Fig 1.
R. rotatoria–conditioned water paralyzes Schistosoma mansoni cercariae.
(A) Life cycle of S. mansoni. Adult parasites, residing in the mammalian host vasculature, lay eggs (not shown). Upon exposure to fresh water, eggs release miracidia, which infect the appropriate snail host. Inside the snail, the parasite reproduces asexually, ultimately producing large numbers of free-swimming infective larvae (cercariae) that can penetrate mammalian skin to continue the life cycle (adapted from [16]). (B and C) Nomarski differential interference contrast microscopy images of R. rotatoria and Philodina acuticornis (arrowhead indicates the rostrum in R. rotatoria, which is lacking in P. acuticornis). Scale bars: 100 μm. (D and E) Maximum intensity projection (5 s, 150 frames) of cercariae motility after treatment with R. rotatoria– or P. acuticornis–conditioned water.
Fig 2.
SPF is a novel tetracyclic alkaloid.
(A) Flowchart for SPF purification. (B) First HPLC plots of R. rotatoria–and P. acuticornis–conditioned water. All fractions were tested for bioactivity; the red arrowhead indicates the only active peak. (C) Second HPLC plot of the bioactive fraction (red arrowhead in panel B). All peaks were tested for bioactivity; the blue arrowhead indicates the only peak containing activity. (D) MS showing the dominant signal of m/z 273.1601 from the peak (blue arrowhead). (E) MS plots showing this signal (asterisk, m/z 273.1601) was only detected in the fraction eluting at 24 to 26 min. (F) Tandem MS acquired from high-resolution Q-TOF analysis. (G and H) NOESY resolved the relative stereochemistry of 3 chiral centers and narrowed it down to 2 possible configurations. HPLC, high-performance liquid chromatography; MS, mass spectrometry; MWCO, molecular weight cut-off; NMR, nuclear magnetic resonance; NOESY, Nuclear Overhauser effect spectroscopy; Q-TOF, quadrupole time-of-flight; SPF, Schistosome Paralysis Factor.
Fig 3.
Structure-activity relationships of SPF and related compounds as measured by cercarial motility assays.
(A–G) Percentage of cercariae (approximately 50) continuing to swim over 3 min after addition of each compound at specified final concentrations. Triplicates were performed. Data are mean ± SD. Serotonin structure in SPF is outlined in red. See S1 Data for corresponding raw data. SPF, Schistosome Paralysis Factor.
Fig 4.
Treating cercariae with SPF, Ht-13-A, or Ht-13-A-pr blocks schistosome infection and alleviates pathology.
(A) Representative livers (post perfusion) from mice (N = 6) exposed to drug-treated cercariae. Livers from mice treated with control and lower drug concentrations were darker in color and contained more granulomas (white spots). With higher drug concentrations, livers had normal morphologies with few or no granulomas; 25 μM SPF treatment was ND because of limited amounts of purified SPF. (B–D) Numbers of adult worms recovered from exposed mice (2 experiments for each drug, 6 mice total for each condition). (E–G) Numbers of schistosome eggs per area (/mm2) from liver sections (4–6 sections per mouse). Data for panels B–G are mean ± SD. Statistics: One-way ANOVA, post Dunnett’s test. See S2 Data for corresponding raw data. ND, not determined; SPF, Schistosome Paralysis Factor.