Fig 1.
Identification of salicylic acid from Plasmodium berghei ANKA.
P. berghei 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 P. berghei ANKA sample. (C) Fragmentation analysis of peaks in (B) (colored in aqua). Collision energy was 20 eV.
Fig 2.
Quantification, in vitro function and genetic manipulation of salicylic acid (SA).
(A) SA concentrations identified by automated quantitative mass spectrometry. Black and white bars show the SA concentrations in fresh cell lysates of Plasmodium berghei ANKA (Pb) purified from infected mouse blood in vivo, and Toxoplasma gondii RH (Tg) cultured in vitro, respectively. The concentrations were calculated based on the weight of parasite lysate. (B) Effect of addition of SA to culture medium on growth of T. gondii 2F. Growth was quantified by β-galactosidase activity after cultivation for 48 h. Each value was normalized using control parasites treated with DMSO. The graph shows the mean of two independent experiments. (C) Effect of addition of SA to culture medium on growth of P. falciparum. The growth rate was monitored by counting parasitemia by Giemsa staining. (D) Western blotting of nahG fused with a cmyc2-tag. HSP-90 was used as an internal control (bottom panel). (E–G) Biological changes of P. falciparum due to SA deficiency in vitro. (E) Secretion of SA into the culture supernatant by P. falciparum transfected with nahG-cmyc2 or GFP (control). (F) Growth effect of intracellular SA depression. Growth kinetics of nahG-containing mutants were compared with the control (gfp-transfectant). (G) SA affected the synthesis of prostaglandin E2 (PGE2) in parasites (p<0.01, Student t-test). All experiments with P. falciparum were performed after synchronization twice with 5% (w/v) D-sorbitol solution (n = 3). Bars indicate the data range (B) or standard error (C, E-G).
Fig 3.
In vivo influence of SA depression.
(A) Survival following infection of mice with nahG- or gfp-expressing parasites. Data are from two independent experiments, n = 13. *: p<0.05. (B, C) Parasite growth (B) and animal weight gain/loss (C). Each graph indicates an individual mouse infected by nahG (red) or gfp (blue)-expressing parasites. (D) Parasitemia of nahG- or gfp-expressing parasites at day 6 post-infection, when clinical signs were most significant. Plasma PGE2 and cytokines were quantified at this time point. (E) Hematocrit levels in mice at 6 days post-infection. Hematocrit did not differ between the groups, whereas the survival curves and clinical behavior shifted at the same time point. Statistics were calculated by Log-rank and general Wilcoxon test (A) and Mann-Whitney U-test (E).
Fig 4.
Parasite SA influences the cerebral malaria outcome.
(A-C) Histological observation of infected mouse cerebellums. (A) Cerebellum infected by nahG-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 gfp-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 nahG- (left upper) and gfp- (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.
Fig 5.
Plasma levels of cytokines, chemokines, and prostaglandin E2 (PGE2) in infected mice.
(A) PGE2. (B-F) Proinflammatory cytokines: IFN-γ (B), TNF-α (C), IL-2 (D), IL-1β (E), and IL-12 (F). (G-I) Anti-inflammatory cytokines: IL-10 (G), IL-4 (H), and IL5 (I). (J) Inflammatory chemokine MCP-1. Plasma from heparin-treated whole blood of mice infected by nahG- or gfp-expressing parasites was used in all experiments. Control sample of IFN-γ was analyzed at the different day and indicated by dashed line. Statistics were calculated by Mann-Whitney U-test. Bonferroni correction was used for multiple tests.