Comparison of growth behavior of YM and <i>PYΔpy01365.</i>

<p>A- Parasitaemia of BALB/c mice infected with 10⁴ parasites on day 0 was taken daily. The average parasitaemia of 5 mice for both YM and <i>PYΔpy01365</i> is represented. Error bars are given for each time point. <b>†</b> Indicates death of animals. B- Average Selective index of 5 BALB/c mice infected with either YM or <i>PYΔpy01365.</i> Parasites smears were analyzed when parasitaemia was in the range of 5–15%. Differences in SI between YM and <i>PYΔpy01365</i>were significant (p<0.01).</p

Transcription of <i>py235</i> in YM and <i>PYΔpy01365</i> parasites.

<p>Analysis of changes of the transcription pattern of different <i>py235</i> members by quantitative reverse transcription - real time-PCR. Analysis of transcription levels of 11 different <i>py235</i> members in YM (red) and <i>PYΔpy01365(NF1</i>) (blue) and <i>PYΔpy01365(NF2</i>) (green). Results are expressed as percent of total <i>py235</i> transcription. * indicates statistically significant differences in the transcription levels of a gene between YM and <i>PYΔpy01365</i> parasites (p<0.05).</p

Differences in expression of Py235 recognized by 25.77 in YM or <i>PYΔpy0136</i> parasites.

<p>A- Immunofluorescence Assays of YM and <i>PYΔpy01365(NF1</i>) and <i>PYΔpy01365(NF2</i>) with the protective monoclonal antibody 25.77 (Py235). Fewer schizonts in <i>PYΔpy01365</i> reacted specifically with the Py235 specific antibody (circled). The specific antibodies that reacted with the schizonts were detected with Alexa Fluor labeled anti-mouse IgG. The fluorescent images (individual stains and merged) and the bright-field are shown. B- Immunofluorescence Assays of YM and <i>PYΔpy01365</i> parasites with mcAb 25.77 (Py235) and a rabbit serum against the rhoptry protein MAEBL. The specific antibodies that reacted with the schizonts were detected with Alexa Fluor labeled goat anti rabbit (or anti-mouse) IgG. The fluorescent images (individual stains and merged) and the bright-field are shown. C- Quantification of the number of schizonts that are MAEBL and Py235 positive. YM and <i>PYΔpy01365</i> parasites were stained with mcAb 25.77 and a rabbit serum against the rhoptry protein MAEBL. A total of 200 MAEBL positive schizonts were counted and their mcAb 25.77 staining was determined. Comparison of double labeled parasites showed a significant difference between YM and <i>PYΔpy01365</i> parasites (p< 0.01).</p

Disruption of <i>py01365</i> using homologous recombination.

<p>A- genomic locus MALPY00360 coding for <i>py01365</i> showing the two regions (blue and red) used for targeting this locus by a double cross-over strategy. Homologous recombination with the linearized plasmid containing the selectable marker flanked by the targeting sequences results in the Py01365 KO locus. Restriction sites used for Southern blot analysis as well as the location of the primer pairs A-F, A-R and B-F, B-R important for PCR screening of both the 5′ and 3′ integration event as well as region used for Southern blot probe are also indicated. B- PCR screening of 5′ (A) and 3′ (B) integration events in both wild type (YM) and knock out (KO) parasites using primers A-F, A-R and B-F, B-R respectively. Both primer pairs are only expected to give a product if integration has occurred. C- Southern blot screening of parasites for correct integration. (1) BstBI digested DNA obtained from wild type (YM) as well as transfected parasites (K1, K2 and K3) and the transfection plasmid (Pl) was analyzed by Southern blot using a <i>PY01365</i> specific probe (region indicated in red). The expected fragment of ∼7.4 kb can be seen in all three transfected parasite lines. (2) Transfected clone K1 and K3 were subsequently cloned out by limiting dilution and again screened by Southern blot. Single parasite clone K1-C1 and K3-C2 were selected for further analysis, and were renamed <i>PYΔpy01365(NF1</i>)and <i>PYΔpy01365(NF2</i>), respectively.</p

Unique PCR primers used for Real Time-PCR.

<p>Unique PCR primers used for Real Time-PCR.</p

Erythrocyte binding assay of parasite culture supernatant from both YM and <i>PYΔpy01365</i>.

<p>Western blot analysis using mcAb 25.77 of equal amounts of parasite culture supernatant as well as proteins bound to erythrocytes from A) YM and <i>PYΔpy01365(NF2</i>) as well as B) YM and <i>PYΔpy01365(NF1</i>).</p

Infection of <i>ex vivo</i> cultured <i>P</i>. <i>falciparum</i> K1 strain in humanized mice.

<p>Humanized mice were infected with <i>ex vivo</i> cultured <i>P</i>. <i>falciparum</i> K1 ring stage parasites and the parasite PCR product (indicated by arrow) was determined using nested PCR at the indicated time points after parasite injection. 3 out of 4 mice (M1, M3 and M4) showed infected human RBCs until the 3^rd cycle. Genomic DNA prepared from blood of an uninfected mouse was used as negative (-ve) control.</p