Insect stage-specific conditional deletion of the <i>pbsub1</i> gene blocks the transition from salivary gland sporozoite to subsequent asexual blood stages.

<p>(A) Double crossover homologous recombination strategy to simultaneously flirt and epitope-tag the <i>pbsub1</i> gene, whilst also introducing a GFP reporter for successful gene excision. A ∼10 kb genomic DNA library clone containing the <i>pbsub1</i> gene was modified by recombineering and Gateway technology to place an <i>FRT</i> site ∼2.3 kb or ∼1.8 kb upstream of the <i>pbsub1</i> gene and directly downstream of an inserted <i>P. berghei hsp70</i> promoter. A second downstream <i>FRT</i> site was inserted in frame with a GFP reporter coding sequence so that, upon excision, the <i>hsp70</i> promoter drives expression of GFP. The final transfection constructs (pJazz-FRTed-pbsub1 and pJazz-FRTed-pbsub1_short), which also contained a hDHFR-yFCU positive-negative selectable marker cassette, were transfected into the <i>P. berghei</i> UIS4/FlpL clone <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003811#ppat.1003811-Combe1" target="_blank">[30]</a> and integrant parasite clones obtained, called condSUB1 (clones A and B) and condSUB1_short. (B) Mosquitos fed on mice infected with condSUB1 clone A parasites were subjected to a temperature shift 18 days post transmission to ensure optimal activity of the FlpL recombinase. Oocysts, salivary glands and sporozoites from these insects displayed strong GFP expression at 26 days, when the insects were allowed to feed on naive mice (bite-back). Resulting blood stage parasites were collected. (C) PCR analysis of blood stage condSUB1 parasites from mouse 1 (M1) and mouse 2 (M2), as well as sporozoites isolated 18 or 26 days following transmission. The primers used (F_out_hsp70 and R_out_GFP; red arrows in panel A, see Table S1 for sequences) produce a 5.4 kb amplicon from the non-excised modified <i>pbsub1</i> locus, or a 600 bp product from the excised locus (panel A). The results indicate that, whereas excision occurred highly efficiently in the 26 day sporozoites to the degree that the non-excised locus was undetectable by PCR, only residual non-excised parasites were capable of establishing a blood stage infection in the bite-back mice. Note that since smaller PCR products are generally produced more efficiently than large amplicons, the PCR result likely exaggerates the degree of excision in the 26 day sporozoites. Microscopic examination of dissected d26 condSUB1 clone A sporozoites showed that the proportion of GFP-positive sporozoites in these experiments was usually ∼90% (data not shown).</p

Excised condSUB1 sporozoites display no defect in hepatocyte invasion or capacity to establish a liver stage infection.

<p>(A) Deletion of the <i>pbsub1</i> gene does not affect the invasive capacity of sporozoites <i>in vitro</i>. HepG2 cell monolayers were infected with equal numbers of condSUB1 (∼85% excised) or control UIS4/FlpL-F sporozoites. 2 h post infection (2 hpi), cell monolayers were washed, fixed, and processed for in-out staining with anti-CSP and anti-GFP antibodies and the nuclear stain Hoechst 33342. Quantification of intracellular parasites was performed by Cellomics automated microscopy and image analysis software. Results are expressed as the mean ± SD (3 experiments, each using n = 3–7 replicate wells) of the number of infected cells as a proportion of those infected with control parasites. There is no significant difference between the means (Student's <i>t</i>-test). (B) Deletion of <i>pbsub1</i> does not reduce levels of liver infection <i>in vivo</i>. Mice were infected by intravenous inoculation of 20,000 condSUB1 (∼85% excised) or control sporozoites. After 40 h, total RNA was prepared from whole livers and levels of 18S ribosomal parasite RNA and mouse hypoxanthine guanine phosphoribosyltransferase (HPRT) mRNA quantified by qRT-PCR. Relative amounts of RNA were calculated using the ABI Prism 7000 SDS 1.2.3 Software, and normalised against the expression levels of the mouse housekeeping gene HPRT. Data are expressed as the mean ± SD (3 experiments each using n = 3–5 mice). There is no significant difference between the means (Student's <i>t</i>-test).</p

Merozoite formation, PV rupture and the expression profile of MSP1 is abnormal in maturing PbSUB1-deficient liver stage schizonts.

<p>Comparative IFA of EEFs obtained following infection of hepatoma cells with either control UIS4/FlpL-F sporozoites or excised condSUB1 (<i>pbsub1</i> KO) clone A sporozoites. Antibodies or dyes used to probe the samples are indicated on the left, whilst typical representative morphologies obtained are shown in the associated microscopic images. Each bar on the histograms refers to a total of 40–60 randomly-selected images which were acquired at each of 5 time points (52, 56, 60, 64 or 68 h) post infection. The classification of these images based on morphology is indicated by the height of the coloured sections of each bar. The colour code for each different morphology is indicated alongside the sets of histograms.</p

PbSUB1 is expressed in exoneme-like subcellular organelles in mature liver stage schizonts.

<p>HepG2 cells infected <i>in vitro</i> with sporozoites of the PbSUB1-HA clone were fixed and examined by IFA, probing with an anti-HA mAb, or anti-GFP antibodies, or antibodies against the PVM protein EXP1. Nuclei were counterstained with 4,6-diamidino-2-phenylindol (DAPI, blue). No HA-specific signal was detected in early schizonts (top row) in which formation of individual merozoites is not yet visible. The images on the second row are shown at higher magnification below in order to better visualize the relative localization of the PbSUB1-specific signal relative to the parasite nuclei and cytosol. Scale bar, 10 µm.</p

Expression of epitope-tagged PbSUB1 in liver stage schizonts of condSUB1 clone A, and detection of a developmental defect associated with <i>pbsub1</i> excision.

<p>HepG2 cells infected <i>in vitro</i> with sporozoites of the indicated clones were fixed at 64 h post infection and probed with anti-GFP (green) or anti-HA (red) antibodies. DAPI-stained nucleic acids are shown in blue, whilst differential interference contrast (DIC) was used to obtain bright field images of the infected cells. The non-excised condSUB1 liver stage schizonts displayed the expected punctate HA signal, indicating the expected epitope tagging of PbSUB1. No HA signal was associated with the excised condSUB1 EEFs; however, mature schizont stages displaying merozoite production could not be found, suggesting a defect in liver stage development. Scale bar, 10 µm.</p

Onset of MSP1 expression is ablated in PbSUB1-deficient EEFs.

<p>HepG2 cells infected with sporozoites of the indicated clones were fixed 48–56 h post infection and probed with antibodies against MSP1 (red), EXP1 (pink), or GFP (green). DAPI-stained nucleic acids are shown in white or blue. MSP1 expression was undetectable in the excised (GFP-expressing) condSUB1 EEFs. Scale bar, 10 µm.</p

PbSUB1-deficient liver stage schizonts are defective in development.

<p>IFA analysis of hepatoma cells 60–64 h post infection with excised or non-excised condSUB1 clone A sporozoites, or control UIS4/FlpL-F sporozoites. Antibodies to the PV protein PbSERA3 (red), GFP (green), the plasma membrane marker MSP1 (red), or the PVM marker EXP1 (pink) were used to probe the samples. Nucleic acids were stained with DAPI (white or blue). Both merozoite production and PVM rupture were defective in the excised (PbSUB1-deficient) condSUB1 EEFs. Identical results were obtained for the condSUB1 clone B parasites (not shown). Note that the non-excised condSUB1 parasites do not express GFP. Scale bar, 10 µm.</p

Effect of GC content on uniformity of coverage.

<p>(a) Genome browser view showing the coverage obtained across a region from <i>P. berghei</i> for both methods. While near constant for ReBuilT, distinct read pile-ups in PCR-BS that appear to track GC content. (b) The GC content was calculated in 300 bp windows and plotted against the normalized informative read count. PCR amplification induces a strong preference for more balanced base compositions. (c) In <i>P. berghei</i> the GC content has a distinct profile across exons (dashed line). ReBuilT coverage is unaffected across this genomic feature, while PCR-BS tracks the GC percentage closely.</p

Schematic describing key differences between the ReBuilT and PCR-BS protocols.

<p>The possible fragments generated by cleavage (indicated with red stars) during bisulfite treatment are illustrated, and annotated to indicate if they remain sequenceable. <b>Left track:</b> adapter ligation precedes bisulfite treatment, after which the few surviving fragments are amplified by PCR. <b>Right track:</b> a single-stranded 3′ selective adapter ligation precedes bisulfite treatment. A primer extension generates dsDNA, which is immobilized on magnetic beads. A second ligation is performed before the non-uracil containing strand is denatured ready for sequencing.</p

Cytosine methylation in <i>P. berghei</i>.

<p>(a) The log2 fold change of 5mC contexts from cytosine contexts within the <i>P. berghei</i> genome. Each bar is annotated with the percentage of 5mC loci within the context. The sequence context varies greatly between PCR-free and amplified samples. ReBuilT data reveals an enrichment in the asymmetric CAH context, and CHG and CC contexts are strongly disfavoured. PCR-BS methylation occurs more often in CG and CHG contexts (H = A, T, G). (b) The distribution of methylation across genomic regions, shown as log2 fold change from the distribution of cytosines. The percentage of 5mC loci in the regions is given above each bar. Methylation in the AT-rich intergenic regions is underrepresented in the PCR-BS dataset. (c) The profile of 5mC levels over exons. Traditional PCR-BS gives a similar profile to ReBuilT, but greatly over-estimates the 5mC levels.</p