Investigation of the Plasmodium falciparum Food Vacuole through Inducible Expression of the Chloroquine Resistance Transporter (PfCRT)

Haemoglobin degradation during the erythrocytic life stages is the major function of the food vacuole (FV) of Plasmodium falciparum and the target of several anti-malarial drugs that interfere with this metabolic pathway, killing the parasite. Two multi-spanning food vacuole membrane proteins are known, the multidrug resistance protein 1 (PfMDR1) and Chloroquine Resistance Transporter (PfCRT). Both modulate resistance to drugs that act in the food vacuole. To investigate the formation and behaviour of the food vacuole membrane we have generated inducible GFP fusions of chloroquine sensitive and resistant forms of the PfCRT protein. The inducible expression system allowed us to follow newly-induced fusion proteins, and corroborated a previous report of a direct trafficking route from the ER/Golgi to the food vacuole membrane. These parasites also allowed the definition of a food vacuole compartment in ring stage parasites well before haemozoin crystals were apparent, as well as the elucidation of secondary PfCRT-labelled compartments adjacent to the food vacuole in late stage parasites. We demonstrated that in addition to previously demonstrated Brefeldin A sensitivity, the trafficking of PfCRT is disrupted by Dynasore, a non competitive inhibitor of dynamin-mediated vesicle formation. Chloroquine sensitivity was not altered in parasites over-expressing chloroquine resistant or sensitive forms of the PfCRT fused to GFP, suggesting that the PfCRT does not mediate chloroquine transport as a GFP fusion protein

Plasmodium falciparum Heterochromatin Protein 1 Marks Genomic Loci Linked to Phenotypic Variation of Exported Virulence Factors

Epigenetic processes are the main conductors of phenotypic variation in eukaryotes. The malaria parasite Plasmodium falciparum employs antigenic variation of the major surface antigen PfEMP1, encoded by 60 var genes, to evade acquired immune responses. Antigenic variation of PfEMP1 occurs through in situ switches in mono-allelic var gene transcription, which is PfSIR2-dependent and associated with the presence of repressive H3K9me3 marks at silenced loci. Here, we show that P. falciparum heterochromatin protein 1 (PfHP1) binds specifically to H3K9me3 but not to other repressive histone methyl marks. Based on nuclear fractionation and detailed immuno-localization assays, PfHP1 constitutes a major component of heterochromatin in perinuclear chromosome end clusters. High-resolution genome-wide chromatin immuno-precipitation demonstrates the striking association of PfHP1 with virulence gene arrays in subtelomeric and chromosome-internal islands and a high correlation with previously mapped H3K9me3 marks. These include not only var genes, but also the majority of P. falciparum lineage-specific gene families coding for exported proteins involved in host–parasite interactions. In addition, we identified a number of PfHP1-bound genes that were not enriched in H3K9me3, many of which code for proteins expressed during invasion or at different life cycle stages. Interestingly, PfHP1 is absent from centromeric regions, implying important differences in centromere biology between P. falciparum and its human host. Over-expression of PfHP1 results in an enhancement of variegated expression and highlights the presence of well-defined heterochromatic boundaries. In summary, we identify PfHP1 as a major effector of virulence gene silencing and phenotypic variation. Our results are instrumental for our understanding of this widely used survival strategy in unicellular pathogens

The Plasmodium food vacuole: protein targeting of transmembrane proteins

© 2011 Dr. Florian EhlgenThe causative agent of severe malaria in humans is Plasmodium falciparum – a parasite whose complex life cycle includes an asexual proliferation within human erythrocytes. Adaptations to the intra-erythrocytic lifestyle have created new and in some cases unique organelles, such as an endosomal/ lysosomal-like organelle, the food vacuole (FV). The only verified function of the FV is haemoglobin degradation and detoxification of the breakdown byproduct, haem. Several anti-malarial drugs interfere with this detoxification and thus kill the parasite. Two multi-spanning membrane proteins in the bounding membrane of the FV, Multidrug Resistant 1 (PfMDR1) and the Chloroquine Resistance Transporter (PfCRT), are associated with resistance against anti-malarial drugs. Protein targeting has been investigated for PfCRT, yet insights from this study are not readily transferable to PfMDR1. In this thesis I investigate the functions of the FV and underlying targeting processes by analysing its membrane proteome. I have expressed PfCRT as a green fluorescence protein (GFP)-tagged fusion and performed further studies on underlying targeting events, and on the biogenesis and development of the FV. I demonstrate that PfCRT targeting is dynamin-dependent and show the hitherto uncharacterised dynamic nature of the FV during intra-erythrocytic life stages. We are confronted with a limited knowledge about the functions and targeting events of membrane proteins to the FV. I have addressed these questions by applying bioinformatic approaches and thereby predicted 74 candidate FV membrane proteins. I have successfully expressed PfMDR1 and four candidate FV membrane proteins as epitope- or GFP-tagged proteins, as well as identifying a sixth likely FV membrane protein using a generated anti-peptide antibody. The subcellular localisation of four candidate proteins confirmed their FV association. Bioinformatic analysis of the putative functions of these revealed two transporter proteins, a pore-forming protein and a subunit of a targeting complex that is involved in retrograde transport from endosomes to the Golgi apparatus in other organisms. Further bioinformatic analysis of the remaining 70 candidate FV membrane proteins revealed eight putative transporter proteins involved in transport of ions, metabolites and amino acids in other organisms (two of which are localised to endosomes and lysosomes). To study the targeting processes of these known and novel FV membrane proteins, I investigated FV membrane trafficking routes using treatment of parasites with the fungal metabolite Brefeldin A, a compound inhibiting early secretory pathway processes. Finally, I have studied the contribution of the adaptin protein (AP) complexes to FV membrane protein targeting. The AP complexes mediate targeting of membrane proteins to endosomes and lysosomes in other organisms. The bioinformatic identification of four distinct AP complexes is described, as well as their subcellular localisation. I show that all four AP complexes are partially associated with the FV, and that AP-1 and AP-2 are strongly linked with the FV membrane. This suggests that AP complexes are involved in targeting of FV membrane proteins. Additionally, I have characterised the four P. falciparum AP complexes in regard to their sensitivity to Brefeldin A

Immunofluorescence microscopy of PfCRT-GFP over-expressing parasites treated with either Brefeldin A or Dynasore.

<p>PfCRT was over-expressed as a GFP-fusion protein using an ATet-inducible expression system and treated with either BFA (5 µg/mL) for 3 h or Dynasore (40 µM/mL) for 2 h. As a control a second parasite population was treated with an equivalent volume of carrier alone (ethanol and DMSO, respectively). Following the BFA treatment, immunofluorescence microscopy was performed on fixed cells with mouse anti-GFP, Alexa-594 goat anti-mouse IgG and DAPI. Representative parasites in trophozoite and schizont stage are shown for control and treated parasites. (<b>A</b>) The control parasites show the restricted FV localisation of PfCRT-GFP (false-coloured in green). (<b>B</b>) BFA treated parasites show an accumulation of fluorescence around the DAPI-stained nuclei, consistent with ER localisation in addition to the FV localisation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038781#pone-0038781-g001" target="_blank">Figure 1</a>). (<b>C</b>) Treatment of parasites with Dynasore resulted in an accumulation of fluorescence around the DAPI-stained nuclei in addition to the FV membrane localisation, similar to the observed effect of BFA treatment (<b>B</b>).</p

IC₅₀ of different <i>P. falciparum</i> transgenic lines and treatments after 48 hours chloroquine treatment.

<p>IC₅₀ of different <i>P. falciparum</i> transgenic lines and treatments after 48 hours chloroquine treatment.</p

Analysis of the <i>de novo</i> biogenesis and development of the FV in live cells using PfCRT-GFP as a marker of the FV membrane.

<p>PfCRT was over-expressed as a GFP-fusion protein using an ATc-inducible expression system. (<b>A</b>) Live DAPI-stained cells were imaged. The earliest detectable and distinct localisation of PfCRT-GFP was observed in mid ring stage parasites, co-localising with a round spherical shape in proximity to the DAPI-stained nucleus. The characteristic dark haemozoin crystal was not yet visible in these parasites. In addition to FV labelling, some fluorescence was detectable at the ER. PfCRT-GFP labelling of the FV membrane was observed throughout the whole intra-erythrocytic life cycle. In accordance with an increase of the haemozoin crystal, the ring-like labelling of the FV membrane expanded as the parasite grew. An unexpected second PfCRT-GFP-labelled sphere was observed in schizont stage parasites. In some parasites this additional structure was associated/attached to the FV membrane (lower schizont panel). (<b>B</b>) An overlay with the corresponding brightfield (BF) image shows that in some parasites the additional PfCRT-GFP enclosed compartment (white arrow) is separate from the FV and surrounds a dark structure, possibly haemozoin.</p

Over-expression of sensitive and resistant form of PfCRT as GFP-fusion protein.

<p>(<b>A</b>) Schematic representation of the inducible expression plasmid pT150KPfCRTGFP. Expression of the transcription activator TATi3 is under the control of the 5' UTR of PTEX150. In the presence of the transcription inhibitor anhydrotetracycline (ATc), TATi3-binding to the Tet operon (TetO) is inhibited and no expression of GFP-tagged PfCRT occurs. In the absence of ATc, TATi3 binds to TetO, initiating the over-expression of PfCRT-GFP. Cam5': 5' UTR of Calmodulin, hDHFR: human dihydrofolate reductase. (<b>B</b>) Western Blot analysis of induced over-expression of sensitive (PfCRT^S) and resistant (PfCRT^R) form of PfCRT as GFP-fusion. Presence of PfCRT^S and PfCRT^R as GFP-fusions, labelled with mouse anti-GFP, is confirmed by the presence of a band at 62 kDa only in the parasites cultured in the absence of ATc. <i>P. falciparum</i> wild-type strain 3D7 represents the negative control. Labelling with rabbit anti-GAPDH shows equal loading in these lanes. (<b>C</b>) Fluorescence microscopy of GFP-fusions of sensitive (PfCRT^S, top) and resistant (PfCRT^R, bottom) form of PfCRT. PfCRT^S and PfCRT^R were over-expressed as GFP-fusion proteins using an ATc-inducible expression system. Live cell images of DAPI-stained infected red blood cells show that both forms of PfCRT localise to the FV membrane. Later stages of the asexual life cycle show ring/dot-like structures (white arrow) within the FV, possibly degraded GFP-fusion proteins.</p