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

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

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

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

The Role of Serine-Type Serine Repeat Antigen in <i>Plasmodium yoelii</i> Blood Stage Development

<div><p></p><p>A key step for the survival of the malaria parasite is the release from and subsequent invasion of erythrocytes by the merozoite. Differences in the efficiency of these two linked processes have a direct impact on overall parasite burden in the host and thereby virulence. A number of parasite proteases have recently been shown to play important roles during both merozoite egress as well as merozoite invasion. The rodent malaria parasite <i>Plasmodium yoelii</i> has been extensively used to investigate the mechanisms of parasite virulence in vivo and a number of important proteins have been identified as being key contributors to pathology. Here we have utilized transcriptional comparisons to identify two protease-like SERAs as playing a potential role in virulence. We show that both SERAs are non-essential for blood stage development of the parasite though they provide a subtle but important growth advantage in vivo. In particular SERA2 appears to be an important factor in enabling the parasite to fully utilize the whole age repertoire of circulating erythrocytes. This work for the first time demonstrates the subtle contributions different protease-like SERAs make to provide the parasite with a maximal capacity to successfully maintain an infection in the host.</p></div

Growth competition between two different parasite lines.

<p>Parasitemia of Balb/C mice with 10⁴ parasites of each parasite line were mixed and injected by iv. on day 0 and recorded daily. The average parasitemia and percentage of gene expression calculated through real-time PCR of 4 mice in each group were presented. A. growth competition of YM and SERA1-ko; B. growth competition of YM and SERA2-ko; C. growth competition of SERA1-ko and SERA2-ko.</p

Western blot analysis using peptide antibody against SERA2.

<p>A. Presence of GFP in different parasite schizont extract (YM, SERA1-tag, SERA2-tag, SERA1-ko C10 and SERA2-ko C2 lines) was detected using primary chicken anti-GFP antibody and secondary <a href="mailto:488@goat" target="_blank">488@goat</a> anti-chicken.IgG (H+L); while presence of Hsp70 in the same membrane was detected by primary anti-Hsp70 and secondary <a href="mailto:549@goat" target="_blank">549@goat</a> anti-mouse lgG (H+L) as loading control at the lower panel. B. Presence of SERA2 in different parasite extract (YM: ring extract, trophozoite extract and schizont extract; SERA2-ko: schizont extract of C1 line and C2 line) was detected using primary peptide antibody 4209 and secondary <a href="mailto:649@goat" target="_blank">649@goat</a> anti-rabbit IgG (H+L); while presence of Hsp70 in the same membrane was detected by primary anti-Hsp70 and secondary <a href="mailto:488@goat" target="_blank">488@goat</a> anti-mouse lgG as loading control at the lower panel. C. Presence of SERA2 in different parasite schizont extract (YM, SERA2-ko C1 and C2 lines, and SERA1-ko C10 and C6 lines) was detected using primary peptide antibody 4209 and secondary <a href="mailto:649@goat" target="_blank">649@goat</a> anti-rabbit IgG (H+L); while presence of Hsp70 in the same membrane was detected by primary anti-Hsp70 and secondary <a href="mailto:549@goat" target="_blank">549@goat</a> anti-mouse lgG as loading control at the lower panel. D. Band intensities corresponding to SERA2 and Hsp70 in the western blot shown above (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060723#pone-0060723-g003" target="_blank">Fig 3C</a>) were measured, and the median intensities for bands corresponding to Hsp70 were used for normalization in individual sample to obtain the relative SERA2 expression, and then the relative SERA2 expression ratio was calculated with one of the YM sample as the reference.</p

Comparison of growth characteristics between wildtype YM and SERA knock-out lines.

<p>A. Parasitemia of Balb/C mice with 10³ parasites injected by iv. on day 0 and recorded daily. The average parasitemia of 5 mice for each group were presented.† denotes the death of animals; B. Survival of Balb/C mice infected with YM or KO lines. Note that there was one outlier mouse infected with SERA1-ko C6 survived beyond post-infection day9 but ultimately died two days later; C. Average selective index of 5 BALB/c mice infected with YM or KO lines. Parasite smears were analyzed when parasitaemia was in the range of 3–13%. Differences in SI between YM and SERA1-ko C6 and SERA2-ko C1 and C2 were significant (p<0.01), indicated by **.</p