Fig 1.
Teredinbacter turnerae T7901 produces a compound with activity against intracellular Toxoplasma gondii.
T. turnerae strains T7901 and T7902 were grown in SBM for 9 days. Supernatants from the bacterial cultures was added to HFF cells infected with T. gondii at 1:100, 1:250 or 1:500 dilutions in cell culture medium and IFAs performed 24 hours later. Upper panels: DIC images, Lower panels: green, rabbit anti-SAG1 antibody, blue: DAPI. Additional representative images are shown in S1 Fig. A. T7901, but not T7902, supernatants inhibited the growth of T. gondii. Scale bar = 16 μm applies to both upper and lower images. B. Left panels: In DMSO treated samples individual tachyzoites are visible within the parasitophorus vacuole. Right panels: In trtE treated samples, the parasite vacuoles remained intact, but individual intact tachyzoites were no longer identifiable in the parasitophorous vacuole. Scale bar = 5 μm applies to both upper and lower images.
Fig 2.
Tartrolon E has potent and rapid parasiticidal activity against intracellular and extracellular Toxoplasma gondii: A. Structure of trtE. B. HFF cells infected with T. gondii RH strain parasites were treated with DMSO vehicle control (left panels) or 60 nM trtE (right panels) for 24 hours. Infected cells were then fixed and processed for IFA. Left images show DIC images, right images show corresponding IFA (red = rabbit anti-T. gondii SAG1 antibody, blue = DAPI). Scale = 5 μm. Images are enlarged from S7 Fig, panels A and B. The morphology of trtE-treated parasites is similar to that seen with T7901 culture supernatant (compare Figs 1 and 2 and S1 and S7). C. Luciferase expressing RH or ME49 strain T. gondii parasites were allowed to infect HFF cells for 24 hours, then the infected cells were treated with two-fold dilutions of trtE purified by method 1 (TrtE-1) or method 2 (TrtE-2). Parasite growth was quantified 24 post-treatment by luciferase expression. Data were analyzed using the log[inhibitor]vs response-Variable slope (four parameter) regression equation in Graphpad Prism and curves compared using the extra sum of squares F-test. One curve adequately fit all the data sets (p = .42) with an estimated EC50 of 3.0 nM (95% CI of 2.5 to 3.5). D. Luciferase expressing RH strain tachyzoites, isolated from their host cells, were treated with two fold dilutions of trtE or DMSO control for 2 hours, then the compound was washed off and the parasites allowed to infect HFF cells for 24 hours. Parasite growth, as evaluated by luciferase expression, was inhibited by trtE treatment of tachyzoites with an EC50 of 3.1 nM (analysis as for C; 95% CI of 2.3–4.3). E: HFF cells were infected with luciferase expressing T. gondii ME49 strain for 24 hours. Infected cells were then treated with 30 nM trtE or DMSO control for 0.5 to 4 hours, at which times the compound was washed off the infected cells. Parasite growth was evaluated by luciferase expression 72 hours after treatment. Parasites were killed after 2 hours of trtE treatment.
Fig 3.
Tartrolon E is active against a broad spectrum of apicomplexan parasites in vitro: A. HCT-8 cells were infected with nanoluciferase expressing-Cryptosporidium parvum oocysts for 24 hours at which time dilutions of trtE were added to the infected cells. Parasite proliferation was evaluated 48 hours post-treatment by quantification of nanoluciferase expression. The data was analyzed as described in the legend for Fig 2C; EC50 = 3.85 nM with a 95% CI of 3.6–4.2 nM. B. C. parvum was allowed to infect HCT8 cells for 8 hours, then the infected cells were treated with 60 nM trtE or DMSO vehicle control for 12 hours. Infected cells were then fixed and processed for IFA. Images on left are DIC, images on right show corresponding IFA (Red: anti-gp15 antibody, blue: DAPI; scale = 5μm). Images shown are enlarged from S10 Fig, panels C and F. DMSO treated parasites formed normal type I meronts (B, upper panel and S10 Fig, panels A-C). There were few discernable parasites in trtE samples, and those parasite vacuoles that could be identified with anti-gp15 antibody did not appear to contain merozoites (B., lower panel and S10 Fig, panels D-F).; C, D. Babesia bovis, B. bigemina (C) and Plasmodium falciparum (D) -infected erythrocytes were incubated with trtE or DMSO vehicle control and proliferation was measured after 72 hours by SYBR green incorporation. Percent proliferation was calculated relative to DMSO treated controls. EC50s were calculated as described in the legend for Fig 2C. (C) B. bovis: EC50 = 15.9 nM (95% CI 10–21 nM) and B. bigemina: EC50 = 9.3 nM (95% CI 7.3–11.8 nM) (D) P. falciparum: EC50 = 105 pM (95% CI 101–110 pM). The antimalarial drug dihydroartemesinin (DHA) is included for comparison, EC50 = 20 pM (95% CI 19–21 pM). E. B. bovis CE11/p2xHA-glmS-gfp-bsd parasites were treated with DMSO (top images) or 50 nM trtE (bottom images) for 24 hours prior to fixation and immunostaining. The infected erythrocytes were labeled with rabbit anti-GFP (green) to visualize the parasite cytoplasm and an anti B. bovis RAP-1 mAb (red). Nuclei are visualized with DAPI. Images of DMSO treated parasites (top panels) show an early invaded erythrocyte (left) and mature meront (right), whereas images of trtE-treated parasites show divided meronts. Scale bars = 5 μm. Phase contrast and IFA-phase overlays of these images are shown in S12 Fig.
Table 1.
EC50s, TC50s and selectivity indices for parasites and their host cells.
Fig 4.
Tartrolon E is effective against Cryptosporidium parvum infection in neonatal mice.
A, B. 8-day-old mice were infected with 5x104 C. parvum oocysts. 48 hours post infection, mice were treated orally, every 12 hours, for 2 days with 2 mg/kg trtE (purification method 1). 5 days post infection mice were euthanized, and intestines processed for histology. A. Left panel: Section of ileum from a control mouse, scale bar = 25 μm. Area in box is enlarged in the upper right corner. Parasites are indicated with arrowheads. Scale bar = 5 μm. Right panel: Section of ileum from a mouse treated with trtE. No parasites were observed in this section. Scale bar = 25 μm. B. Slides with histological sections of the intestine from DMSO and trtE treated mice were generated and infection intensity was scored in the jejunum, ileum, caecum and colon by an investigator blinded to sample treatment. Scores for each section of the intestine were added together to give a total infection score for each animal. TrtE significantly reduced the number of parasites in the intestine (Mann-Whitney non-parametric t-test; ****p<0.0001). C. Neonatal mice were infected and treated as described for the first experiment but were dosed with three concentrations of trtE (purification method 2). Infection was evaluated by quantitative real-time PCR of intestinal tissue. Comparisons between control and experimental groups were made using a one-way ANOVA with Dunnett’s Multiple Comparisons Test in Graphpad Prism. **p = 0.0023, ****p<0.0001.