An in silico down-scaling approach uncovers novel constituents of the Plasmodium-containing vacuole

Abstract

During blood stage development the malaria parasite resides in a membrane-bound compartment, termed the parasitophorous vacuole (PV). The reasons for this intravacuolar life style and the molecular functions of this parasite-specific compartment remain poorly defined, which is mainly due to our limited knowledge about the molecular make-up of this unique niche. We used an in silico down-scaling approach to select for Plasmodium-specific candidates that harbour signatures of PV residency. Live co-localisation of five endogenously tagged proteins confirmed expression in the PV of Plasmodium berghei blood and liver stages. ER retention was ruled out by addition of the respective carboxyterminal tetrapeptides to a secreted reporter protein. Although all five PV proteins are highly expressed, four proved to be dispensable for parasite development in the mammalian and mosquito host, as revealed by targeted gene deletion. In good agreement with their redundant roles, the knockout parasites displayed no detectable deficiencies in protein export, sequestration, or PV morphology. Together, our approach improved the catalogue of the Plasmodium PV proteome and provides experimental genetics evidence for functional redundancy of several PV proteins.

Figure 1

In silico identification of Plasmodium PV protein candidates. (a) Plasmodium protein targeting in infected erythrocytes via the secretory pathway. Depicted are schematic representations of proteins with different targeting information and their expected localisation patterns during blood stage development. SP, signal peptide; PEXEL, Plasmodium export element; API, apicoplast transit peptide; TM, transmembrane domain; RS, endoplasmic reticulum retention signal; GPI, glycosylphosphatidylinositol anchor. (b) Algorithm for the in silico identification of PV candidates. Shown is a schematic representation of the selection procedure. The blue arrow denotes the sequence of events. Individual steps are shown in yellow. Venn diagrams indicate whether the relative complement or the intersection of two steps was used. SP-containing Plasmodium proteins were selected in P. falciparum and P. berghei. Proteins containing additional targeting information were removed sequentially. Proteins with annotated functions outside the PV, e.g. mitochondrial or nuclear proteins, were removed manually. Note that several selected proteins were assigned a predicted transmembrane (TM) domain at the amino-terminus due to the hydrophobicity of the SP. Next, genes showing very weak expression in asexual blood stages or peak expression in schizonts, gametocytes and/or ookinetes were excluded. Finally, previously neglected proteins specific to the Apicomplexa or to the genus Plasmodium were selected, including PV1 and 2, which served as positive controls for experimental validation. Accession codes of the final P. berghei PV candidates are shown in the green box. 1, Predicted with SignalP; 2, predicted with TMHMM; 3, Plasmodium export element; 4, host targeting motif ; 5, predicted with ExportPred. 6, predicted with PlasmoAP; 7, predicted with big-Pi; 8, transcript abundance determined by RNA sequencing. (c) Representation of the applied search algorithm and the associated numbers of proteins.

Figure 2

Validation of novel PV proteins. (a) Strategy for the generation of transgenic parasite lines expressing endogenous PV proteins (PV_X) fused to mCherry-3xMyc (tag) by single homologous integration. In addition recombinant parasites contain the drug-selectable hDHFR-yFcu cassette (drug cassette) and the GFP^PV cassette. Wild-type (WT) and integration-specific (INT) primer combinations (Supplementary Table S1) are indicated by arrows and expected fragments by dotted lines. Note that PV1 and PBANKA_1212100 were tagged using another strategy based on double homologous recombination (see Supplementary Fig. S1). (b) Live fluorescence microscopy of selected PV candidates. Numbers indicate gene accession codes (without the ‘PBANKA_’ prefix). Shown are transgenic P. berghei blood stage parasites expressing the endogenous candidate genes fused to mCherry-3xMyc (red, top) and the marker protein GFP^PV (green, lower top) as well as a merge of both fluorescent protein signals (upper bottom) and a merge of differential interference contrast images (DIC) with Hoechst 33342 nuclear dye (DNA, blue, bottom). The known PV proteins PV1 and PV2 are included as positive controls. , PV protrusions. Candidates showing no PV localisation are depicted in Supplementary Fig. S1. Wild-type (WT) and integration-specific (INT) diagnostic PCRs of the parental transgenic parasite lines, as indicated in a and Supplementary Fig. S1, are shown above and indicate proper genomic integration of the targeting constructs. For full size images of DNA gels, see Supplementary Fig. S5. (c,d) Assessment of ER retention. (c) Schematic representation of the vectors used for ER retention testing. The GFP^PV cassette, consisting of the signal peptide of BiP (purple) fused to GFP (green), was appended with the last four amino acids of the PV candidates (yellow) and expressed from the silent intergenic locus on chromosome 6 (SIL6) under the control of the HSP70 promoter. (d) Live fluorescence microscopy of transgenic P. berghei parasites expressing ER retention testing constructs. Shown are the GFP signal (green, top), differential interference contrast images (DIC, middle), and a merge with Hoechst 33342 nuclear dye (DNA, blue, bottom). The white labelling indicates the last four amino acids of the PV candidates.

Figure 3

PV1-5 are not associated with the parasite plasma membrane. Shown are transgenic Plasmodium berghei blood stage parasites expressing the endogenous PV proteins fused to mCherry-3xMyc (red, top) and GFP^PV (green, lower top), a merge of both fluorescent protein signals (middle), differential interference contrast images (DIC, upper bottom) as well as a merge of all signals with Hoechst 33342 nuclear dye (DNA, blue, bottom). Depicted are ruptured schizonts. Note that during this stage the PV proteins predominantly localise to the digestive vacuole and not to the surface of the parasites. , PV1-mCherry-fluorescent dot associated with an emerging merozoite.

Figure 4

PV protein expression during liver stage development. Transgenic Plasmodium berghei parasites expressing fluorescently labelled PV1, 2, 3, 4, or 5 were imaged live throughout in vitro liver stage development. Representative parasites were recorded 24, 48, and 68 hours after sporozoite inoculation. Shown are the fluorescent signals of the mCherry-3xMyc-tagged PV proteins (red, top), the marker protein GFP^PV (green, middle), as well as a merge of both fluorescent protein signals with Hoechst 33342 nuclear dye (DNA, blue, bottom).

Figure 5

PV5 is essential during blood infection, whereas PV1–4 are dispensable. (a) Replacement strategy to delete the genes encoding PV proteins of Plasmodium berghei. The loci were targeted with replacement plasmids containing the 5′ and 3′ regions flanking the respective open reading frame, a cytoplasmic GFP expression cassette, and the drug-selectable hDHFR-yFcu cassette (drug cassette). For PV visualization, GFP was exchanged for GFP^PV (see also Supplementary Fig. S3). Wild-type (WT) and integration-specific primer combinations (INT) are indicated by arrows and expected fragments by dotted lines. (b) For each target gene, diagnostic PCRs of the WT locus (top) and of the drug-selected and isolated parasites (bottom) are shown using the primer combinations depicted in a. The green frames denote the successful isolation of loss-of-function mutants. The red frame indicates that three independent transfection experiments did not result in the recovery of a PV5 deletion strain. For full size images of DNA gels, see Supplementary Fig. S5.

Figure 6

Normal PV morphology and protein export competence in the absence of PV1, 2, 3, or 4. (a) PV morphology remains unaltered in the absence of PV1, 2, 3, or 4. Shown are WT or PV1, 2, 3, or 4 gene deletion mutants expressing GFP^PV. Depicted are the fluorescent signal of GFP^PV (green, top) and a merge of differential interference contrast images (DIC) with Hoechst 33342 nuclear dye (DNA, blue, bottom). Shown are representative micrographs of asexual blood stages during the ring, trophozoite and schizont stages, as well as selected PV features. (b) Export of EMAP1 is not impaired in the absence of PV1, 2, 3, or 4. Depicted are representative micrographs of WT or PV knockout parasites expressing the exported mCherry-tagged erythrocyte membrane-associated protein 1 (EMAP1). Shown are a merge of EMAP1 (red) with the cytoplasmic GFP fluorescence of the PV knockout mutants (green, left) and a merge of DIC images with Hoechst 33342 nuclear dye (DNA, blue, right). (c) Quantification of EMAP1 export. The extra-parasitic EMAP1-mCherry fluorescence was normalized to the overall EMAP1-mCherry fluorescence of the infected red blood cell. Lines show mean values. n.s., non-significant; One-way ANOVA and Tukey’s multiple comparison test, n = 30. (d) Normal sequestration in the absence of PV1, 2, 3, or 4. Purified WT, pv1⁻, pv2⁻, pv3⁻, and pv4⁻ schizonts were injected intravenously into naïve mice and the number of circulating multi-nucleated parasites was determined 22 hours later. The ptex88⁻ line was reported to exhibit reduced sequestration in vivo and served as a positive control. Shown are mean values (±SD). ***P < 0.001; n.s., non-significant; One-way ANOVA and Tukey’s multiple comparison test, n = 3.

Figure 7

PV1–4 do not promote parasite fitness during life cycle progression. (a–d) Intravital competition assay demonstrates normal blood propagation in the absence of PV1 (a), PV2 (b), PV3 (c), or PV4 (d). 500 mCherry-fluorescent Berred WT and 500 GFP-fluorescent knockout blood stage parasites were co-injected into NMRI mice and peripheral blood was analysed daily by flow cytometry. Mean values (±SD) are shown for each time point. n.s., non-significant; Two-way ANOVA, n = 3. (e) PV1 is expressed during the oocyst stage. Shown are transgenic P. berghei parasites expressing the endogenous PV proteins fused to mCherry-3xMyc (red, top) as well as a merge of differential interference contrast images (DIC) with Hoechst 33342 nuclear dye (DNA, blue, bottom). Depicted are 10 day old oocysts. (f) Normal maturation of PV knockout parasites in the mosquito and in hepatocytes. Representative live fluorescence micrographs of Berred wild-type (WT) or PV knockout parasites during the oocyst and liver stages. Shown is a merge of the parasite’s cytoplasmic fluorescence (WT, mCherry, red; PV knockouts, GFP, green) and differential interference contrast images (DIC). Midgut-associated oocysts were recorded 10 days after the blood meal, liver stages were recorded 24, 48 and 72 hours after sporozoite inoculation.