<i>UIS3</i> and <i>UIS4</i> gene expression is dramatically reduced in <i>SLARP</i>-deficient parasites.

<p>(A) Quantitative RT-PCR analysis of gene expression in infected HepG2 cell cultures, 6 hours post-infection with WT or <i>slarp</i>(-) sporozoites. Relative gene expression was normalized to the <i>GFP</i> expression level, and is expressed as the log2 of the ratio <i>slarp</i>(-)/WT (mean of three independent experiments+/−SD). (B) Air-dried WT and <i>slarp</i>(-) salivary gland sporozoites were stained with anti-UIS4 antibodies (red) before examination by confocal fluorescence microscopy, using the same settings as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000086#ppat-1000086-g005" target="_blank">Fig. 5A</a>. Bar = 5 µm. (C) Quantitative RT-PCR analysis of gene expression in <i>slarp</i>(-) salivary gland sporozoites. Relative gene expression was normalized to the <i>GFP</i> expression level, and is expressed as the log2 of the ratio <i>slarp</i>(-)/WT (mean of three independent experiments+/−SD).</p

Targeted gene disruption of <i>P. berghei SLARP</i>.

<p>(A) Replacement strategy to generate the <i>slarp(-)</i> parasites. The wild-type (WT) <i>PbSLARP</i> genomic locus was targeted with a <i>Sac</i>II/<i>Kpn</i>I-linearized replacement plasmid containing 5’ and 3’ regions of the <i>SLARP</i> open reading frame (ORF) and the <i>Toxoplasma gondii dhfr/ts</i>-positive selectable marker. Upon a double crossover event, the <i>SLARP</i> gene is replaced by the selectable marker. Replacement- and wild type- specific test primer combinations and expected fragments (test1; test2; wt) are indicated. (B) Replacement-specific PCR analysis. Confirmation of the predicted gene targeting is achieved by specific primer combinations (test 1 and test 2), which can only amplify a signal from the recombinant locus. A wild type-specific PCR reaction confirms the absence of residual wild-type parasites in the clonal <i>slarp(-)</i> populations. (C) Depletion of <i>SLARP</i> transcripts in <i>slarp(-)</i> parasites. cDNA from wild-type and <i>slarp(-)</i> salivary gland sporozoites was amplified by PCR. Note the absence of a <i>SLARP</i>–specific signal in <i>slarp</i>(-) clones compared to a control transcript (<i>TRAP</i>), confirming the successful depletion of <i>SLARP</i>.</p

Alignment of <i>P. falciparum</i> LplA1 and <i>P. berghei</i> LplA1.

<p>The deduced amino acid sequences of PF13_0083 and PB 000283.02.0 were aligned using ClustalW. The identity between the two sequences was determined to be 70.3% while the similarity is 78%. The predicted sizes of the two proteins are almost identical with 47.94 kDa for PfLplA1 and 47.97 kDa for <i>Pb</i>LplA1. Identical residues are labelled in yellow; homologous residues are labelled in green. Consensus: gives the consensus amino acid sequence of both proteins.</p

<i>PbLplA1</i> is essential for propagation of asexual stages.

<p><i>A.</i> Insertion strategy to generate <i>LplA1</i> null mutants. The <i>PbLplA1</i> genomic locus (I.) is targeted with a <i>Hpa</i>I-linearized targeting vector (b3D-LplA1-int1, II.) containing 5′ and 3′ truncations of the <i>LplA1</i> open reading frame and the <i>T. gondii DHFR/TS</i> positive selectable marker. Upon a single cross-over event, the region of homology is duplicated, resulting in two truncated, non-expressed <i>LplA1</i> copies in the recombinant locus (III.). Wild-type, plasmid, and integration-specific primer combinations are indicated by arrows and sizes of expected fragments are shown. <i>B.</i> Genotyping indicates absence of successful integration. While episomal plasmid (primer pair b3D-1 and b3D-2 amplifying a 1.63 kb pair; lane 3) and the endogenous <i>LplA1</i> gene (primer pair Pb-10 and Pb-11 amplifying a 2.48 kb fragment; lane 4) are amplified, no integration-specific bands (lane 1 and 2: 3′- and 5′-specific integrations, respectively) using the primer pairs b3D-1 and Pb-11 to amplify a 2.38 kb 3′-fragment and Pb-10 and b3D-2 to amplify a 2.45 kb 5′-fragment were obtained from each of 3 transfected parasite populations.</p

<i>PbLplA1</i> is susceptible to gene targeting.

<p><i>A.</i> Control integration strategy to recover one functional <i>LplA1</i> gene copy. The targeting vector (b3D-LplA1-int2, II.) contains the endogenous stop codon and 3′ untranslated regions of the endogenous <i>PbLplA1</i> but lacks the promoter and the start codon. Upon a single cross-over event, one functional <i>LplA1</i> copy that is driven by the endogenous promoter and a 5′ truncated copy of the gene are generated (III.). Wild-type, plasmid, and integration-specific primer combinations are indicated by arrows and sizes of expected fragments are shown. <i>B.</i> Genotyping indicates successful integration of the control targeting construct. Both integration-specific PCRs (lane 1, 3′ specific integration using primer pair b3D-1 and Pb-11 and lane 2, 5′ specific integration using primer pair Pb-10 and b3D-2) amplified fragments of the expected sizes and their authenticity was verified by nucleotide sequencing. The population still contains episomal plasmid (lane 3; primer pair b3D-1 and b3D-2 amplifying a 2.35 kb band which was subcloned and sequenced) and residual wild-type parasites (lane 4; primer pair Pb-10 and Pb11 fragment was amplified and subcloned and sequenced).</p

The P. falciparum LplA1 locus cannot be disrupted.

<p><i>A</i>. Schematic representation of endogenous <i>PfLplA1</i> gene locus (I.), transfection plasmid pHH1-<i>LplA</i>-KO (II.) and <i>PfLplA1</i> gene locus after single cross-over recombination (III.). The restriction enzyme used for diagnostic digest is shown (<i>Nde</i>I) and the expected sizes of diagnostic bands after hybridization with <i>PfLplA1</i> or <i>hDHFR</i> (human dihydrofolate reductase) probes are indicated. <i>B</i>. Southern blot analyses of <i>P. falciparum</i> transfected with pHH1-<i>LplA1</i>-KO. Genomic DNA of wild-type D10 (lane 1) and <i>PfLplA1</i>-KO after 3 selection cycles with WR99210 (lane 2) was digested with <i>Nde</i>I and the Southern blot was probed with the <i>P. falciparum LplA1</i> ORF. The 1.9 kb band that corresponds to endogenous <i>PfLplA1</i> is visible in both lanes, whereas the transfection plasmid pHH1-<i>LplA1</i>-KO (6 kb band) only is recognised by the probe in the transfectants. No other bands are visible that are diagnostic for the integration of the <i>PfLplA1</i> gene locus.</p

Remodelling of the PVM is impaired in <i>SLARP</i>-deficient parasites.

<p>(A) HepG2 cells were infected with WT or <i>slarp</i>(-) sporozoites in the presence of FITC-dextran and cultured for 6 hours before staining with anti-CSP (green), anti-UIS4 (red) and filipin (blue), followed by analysis by confocal fluorescence microscopy. Bar = 5 µm. Note that sporozoites inside dextran-positive cells were not stained by anti-UIS4 or filipin. (B) The percentage of UIS4 and filipin stained sporozoites was determined by examination of at least 50 dextran-negative infected cells for each population. (C) HepG2 cells were infected with WT or <i>slarp</i>(-) sporozoites and cultured for 48 hours before staining with anti-HSP70 (green) and anti-EXP1 (red) antibodies and analysis by confocal fluorescence microscopy. Bar = 10 µm. (D) The percentage of HSP70 and EXP1 labelled parasites was determined by examination of at least 50 GFP-positive 48h LS for each population.</p

Genotypic analysis of P. falciparum transfected with the complementation construct pHH1-LplA1-KOkon.

<p><i>A</i>. Schematic representation of endogenous <i>PfLplA1</i> gene locus (I.), transfection plasmid pHH1-<i>LplA1</i>-KOkon (II.) and <i>PfLplA1</i> gene locus after single cross-over recombination (III.). The restriction enzyme used for diagnostic digest is shown (<i>Nde</i>I) and the expected sizes of diagnostic bands after hybridization with <i>PfLplA1</i> or <i>hDHFR</i> are indicated. <i>B</i>. Southern blot analyses of transfected parasite lines. Genomic DNA of wild-type and <i>LplA1</i>-KOkon parasites was digested with <i>Nde</i>I and probed with the <i>PfLplA1</i> ORF (left panel). In all parasite lines analysed, the endogenous gene is present (1.9 kb band) and in lanes 3 and 4 two additional DNA fragments are detected by the probe (in lane 2 less DNA was loaded on the gel so that the plasmid band is hardly visible). The faint 6 kb band is diagnostic for the presence of the transfection plasmid (see scheme), but the band of ∼9 kb (*) cannot be assigned to any specific integration event. Lane 1, wild-type D10; lane 2, <i>PfLplA</i>-KOkon, cycle 1; lane 3, <i>PfLplA</i>-KOkon, cycle 2; lane 4, <i>PfLplA</i>-KOkon, cycle 3. The same blot was stripped and re-probed with the probe specifically detecting the selectable marker <i>hDHFR</i> and apart from faint plasmid bands at 6 kb in lanes 2, 3 and 4 two additional bands of ∼9 kb (*) and 0.5 kb (*) are detected. Lane 1, wild-type D10; lane 2, <i>PfLplA</i>-KOkon, cycle 1; lane 3, <i>PfLplA</i>-KOkon, cycle 2; lane 4, <i>PfLplA</i>-KOkon, cycle 3. <i>C.</i> Genotyping by pulsed field gel electrophoreses. Chromosomes of wild-type <i>P. falciparum</i> D10 (lane 1) and parasites transfected with the pHH1-<i>LplA1</i>-KOkon construct in cycle 0 (lane 2) and cycle 3 (lane 3) were analysed by PFGE and the blots were probed with the <i>PfLplA1</i> open reading frame (left panel) or with the <i>hDHFR</i> open reading frame (right panel) present in the transfection plasmid. The <i>PfLplA1</i> probe detected the endogenous <i>PfLplA1</i> gene locus on chromosome 13 in wild-type and transfected parasites. However, the probe also detected a strong signal at the bottom of the blot where the non-separated chromosomes <10 are running. The <i>hDHFR</i> probe similarly generated signals on chromosome <10, but there is no sign of integration into the <i>LplA1</i> gene locus on chromosome 13.</p

Analysis of <i>P. falciparum</i> harbouring pHrBI-<i>PbLplA1</i>.

<p><i>A</i>. Southern blot of <i>P. falciparum</i> transfected with pHrBI-<i>PbLplA1</i> before transfection (lane 1) and after transfection (lanes 2 and 3). The DNA was digested with <i>Nde</i>I and the blot was probed with the <i>PfLplA1</i> ORF. The DNA fragment recognised by the probe corresponds to the endogenous <i>PfLplA1</i> gene. The <i>PfLplA1</i> probe did not cross-react with the transfected plasmid pHrBI-<i>PbLplA1</i>. <i>B</i>. To verify the presence of the transfected plasmid, the blot was re-probed with a probe specifically recognising the blasticidin S deaminase ORF present on the plasmid as selectable marker. The probe hybridised with a DNA fragment diagnostic for the transfected plasmid (∼9.0 kb). <i>C</i>. Western blot of <i>P. falciparum</i> expressing PbLplA1. Lane 1, non-transfected parasites; Lane 2, <i>P. falciparum</i> transfected with pHrBI-<i>PbLplA1</i>. Equal loading was verified by re-probing the blot with a polyclonal antibody directed against the 1-Cys peroxiredoxin of <i>P. falciparum</i>. The polyclonal antiserum raised against <i>Pf</i>LplA1 recognised a protein of ∼45 kDa in the wild-type parasites which corresponds roughly to the expected size of <i>Pf</i>LplA1 (predicted size 47.9 kDa). In the transfected parasite line carrying the pHrBI-<i>PbLplA1</i> plasmid two proteins of very similar size were detected by the antibody – presumably one corresponds to the endogenous <i>Pf</i>LplA1 while the second band corresponds to <i>P. berghei</i> LplA1. This is surprising as the predicted sizes of both proteins are virtually identical. However, it is possible that the cleavage sites of the mitochondrial targeting peptides are distinct generating proteins that are just distinguishable by SDS-PAGE. <i>D</i>. Western blot of <i>P. berghei</i> lysate (lane 1: 1×10⁶ parasites and lane 2: 0.2×10⁷ parasites) using <i>P. falciparum</i> rabbit anti-LplA1 polyclonal antibody. A band of ∼45 kDa (similar size to the protein identified in the double transfected parasites as well as the wild-type parasites only transfected with pHrBI-<i>PbLplA1</i>) reacts strongly with the heterologous antiserum.</p

<i>SLARP</i> is conserved in <i>Plasmodium</i> species.

<p>(A) Genomic organization of representative <i>Plasmodium SLARP</i> genes. Shown are the <i>SLARP</i> gene loci for <i>P. falciparum</i> (Pf<i>SLARP</i>/PF11_0480), <i>P. vivax</i> (Pv<i>SLARP</i>/Pv092945), <i>P. knowlesi</i> (Pk<i>SLARP</i>/PKH_094440), <i>P. yoelii</i> (Py<i>SLARP</i>/PY03269, PY03923, Genbank accession no. EU579525) and <i>P. berghei</i> (Pb<i>SLARP</i>/PB000542.00.0, PB000547.01.0, Genbank accession no. EU579524). Sequence identity of the corresponding gene products are displayed on the right in comparison to <i>Pf</i>SLARP. (B) Alignment of the C-terminal end of <i>Plasmodium</i> SLARP proteins. Conserved residues are shaded in grey. The boxes indicate potential nuclear localization sequences (NLS) identified with PSORT II (<a href="http://psort.nibb.ac.jp" target="_blank">http://psort.nibb.ac.jp</a>).</p