Changes in Parasite Virulence Induced by the Disruption of a Single Member of the 235 kDa Rhoptry Protein Multigene Family of Plasmodium yoelii

Invasion of the erythrocyte by the merozoites of the malaria parasite is a complex process involving a range of receptor-ligand interactions. Two protein families termed Erythrocyte Binding Like (EBL) proteins and Reticulocyte Binding Protein Homologues (RH) play an important role in host cell recognition by the merozoite. In the rodent malaria parasite, Plasmodium yoelii, the 235 kDa rhoptry proteins (Py235) are coded for by a multigene family and are members of the RH. In P. yoelii Py235 as well as a single member of EBL have been shown to be key mediators of virulence enabling the parasite to invade a wider range of host erythrocytes. One member of Py235, PY01365 is most abundantly transcribed in parasite populations and the protein specifically binds to erythrocytes and is recognized by the protective monoclonal antibody 25.77, suggesting a key role of this particular member in virulence. Recent studies have indicated that overall levels of Py235 expression are essential for parasite virulence. Here we show that disruption of PY01365 in the virulent YM line directly impacts parasite virulence. Furthermore the disruption of PY01365 leads to a reduction in the number of schizonts that express members of Py235 that react specifically with the mcAb 25.77. Erythrocyte binding assays show reduced binding of Py235 to red blood cells in the PY01365 knockout parasite as compared to YM. While our results identify PY01365 as a mediator of parasite virulence, they also confirm that other members of Py235 are able to substitute for PY01365

Role of a specific member of 235 kDa multigene family (Py235) of the rodent malaria parasite Plasmodium yoelii in parasite mediated virulence

The mechanism by which the invasive form of the malaria parasites, the merozoite, selects and successfully invades an erythrocyte involves numerous receptor-ligand interactions. Recognition of specific erythrocyte receptor by the merozoite is mediated by at least two gene families: the Reticulocyte binding protein Homologues (RHs) and the Erythrocyte Binding Ligands (EBLs). In the rodent malaria parasite Plasmodium yoelii, members of the RHs termed Py235 (Plasmodium yoelii 235 kDa rhoptry protein) are implicated in host cell selection and invasion. In P. yoelii, Py235 as well as a single member of EBL have been shown to be key mediators of virulence enabling the parasite to invade a wider range of host erythrocytes. Previous studies using both quantitative RT-PCR and mass spectrometry have identified Py01365 as the dominant Py235 member expressed in the virulent P. yoelii YM strain. Based on these findings, py01365 was disrupted and its overall impact on parasite virulence and Py235 expression levels was investigated. We found that disruption of py01365 (PYΔpy01365) resulted in significantly lower parasitaemias, increased host cell selectivity and longer survival of the host, identifying this member of Py235 as a key mediator of virulence in the virulent P. yoelii YM strain. Moreover, the analysis of the impact of the disruption of py01365 on the transcription levels of the other py235 members showed no significant change in the overall py235 transcription pattern in PYΔpy01365 as compared to YM. We observed that the protective mcAb 25.77, which had recently been shown to recognize Py01365, reacted specifically with a large number of schizonts in the PYΔpy01365 by Immunofluorescence microscopy, suggesting that mcAb 25.77 was still able to recognize other members of Py235.Master of Scienc

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