Additional file 1 of Characterization of a nuclear transport factor 2-like domain-containing protein in Plasmodium berghei

Additional file 1: Figure S1. Schematic representation of the gene-targeting vector used to disrupt PBANKA_1019700, PBANKA_1101300 (SBP1) and PBANKA_0519900. Figure S2. Generation of parasites to investigate the localization of PBANKA_1019700.Figure S3. Generation of parasites to investigate the localization of PBANKA_0519900::GFP and PBANKA_1359300::GFP

IFNGR1 signaling is associated with adverse pregnancy outcomes during infection with malaria parasites

<div><p>Complicated/severe cases of placental pathology due to <i>Plasmodium falciparum</i> and <i>P</i>. <i>vivax</i>, especially adverse pregnancy outcomes during <i>P</i>. <i>vivax</i> infection, have been increasing in recent years. However, the pathogenesis of placental pathology during severe malaria is poorly understood, while responses against IFN-γ are thought to be associated with adverse pregnancy outcomes. In the present study, we explored the role of IFN-γ receptor 1 (IFNGR1) signaling in placental pathology during severe malaria using luciferase-expressing rodent malaria parasites, <i>P</i>. <i>berghei</i> NK65 (<i>Pb</i>NK65L). We detected luciferase activities in the lung, spleen, adipose tissue, and placenta in pregnant mice, suggesting that infected erythrocytes could accumulate in various organs during infection. Importantly, we found that fetal mortality in IFNGR1-deficient mice infected with <i>Pb</i>NK65L parasites was much less than in infected wild type (WT) mice. Placental pathology was also improved in IFNGR1-deficient mice. In contrast, bioluminescence imaging showed that parasite accumulation in the placentas of IFNGR1-deficient pregnant mice was comparable to that in WT mice infected with <i>Pb</i>NK65L. These findings suggest that IFNGR1 signaling plays a pivotal role in placental pathology and subsequent adverse pregnancy outcomes during severe malaria. Our findings may increase our understanding of how disease aggravation occurs during malaria during pregnancy.</p></div

CD8⁺ T cells and F4/80⁺ cells are involved in the development of placental pathology during malaria.

<p>(A) Intervillous space in the labyrinth of placentas on day 6 p.i. The labyrinth region of the intervillous space is shown at higher magnification in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185392#pone.0185392.g004" target="_blank">Fig 4B</a>. (B) Fetal weight on day 6 p.i. (C) The proportion of CD4⁺ cells in the CD3⁺ gate in placentas on day 6 p.i. (D) The proportion of CD8⁺ cells in the CD3⁺ gate in placentas on day 6 p.i. (E) The proportion of F4/80⁺ cells in the CD11b⁺ gate in placentas on day 6 p.i. Asterisks indicate a significant difference (<i>p</i> < 0.05). Results are expressed as means ± SD of three mice. Experiments were performed in duplicate with similar results.</p

The effect of IFNGR1 deficiency on the localization of infected erythrocytes in pregnant mice.

<p><i>Ex vivo</i> bioluminescent images of luciferase expression in organs of WT or IFNGR1-KO mice following infection with <i>Pb</i>NK65L. Nonpregnant (NP) and pregnant mice (PG) on day 12 post-mating were infected with 1 × 10⁴ infected erythrocytes of <i>Pb</i>NK65L. D-luciferin (50 mg) was injected into the tail vein of NP and PG mice and the organs of mice from each group were removed on day 3 p.i. (A and B) or day 6 p.i. (C and D). (A and C) Bioluminescent images of luciferase activity in the organs of mice from each group. Representative data are shown. (B and D) The bioluminescent signal from each organ was quantified using the Living Image software. Asterisks indicate a statistically significant difference (<i>p</i> < 0.05). Results are expressed as means ± SD of three mice. Experiments were performed in duplicate with similar results.</p

The effect of IFNGR1 deficiency on the outcome of infection with <i>Pb</i>NK65L during pregnancy.

<p>Female wild type (WT) and IFN-γ receptor 1-deficient (IFNGR1-KO) mice were put together with male WT mice for 1 day. Mice on day 12 post-mating were infected with 1 × 10⁴ infected erythrocytes of <i>Pb</i>NK65L. (A) Course of parasitemia in WT and IFNGR1-KO mice. Parasitemia was observed in nonpregnant (circles) and pregnant (triangles) mice infected with <i>Pb</i>NK65L. (B-D) Blood or plasma were obtained from uninfected wild type mice (Uninfected WT), WT mice infected with <i>Pb</i>NK65L (Infected WT), or IFNGR1-KO mice infected with <i>Pb</i>NK65L (Infected KO) mice on day 6 p.i. (B) Hematocrit in nonpregnant and pregnant mice on day 6 p.i. (C) Plasma IFN-γ levels in nonpregnant and pregnant mice on day 6 p.i. (D) Plasma IL-10 levels in nonpregnant and pregnant mice on day 6 p.i. Asterisks indicate a significant difference (<i>p</i> < 0.05). Results are expressed as means ± SD of three mice. Experiments were performed in triplicate with similar results.</p

Role of IFNGR1 signaling in placental inflammation during <i>Pb</i>NK65L infection.

<p>Representative hematoxylin and eosin (H&E)-stained placental sections are shown. Placentas were obtained from uninfected pregnant mice (Uninfected WT, top panels), pregnant WT mice infected with <i>Pb</i>NK65L (infected WT, middle panels), or pregnant IFNGR1-KO mice infected with <i>Pb</i>NK65L (Infected KO, bottom panels) mice on day 6 p.i. (A) Low magnification images of placenta. (B) Higher magnification of labyrinth region (black boxes (i) in A). The scale bars indicate 100 μm. (C) Higher magnification of maternal blood vessel (black boxes (ii) in A). The scale bars indicate 40 μm. (D) Higher magnification of fetal blood vessel (black boxes (iii) in A). The scale bars indicate 40 μm. Regions indicated by white boxes in B–D were enlarged to inset boxes. Arrows indicate <i>Pb</i>NK65L-infected erythrocytes. Experiments using three to five mice were performed in triplicate with similar results, and representative data are shown.</p

The increase in susceptibility of mice to infection with <i>Pb</i>NK65L during pregnancy.

<p>Female C57BL/6 (B6) mice on day 12 post-mating were infected with 1 × 10⁴ infected erythrocytes of luciferase-expressing <i>P</i>. <i>berghei</i> NK65 (<i>Pb</i>NK65L). (A) Course of parasitemia in nonpregnant and pregnant mice infected with <i>Pb</i>NK65L. (B) Hematocrit on days 0, 5, and 7 post-infection (p.i). (C) Plasma IFN-γ levels on day 5 p.i. (D) Plasma IL-10 levels on day 5 p.i. Asterisks indicate a statistically significant difference (<i>p</i> < 0.05). Results are expressed as means ± SD of three to five mice. Experiments were performed in duplicate with similar results.</p

Growth-arresting effects of 2-AAPA and BCNU on synchronized <i>P</i>. <i>falciparum</i>.

<p>Parasites synchronized at the ring stage were cultured for 24 h in the presence of graded concentrations of 2-AAPA <b>(a)</b> and BCNU <b>(b)</b>. Each developmental stage was counted after Giemsa staining. *Significant difference versus no 2-AAPA or BCNU.</p

Growth-arresting effects of DIDS on asynchronous and synchronized <i>P</i>. <i>falciparum</i>.

<p><b>(a)</b> Asynchronous parasites were cultured for 95 h in the presence of graded concentrations of DIDS. The IC₅₀ was 25.21 ± 4.34 μM. <b>(b)</b> Parasites synchronized at the ring stage were cultured for 24 h in the presence of graded concentrations of DIDS. Each developmental stage and parasitemia were counted after Giemsa staining. % of pyknotic forms are not significantly different at various concentrations versus no DIDS.</p

Profiling molecular factors associated with pyknosis and developmental arrest induced by an opioid receptor antagonist and dihydroartemisinin in <i>Plasmodium falciparum</i>

<div><p>Malaria continues to be a devastating disease, largely caused by <i>Plasmodium falciparum</i> infection. We investigated the effects of opioid and cannabinoid receptor antagonists on the growth of intraerythrocytic <i>P</i>. <i>falciparum</i>. The delta opioid receptor antagonist 7-benzylidenenaltrexone (BNTX) and the cannabinoid receptor antagonists rimonaband and SR144528 caused growth arrest of the parasite. Notably BNTX and the established antimalarial drug dihydroartemisinin induced prominent pyknosis in parasite cells after a short period of incubation. We compared genome-wide transcriptome profiles in <i>P</i>. <i>falciparum</i> with different degrees of pyknosis in response to drug treatment, and identified 11 transcripts potentially associated with the evoking of pyknosis, of which three, including glutathione reductase (PfGR), triose phosphate transporter (PfoTPT), and a conserved <i>Plasmodium</i> membrane protein, showed markedly different gene expression levels in accordance with the degree of pyknosis. Furthermore, the use of specific inhibitors confirmed PfGR but not PfoTPT as a possible factor contributing to the development of pyknosis. A reduction in total glutathione levels was also detected in association with increased pyknosis. These results further our understanding of the mechanisms responsible for <i>P</i>. <i>falciparum</i> development and the antimalarial activity of dihydroartemisinin, and provide useful information for the development of novel antimalarial agents.</p></div