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

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

dentification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined T1D+T2D GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 subjects with diabetes (18,582 with DKD). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, P = 4.5 x 10(-8)) associated with microalbuminuria in European T2D case subjects. However, no replication of this signal was observed in Asian subjects with T2D or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously reported DKD signals, except for those at UMOD and PRKAG2, both associated with estimated glomerular filtration rate. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk variant discovery for DKD.Peer reviewe

Tissue perfusion diagnostic classification using a spatio-temporal analysis of contrast ultrasound image sequences.

The analysis of tissue perfusion in myocardial contrast echocardiography (MCE) remains a qualitative process dependent on visual inspection by a clinician. Fully automatic techniques that can quantify tissue perfusion accurately has yet to be developed. In this paper, a novel spatio-temporal technique is described for segmenting the myocardium into differently perfused regions and obtaining quantitative perfusion indices, representing myocardial blood flow and blood flow reserve. Using these indices, Myocardial segments in 22 patients were classed as either healthy or diseased, and results compared to coronary angiogram analysis and an experienced clinician. The results show that the automatic method works as well as a human at detecting areas of ischaemia, but in addition localizes the spatial extent of each perfused region as well. To our knowledge this is the first reported spatio-temporal method developed and evaluated for MCE assessment

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