Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome

<div><p>The apicomplexan parasite <i>Toxoplasma gondii</i> produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite <i>Plasmodium</i> spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in <i>P</i>. <i>berghei</i> ANKA and <i>T</i>. <i>gondii</i> cell lysates. However, addition of salicylic acid to <i>P</i>. <i>falciparum</i> and <i>T</i>. <i>gondii</i> culture had no effect. We transfected <i>P</i>. <i>falciparum</i> 3D7 with the <i>nahG</i> gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting <i>Pseudomonas</i> sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E₂, which potentially modulates host immunity as an adaptive evolution of <i>Plasmodium</i> spp. To investigate the function of salicylic acid and prostaglandin E₂ on host immunity, we established <i>P</i>. <i>berghei</i> ANKA mutants expressing <i>nahG</i>. C57BL/6 mice infected with <i>nahG</i> transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The <i>nahG</i>-transfectant also significantly increased the mortality rate of mice. Prostaglandin E₂ reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the <i>nahG</i> transfectant. Thus, salicylic acid of <i>Plasmodium</i> spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E₂.</p></div

Identification of salicylic acid from <i>Plasmodium berghei</i> ANKA.

<p><i>P</i>. <i>berghei</i> ANKA was purified from infected mice blood, and salicylic acid (SA) was extracted, and analyzed by LC-triple TOF mass spectrometry. (A) Structural formula of SA. (B) LC chromatogram of SA standard (control) and <i>P</i>. <i>berghei</i> ANKA sample. (C) Fragmentation analysis of peaks in (B) (colored in aqua). Collision energy was 20 eV.</p

Parasite SA influences the cerebral malaria outcome.

<p>(A-C) Histological observation of infected mouse cerebellums. (A) Cerebellum infected by <i>nahG</i>-expressing parasites. Note the sequestrated leukocytes in microvessels. The inset image shows a higher magnification of the boxed portion. Phagocytized hemozoin is observed (arrowhead). (B) Brain of a mouse infected with <i>gfp</i>-expressing parasites. Slight microbleeding was observed, but no sequestrated vessels were found. (C) Brain of an uninfected control. Sections were stained by hematoxylin and eosin. (D) Evans blue leakage analysis of the severity of cerebral malaria. Photographs of brains from mice infected with <i>nahG</i>- (left upper) and <i>gfp-</i> (left middle) expressing parasites and uninfected controls (left bottom), and quantification of dye leakage (right). Mice (n = 5) were sacrificed at 6 days post-infection. Solid line, p<0.01; dashed line, p<0.05. C57BL/6 mice at 6 days post-infection were used for all experiments. Bar: 50 μm.</p

Isoprenoid biosynthetic pathways and known inhibitors in various organisms.

<p>Broken arrows indicate blocks in the biosynthesis due to specific inhibitors. “R” indicates various functional groups specific to individual compounds. CPPS, copalyl-diphosphate synthase (EC 5.5.1.13); KO, <i>ent</i>-kaurene oxidase (EC 1.14.13.78); CAS, cycloartenol synthase (EC 5.4.99.8); LS, lanosterol synthase (EC 5.4.99.7); KS, <i>ent</i>-kaurene synthase (EC 4.2.3.19); PP, pyrophosphate.</p

Transmission electron microscopy of parasitized erythrocytes treated with inhibitors.

<p>(A) Sections through an erythrocyte containing a trophozoite-stage parasite exposed to 0.1% DMSO for 6 h, 50 µM INA for 6 h or 250 µM AMO-1618 for 8 h, respectively. (B) Asynchronized parasites were treated with 0.1% DMSO (a–d) or 50 µM INA for 6 h (e–h). Sections from representative stages during intraerythrocytic development: ring- (a and e), early trophozoite- (b and f), mature trophozoite- (c and g) and schizont-stage parasites (d and h) are shown. Nuclei (n), food vacuoles (fv), merozoites (m), nuclei of merozoites (mn) and abnormal gaps between the nuclei and the nuclear envelopes (arrowheads) are indicated. Scale bar is indicated at the bottom of the images.</p

Effect of INA and AMO-1618 on intraerythrocytic development of <i>P. falciparum</i>.

<p>Tightly synchronized parasite cells that have undergone 5% sorbitol treatment and percoll density gradient centrifugation (window period: 4 h) were treated with 1 µl/ml DMSO, 50 µM INA or 250 µM AMO-1618 at different parasite stages: after 0 h (ring, A), 20 h (immature trophozoite, B), 28 h (mature trophozoite, C) and 36 h (schizont, D). Cultures were examined after 4, 8 and 12 h using Giemsa thin blood smears. Scale bar: 3 µm; all images without a scale bar are displayed at the same scale as the left uppermost image in (A).</p

ED₅₀ of the gibberellin biosynthetic inhibitors.

<p>Growth inhibitory effects of gibberellin biosynthetic inhibitors and enantiomers of INA to <i>P. falciparum</i> and <i>T. gondii</i> are shown. Values are the mean ± standard deviations (SD) from three independent experiments, with each treatment duplicated twice. N.D.; not determined.</p

GC-MS analysis of isoprenoids in <i>P. falciparum in vitro</i> cultures.

<p>(A) Total ion chromatograms of extracts from unparasitized erythrocytes (Unparasitized), parasites treated with 0.1% DMSO, 250 µM AMO-1618, and 50 µM INA for 6 h. Triangles and circles indicate the peaks of geranylgeraniol mixed as an internal control (retention time = 6.57 min) and cholesterol (11.23 min), respectively. These compounds were identified by direct comparison with authentic samples. (B) Quantification of cholesterol in each treated sample. Cholesterol amount was calculated from the peak areas and normalized relative to the ratio of the internal control geranylgeraniol. Values are means and SD of triplicate measurements of a representative experiment.</p

Fluorescence microscopy of INA-treated parasites.

<p>Infected erythrocytes were stained with (A) acridine orange, (B) Nile Red, (C) rhodamine 123 and (D) LysoTracker™ Red DND-99. INA was introduced at: (A) 100 µM for 9 h, (B) 50 µM for 6 h, and (C and D) 50 µM for 4 h. 100 µg/ml acridine orange was applied to thin blood smears made from intraerythrocytic parasites treated with INA. For other fluorescence dyes, <i>P. falciparum</i> cultures were incubated with probes for 1 h at the following concentrations: Nile Red, 1 µg/ml; rhodamine 123, 10 ng/ml; and LysoTracker® Red DND-99, 75 nM. Cells were not washed prior to fluorescence microscopy to minimize damage due to osmotic changes. Scale bar, 3 µm. An arrow indicates a lipid body in (B).</p

Effects of osmotic pressure to intraerythrocytic parasites treated with INA.

<p>(A) Effects of hyperosmotic stress in INA-treated parasites. <i>P. falciparum</i> cultures treated with 50 µM INA were observed in various dilution ratios of PBS for 8 h. Parasitemia was determined by Giemsa-stained thin blood smears. Student's <i>t</i>-test: *P>0.005. Values are means ± SD of n = 6 in two independent experiments. Data were normalized relative to control cultures (1× PBS) in DMSO- and INA-treated samples, respectively. Representative parasite morphologies are shown for each treatment. Scale bar, 3 µm. (B) Effects of hyposmotic stress, experimentally induced by the addition of water to the culture medium of <i>P. falciparum</i>, after 8 h of 50 µM INA treatment. N = 6 smears each in two independent experiments.</p