Phenotypic Dissection of a <i>Plasmodium</i>-Refractory Strain of Malaria Vector <i>Anopheles stephensi</i>: The Reduced Susceptibility to <i>P. berghei</i> and <i>P. yoelii</i>

<div><p>Anopheline mosquitoes are the major vectors of human malaria. Parasite-mosquito interactions are a critical aspect of disease transmission and a potential target for malaria control. Current investigations into parasite-mosquito interactions frequently assume that genetically resistant and susceptible mosquitoes exist in nature. Therefore, comparisons between the <i>Plasmodium</i> susceptibility profiles of different mosquito species may contribute to a better understanding of vectorial capacity. <i>Anopheles stephensi</i> is an important malaria vector in central and southern Asia and is widely used as a laboratory model of parasite transmission due to its high susceptibility to <i>Plasmodium</i> infection. In the present study, we identified a rodent malaria-refractory strain of <i>A. stephensi mysorensis</i> (Ehime) by comparative study of infection susceptibility. A very low number of oocysts develop in Ehime mosquitoes infected with <i>P. berghei</i> and <i>P. yoelii</i>, as determined by evaluation of developed oocysts on the basal lamina. A stage-specific study revealed that this reduced susceptibility was due to the impaired formation of ookinetes of both <i>Plasmodium</i> species in the midgut lumen and incomplete crossing of the midgut epithelium. There were no apparent abnormalities in the exflagellation of male parasites in the ingested blood or the maturation of oocysts after the rounding up of the ookinetes. Overall, these results suggest that invasive-stage parasites are eliminated in both the midgut lumen and epithelium in Ehime mosquitoes by strain-specific factors that remain unknown. The refractory strain newly identified in this report would be an excellent study system for investigations into novel parasite-mosquito interactions in the mosquito midgut.</p></div

Recombinant EBA-175 and RH5 antigens are stable, pure and expressed in the correct conformation.

<p>The non-reduced (Lane 1 of panel <b>A</b>) and the reduced elution (Lane 2 of panel <b>A</b>; both visualized by Coomassie staining) of region II of EBA-175 synthesized using the yeast expression system <i>Pichia pastoris</i>, and the binding of this recombinant to normal erythrocytes (panel <b>B</b>), confirm correct expression and conformation of the EBA-175_RII antigen with the expected product of ∼80 kDa (indicated by the arrows in Lane 2 of panel <b>A</b> and panel <b>B</b>). The elution of full length RH5 synthesized using the wheat-germ synthesis (panel <b>C</b>, indicated by the arrow), and binding of rRH5 (panel <b>D</b>) to normal erythrocytes indicates functional conformity of this recombinant antigen, as shown by the presence of a single product at the expected size of ∼63 kDa (indicated by arrows in both <b>C</b> and <b>D</b>).</p

Independent Antibody Interaction and Co-Operative Antibody Interaction Models.

<p><b><i>Independent Antibody Interaction Model</i></b> (Panel <b>A</b>): If the ligand-receptor interactions are independent of each other, invasion by either EBA-175 (Route 1) or by RH5 (Route 2) is not affected by the other. Thus, in the presence of both anti-EBA-175_RII and anti-rRH5 antibodies, the expected inhibition from the combination anti-sera is ADDITIVE (synergistic) when compared to the inhibition from the individual anti-sera. <b><i>Co-Operative Antibody Interaction Model</i></b> (Panel <b>B</b>): If the ligand-receptors act in a co-operative method, then invasion by EBA-175 (Route 1) and RH5 (Route 2) are not independent of each other. Thus, in the presence of anti-EBA-175_RII and anti-rRH5 antibodies, the expected inhibition from the combination anti-sera is only as effective as the most active individual antibody. <b><i>Antibody Steric Hindrance</i></b> (Panels <b>C</b> and <b>D</b>): Data suggests that EBA-175 abundance is greater than RH5 and is possibly released before RH5 (Ord et al, unpublished observations). At LOW antibody concentrations (<b>C</b>), there is no possible hindrance of RH5 by EBA-175, and all available antibodies are able to bind to their respective ligands independently. This is observed as growth inhibition with the combination anti-sera being more effective than that observed with the individual antibodies, i.e., it follows the Independent Antibody Interaction Model (<b>A</b>). Conversely, at HIGH antibody concentrations (<b>D</b>), anti-EBA-175 antibodies are able to bind to available EBA-175 ligands but they sterically hinder some RH5 antibody/ligand interactions, leaving some RH5 ligands available for invasion through the RH5 ligand/receptor pathways. This is observed as growth inhibition with the combination anti-sera being only as effective as anti-EBA-175_RII sera alone, i.e. it follows the Co-Operative Antibody Interaction Model (<b>B</b>).</p

Anti-sera against the hybrid vaccine show synergistic effects at low concentrations.

<p>The invasion inhibition of both combination sera are greater than those obtained from the individual sera at the lowest concentrations used, 1 µg/mL. The combinations contain 50% of each individual immunogen (in the case of the combination vaccination, anti-EBA-175_RII/rRH5) or sera (in the case of the in-tube combination, anti-EBA-175_RII+anti-rRH5). However, by 100 µg/mL, the synergistic effects of the combinations are no longer apparent, and the inhibition from the combinations is equivalent to ∼50% contribution from the two individual sera (anti-EBA-175_RII shown by black bars, anti-rRH5 shown by blue bars, anti-EBA-175_RII/rRH5 shown by red bars, anti-EBA-175_RII+anti-rRH5 shown by green, control IgG shown by white bars).</p

Dd2 parasites are not wholly dependent on the EBA-175/GPA pathway of invasion.

<p>In the presence of anti-EBA-175_RII antibodies (<b>A</b>; black bars), inhibition when using the Dd2 strain is only 60% (Ab concentration of100 µg/mL), compared to 55% inhibition from 3D7, suggesting alternative SA dependent pathways are utilized by Dd2, such as EBL-1/GPB or EBA-140/GPC (for <b>A</b>, <b>B</b> and <b>C</b>, Dd2 with its control IgG shown as solid white bars). Although anti-rRH5 antibodies (<b>B</b>; blue bars), should only block a SA independent pathway, there is still greater inhibition with Dd2 compared to 3D7. In the presence of both antibodies (<b>C</b>; anti-EBA-175_RII/rRH5 shown as red bars and anti-EBA-175_RII+anti-rRH5 shown as green bars, respectively), there is a significant difference in the ability of Dd2 to invade compared to 3D7, especially when individual antibodies are combined.</p

Invasion Inhibition of 3D7 with anti-EBA-175_RII and anti-rRH5 antibodies.

<p>Anti-EBA-175_RII (solid black line in panel <b>A</b>) and anti-rRH5 antibodies (solid blue line in panel <b>B</b>) inhibit invasion of 3D7 in a linear correlation to a similar extent. The two varying combinations used, anti-EBA-175_RII/anti-rRH5 and anti-EBA-175_RII+anti-rRH5 (solid red and solid green lines, respectively, in <b>C</b>), also showed the same positive correlation between increased antibody concentration and % inhibition (the color key for each antibody is conserved from <b>A</b>, <b>B</b>, and <b>C</b>). Percentage invasion inhibition from purified mouse IgG used as a control is shown as dashed black line (<b>A</b>, <b>B</b>, <b>C</b>).</p

Enzymatic (neuraminidase) treatment of erythrocytes eliminates the inhibitory effects of anti-EBA-175_RII antibodies but has only a mild effect on anti-rRH5 sera.

<p>Invasion inhibition assays with 3D7 and neuraminidase-treated cells (100 µg/mL antibody used) show that any inhibition due to the presence of anti-EBA-175_RII antibodies is masked by the removal of ligands with sialic acid compared to untreated cells (<b>A</b>; anti-EBA-175_RII and IgG shown as solid black and solid white bars, respectively). Anti-rRH5 alone (<b>B</b>; solid blue bars), or in combination (<b>C</b>; anti-EBA-175_RII/rRH5 and anti-EBA-175_RII+anti-rRH5 shown as solid red and solid green bars, respectively) is still able to significantly inhibit growth in treated cells as a sialic acid independent pathway is utilized by the RH5 antigen.</p

PfAtg8 localizes to the apicoplast.

<p><i>P. falciparum</i> FCR3 (A–E) and <i>P. falciparum</i> 3D7 transfected with ACP-GFP (F–H) were stained with the indicated organelle markers and visualized by confocal microscopy (because ACP-GFP was not uniformly expressed, some merozoites displayed only faint GFP signals). Anti-PfAtg8 antibody #1 was used in (A–F), and anti-PfAtg8 antibody #2 was used in (G). Apical membrane antigen 1 (AMA1) as a microneme marker (A), rhoptry-associated protein 1 (RAP1) as a rhoptry body marker (B), rhoptry neck protein 2 (RON2) as a rhoptry neck marker (C), the ring-infected erythrocyte surface antigen (RESA) as a dense granule marker (D), MitoTrackerRed CMXRos as a mitochondria marker (E), ACP-GFP (F–H) and the organellar histone-like protein PfHU (H) as an apicoplast marker were used. Scale bar, 1 μm.</p

Autophagy-Related Atg8 Localizes to the Apicoplast of the Human Malaria Parasite <em>Plasmodium falciparum</em>

<div><p>Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite <em>Plasmodium falciparum</em> (PfAtg8). PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although <em>Plasmodium</em> parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.</p> </div

PfAtg8 is associated with the apicoplast membrane.

<p>(A) <i>P. falciparum</i> FCR3 parasites at the schizont stage were analyzed by immunoelectron microscopy (immunogold and silver enhancement method) with an antibody against PfAtg8 (#1). (a) A schizont in an erythrocyte. (b) A magnified image of the area indicated in (a). (c) Another typical image of a PfAtg8-positive structure. (B) <i>P. falciparum</i> transfectant expressing ACP-GFP was analyzed as in panel (A) with an antibody against GFP. A, apicoplast; Mt, mitochondrion. Scale bars, (A, a) 1 μm, (A, b and c, and B) 200 nm.</p