Dataset Information

Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

ABSTRACT: Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

SUBMITTER: Fougere A

PROVIDER: S-EPMC5113031 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

Fougère Aurélie A Jackson Andrew P AP Bechtsi Dafni Paraskevi DP Braks Joanna A M JA Annoura Takeshi T Fonager Jannik J Spaccapelo Roberta R Ramesar Jai J Chevalley-Maurel Séverine S Klop Onny O van der Laan Annelies M A AM Tanke Hans J HJ Kocken Clemens H M CH Pasini Erica M EM Khan Shahid M SM Böhme Ulrike U van Ooij Christiaan C Otto Thomas D TD Janse Chris J CJ Franke-Fayard Blandine B

PLoS pathogens 20161116 11

Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understa ...[more]

PMID: 27851824

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Project description:During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to in vitro-cultivated Plasmodium berghei blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved Plasmodium proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in P. berghei. IMPORTANCE Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In Plasmodium parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent P. berghei parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins.

Project description:BackgroundThe Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.MethodsBy using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.ResultsThe high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.ConclusionsThe PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

Dataset Information

Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

Publications

Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

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