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

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

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

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

Isolated merozoites maintain surface coat integrity.

<p>A) E64-treated schizonts were filtered to release free merozoites and haemozoin crystals, and the filtrate was passed over magnetic columns. Merozoite purification was confirmed by Giemsa-stained smears of i) filtrate, ii) retained haemozoin and iii) purified merozoites. B) Merozites were stained with EtBr and enumerated by flow cytometry. C) Washed Merozoites retained surface proteins MSP-3, MSP-6 and AMA-1 by western blot (S: purified Schizonts; M: filtered merozoites; SM: EtBr stained merozoites). D) Merozoite surface proteins are maintained during merozoite isolation and wash steps as shown by surface localisation of MSP-3 and MSP1₁₉ by immunofluorescence microscopy. Antigens were stained with Alexa-594 and the nucleus with DAPI (panels in order Alexa549; DAPI; brightfield; Alexa549/DAPI; merge).</p

Phagocytosis by THP-1 cells is antibody and Fc Receptor dependent.

<p>A) EtBr stained merozoites were incubated with i) non-immune plasma or ii) immune PNG plasma, and were added to THP-1 cells. THP-1 cells were gated by forward and side scatter, and EtBr fluorescence was determined by flow cytometry. A non-immune control sample was used to set the EtBr positive gate. B) THP-1 cells were stained with anti-CD14 antibody and treated with trypan blue (TB) buffer which quenched all surface FITC fluorescence (black:THP-1 cells only, white: CD14-FITC stained THP-1, grey: CD14-FITC stained THP-1 with TB buffer). C) FITC stained merozoites were opsonised with i) non-immune or ii) immune PNG plasma and added to THP-1 cells. FITC fluorescence was measured by flow cytometry before and after quenching, and phagocytosed merozoites were resistant to quenching (black:THP-1 cells only, white: fluorescence with FITC stained merozoites, grey: fluorescence with FITC stained merozoites after TB buffer). D) The % phagocytosis measured for EtBr stained merozoites was equivalent to FITC stained merozoites after quenching. Opsonised EtBr stained and FITC stained merozoites were added to THP-1 cells at 3∶1 and 10∶1 merozoite:THP-1 ratios. E) Phagocytosis is active and Fc Receptor dependent. THP-1 cells were treated with cytochalasin D (CytoD) or blocked with non-specific IgG prior to addition to immune plasma opsonised merozoites in the phagocytosis assay. Each point represents the mean ± standard error. <b>*</b>, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.005.</p

Merozoite opsonisation proceeds rapidly at low antibody concentrations.

<p>Merozoites were added to wells containing THP-1 cells simultaneously (white bars), or after 40 mins of preincubation (grey bars), with varying dilutions of an immune plasma pool. % phagocytosis was determined by flow cytometry.</p

Efficient merozoite phagocytosis at low antibody concentrations.

<p>A) Increasing numbers of EtBr labeled merozoites were incubated with a pool of immune plasma from PNG children and then added to THP-1 cells. The % phagocytosis was determined by flow cytometry. The ratio of 4∶1 was chosen for subsequent assays, and is indicated by the arrow above. B) Titration of a pool of immune plasma from PNG children for opsonising activity using the 4∶1 merozoite:THP-1 cell ratio. % phagocytosis was determined by flow cytometry. The chosen dilution, 1/30,000, is indicated by an arrow above.</p

The importance of polymorphisms in the C1-L region of 3D7 for vaccine escape. A.

<p>Plasmid design and integration. The C1-L of 3D7 and FVO AMA1 differ by 5 amino acid (aa) residues located at positions 196, 197, 200, 204 and 206. The hybrid 3F3 AMA1 (3D7 allele with the FVO C1-L sequence) was transfected into W2Mef parental parasites. The single-crossover event for allelic replacement of the wild type (WT) AMA1 with 3F3 is illustrated. <b>B.</b> Southern blot. Genomic DNA from parental W2Mef and transfected parasites was digested with restriction enzymes as indicated and hybridised with an AMA1 probe. Expected sizes for WT, non-integrated plasmid and integrated 3F3-AMA1 are shown in kilobases (kb). <b>C.</b> Phenotypic analysis of transgenic parasites expressing the 3F3-AMA1 hybrid. Transgenic W2Mef parasites expressing 3D7-AMA1 (W2-3D7), FVO-AMA1 (W2-FVO) or the hybrid 3F3-AMA1 (W2-3F3) were tested for their susceptibility to growth inhibition with the R1 peptide (final concentration 100 mg/ml) or the monoclonal antibody 1F9 (final concentration 0.2 mg/ml). <b>D.</b> Differential growth inhibition of transgenic parasite lines by polyclonal rabbit antibodies to AMA1; anti-W2Mef #1 and anti-FVO#2 rabbit sera were tested at a final dilution of 1∶10, all other antibodies listed were tested at a final concentration of 2 mg/ml IgG. Columns represent the mean parasite growth inhibition achieved in two separate assays tested in triplicate wells. <b>*</b> indicate a significant difference in inhibition when compared to the W2-3D7 reference line, P&lt;0.05 by t-test.</p

Phylogenetic analysis of AMA1 sequences.

<p><b>A.</b> Phylogenetic tree of the AMA1 alleles expressed by 18 different isolates examined in this study in relation to 250 other AMA1 alleles obtained from the public database. Analysis was based on the ectodomain sequence. <b>B.</b> Phylogenetic tree of the AMA1 alleles expressed by the 18 different isolates used in this study, based on the AMA1 ectodomain sequence (amino acids 25–456). The AMA1 sequences of HCS-E5 and CSL-2 were found to be identical. <b>C.</b> Phylogenetic tree of the AMA1 alleles expressed by isolates used in this study, based on the C1-L sequence of AMA1 (amino acids 196–207). Translated ectodomain and C1-L protein sequences were aligned and phylogenetic trees constructed using ClustalW2.</p