; consistent with this hypothesis, substantial concentration differences of multiple fatty acids were detected between serum- and AlbuMAX-supplemented media. Mass spectroscopy analysis distinguished the lipid profiles of gametocyte- and asexual stage-parasite membranes. Delivery of various combinations of unsaturated fatty-acid-containing phospholipids to AlbuMAX-supported gametocyte cultures improved gametocyte production to the levels achieved with human-serum-supplemented media. Maturing gametocytes readily incorporated externally supplied d5-labeled glycerol with fatty acids into unsaturated phospholipids. Phospholipids identified in this work thus may be taken up from extracellular sources or generated internally for important steps of gametocyte development. Further study of polyunsaturated fatty-acid metabolism and phospholipid profiles will improve understanding of gametocyte development and malaria parasite transmission.

SUBMITTER: Tanaka TQ

PROVIDER: S-EPMC6679406 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Polyunsaturated fatty acids promote Plasmodium falciparum gametocytogenesis.

Tanaka Takeshi Q TQ Tokuoka Suzumi M SM Nakatani Daichi D Hamano Fumie F Kawazu Shin-Ichiro SI Wellems Thomas E TE Kita Kiyoshi K Shimizu Takao T Tokumasu Fuyuki F

Biology open 20190716 7

The molecular triggers of sexual differentiation into gametocytes by blood stage Plasmodium falciparum, the most malignant human malaria parasites, are subject of much investigation for potential transmission-blocking strategies. The parasites are readily grown in vitro with culture media supplemented by the addition of human serum (10%) or by a commercially available substitute (0.5% AlbuMAX). We found better gametocytemia with serum than AlbuMAX, suggesting suboptimal concentrati ...[more]

PMID: 31221627

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Project description:BackgroundThe maturation of Plasmodium falciparum gametocytes in the human host takes several days, during which the parasites need to efficiently evade the host immune system. Like asexual stage parasites, immature gametocytes can sequester at various sites in the human body, and only mature sexual stages are found in the circulation. Although the fundamental mechanisms of gametocyte immune evasion are still largely unknown, candidate molecules that may be involved include variant antigens encoded by multigene families in the P. falciparum genome, such as the PfEMP1, STEVOR and RIFIN proteins. While expression of the former two families in sexual stages has been investigated earlier, we report here RIFIN expression during gametocytogenesis.Methodology/principal findingsVariants of two previously characterized RIFIN subfamilies (A- and B-type RIFINs) were found to be synthesized in gametocytes. Immunofluorescence experiments showed A-type RIFINs to be accumulated in a crescent-shaped pattern of discrete punctate structures at the infected erythrocyte membrane, while members of the B-type family were associated with the parasite. Transcription analysis demonstrated the existence of diverse transcriptional regulation patterns during sexual differentiation and indicated variant-specific regulation of B-type RIFINs, in contrast to group-specific regulation for A-type RIFINs. Phylogenetic analysis of 5'-upstream regions showed that the rif-gene family falls into five defined clusters, designated rups (rifupstream) A1, A2, AB, B and C. In trophozoites and early gametocytes, rif variants of the rupsA2-type were preferentially expressed.Conclusions/significanceIn this work we demonstrate the expression dynamics of the rif-gene family during sexual differentiation and present indications for subgroup specific regulation patterns. Therefore, our data provide a first foundation and point to new directions for future investigations of the potential role of RIFINs in gametocyte immune evasion.

Project description:Palmitoylation is the post-translational reversible addition of the acyl moiety, palmitate, to cysteine residues of proteins and is involved in regulating protein trafficking, localization, stability and function. The Aspartate-Histidine-Histidine-Cysteine (DHHC) protein family, named for their highly conserved DHHC signature motif, is thought to be responsible for catalysing protein palmitoylation. Palmitoylation is widespread in all eukaryotes, including the malaria parasite, Plasmodium falciparum, where over 400 palmitoylated proteins are present in the asexual intraerythrocytic schizont stage parasites, including proteins involved in key aspects of parasite maturation and development. The P. falciparum genome includes 12 proteins containing the conserved DHHC motif. In this study, we adapted a palmitoyl-transferase activity assay for use with P. falciparum proteins and demonstrated for the first time that P. falciparum DHHC proteins are responsible for the palmitoylation of P. falciparum substrates. This assay also reveals that multiple DHHCs are capable of palmitoylating the same substrate, indicating functional redundancy at least in vitro. To test whether functional redundancy also exists in vivo, we investigated the endogenous localization and essentiality of a subset of schizont-expressed PfDHHC proteins. Individual PfDHHC proteins localized to distinct organelles, including parasite-specific organelles such as the rhoptries and inner membrane complex. Knock-out studies identified individual DHHCs that may be essential for blood-stage growth and others that were functionally redundant in the blood stages but may have functions in other stages of parasite development. Supporting this hypothesis, disruption of PfDHHC9 had no effect on blood-stage growth but reduced the formation of gametocytes, suggesting that this protein could be exploited as a transmission-blocking target. The localization and stage-specific expression of the DHHC proteins may be important for regulating their substrate specificity and thus may provide a path for inhibitor development.

Project description:BackgroundThe malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for identifying and evaluating transmission in the laboratory is standard membrane feeding assays (SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to mosquitoes as part of a blood-meal. However, propagation of "infectious" gametocytes requires 10-12 days with considerable physico-chemical demands imposed on host RBCs and thus, "fresh" RBCs that are ≤ 1-week old post-collection are generally recommended. However, in addition to the costs, physico-chemical characteristics unique to RBC donors may confound reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs ("cryo-preserved RBCs") is accepted by European and US FDAs as an alternative to refrigeration (4 °C) for preserving RBC "quality" and while cryo-preserved RBCs have been used for in vitro cultures of other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to support parasite development for > 4 days.ResultsUsing the standard laboratory strain, P. falciparum NF54, 11 SMFAs were performed with RBCs from four separate donors to demonstrate that RBCs cryo-preserved in the gaseous phase of liquid nitrogen (- 196 °C) supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensi mosquitoes. Overall levels of sporogony in the mosquito, as measured by oocyst and sporozoite prevalence, as well as oocyst burden, from each of the four donors thawed after varying intervals of cryopreservation (1, 4, 8, and 12 weeks) were comparable to using ≤ 1-week old refrigerated RBCs. Lastly, the potential for cryo-preserved RBCs to serve as a suitable alternative substrate is demonstrated for a Cambodian isolate of P. falciparum across two independent SMFAs.ConclusionsBasic guidelines are presented for integrating cryo-preserved RBCs into an existing laboratory/insectary framework for P. falciparum SMFAs with significant potential for reducing running costs while achieving greater reliability. Lastly, scenarios are discussed where cryo-preserved RBCs may be especially useful in enhancing the understanding and/or providing novel insights into the patterns and processes underlying human-to-mosquito transmission.

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Polyunsaturated fatty acids promote Plasmodium falciparum gametocytogenesis.

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Polyunsaturated fatty acids promote <i>Plasmodium falciparum</i> gametocytogenesis.

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