Fig 1.
The TRPV1 activator capsaicin stimulates a significant Ca2+ influx in CHO cells expressing schistosome TRPA1-like channels.
A. Montage showing the response of CHO cells co-transfected with SmTRPA and pGP-CMV-GCaMP6f to 10 μM capsaicin over time. Number in upper left corner of each picture represents elapsed time (in minutes:seconds). Capsaicin was applied at frame 6 (0:19). Calibration bar = 85 μm. B. Traces of averaged GCaMP6f fluorescence intensity change in response to 10 μM capsaicin in cells transfected with empty vector (pcDNA3.1/zeo(+); orange), rat TRPV1 (green), SmTRPA (purple), or ShTRPA (blue). The black trace is a positive control indicating the averaged response of randomly selected cells (n = 90, 5 independent transfections) from each group to 1 μM ionomycin, a Ca2+ ionophore. Capsaicin was applied at 5 min. C. Normalized maximal GCaMP6f fluorescence intensity in response to 10 μM capsaicin in cells transfected with: empty vector (Vector; n = 62, 3 independent transfections); SmTRPA (n = 131 cells, 6 independent transfections); ShTRPA (n = 112, 6 independent transfections); rat TRPA1 (rTRPA1, n = 99, 3 independent transfections); and rat TRPV1 (rTRPV1, n = 41, 3 independent transfections). ***, P < 0.0001, unpaired, two-tailed t-test vs. empty vector data. Responses of cells transfected with SmTRPA or ShTRPA to capsaicin are also significantly higher (P <0.0001) than responses to DMSO (S1 Fig). D. Dose response of expressed SmTRPA to capsaicin. Normalized maximal GCaMP6f fluorescence in response to different concentrations of capsaicin is shown for CHO cells transfected with SmTRPA. Capsaicin concentrations tested are 5 μM (n = 53), 10 μM (n = 34), 30 μM (n = 121), 70 μM (n = 90). Control (n = 39) is 0.01% DMSO. In this experiment, all cells tested were from a single transfection, which likely accounts for the differences from the maximal fluorescence value at 10 μM shown in panel C, which represents results from multiple transfections.
Fig 2.
The TRPV1 activator olvanil stimulates an increase in intracellular Ca2+ in cells transfected with SmTRPA or ShTRPA and increases motor activity in adult S. mansoni.
A. Traces of averaged GCaMP6f fluorescence intensity change in response to 10 μM olvanil in cells transfected with empty pcDNA3.1/zeo(+) (orange), SmTRPA (purple), or ShTRPA (blue). Olvanil was applied at 5 min. B. Normalized maximal GCaMP6f fluorescence intensity in response to 10 μM olvanil in cells transfected with: empty vector (n = 67, 3 independent transfections); SmTRPA (n = 23, 4 independent transfections); ShTRPA (n = 177, 4 independent transfections). *, P < 0.05, ***, P < 0.0001, unpaired, two-tailed t-test vs. empty vector data. C, D. Measurement of motor activity in adult (~7 weeks post infection) S. mansoni males (C) or females (D) that were exposed in culture to 10 μM olvanil. Motility was assayed before and after addition of compound. "Control" contained no added compounds, and the "DMSO" sample contained 0.1% DMSO. Motility was analyzed as described [39], with each individual tested worm serving as its own control, and data normalized to Control response for each worm. *, P < 0.05, **, P < 0.01, paired, two-tailed t-test against Control, prior to normalization.
Fig 3.
The TRPA1 activator AITC activates SmTRPA but not ShTRPA.
A. Traces of averaged GCaMP6f fluorescence intensity change in cells transfected with empty vector (pcDNA3.1/zeo(+); orange), rat TRPA1 (green), SmTRPA (purple), or ShTRPA (blue). B. Normalized maximal GCaMP6f fluorescence intensity in response to 20 μM AITC in cells transfected with empty vector (Vector, n = 149, 3 independent transfections); SmTRPA (n = 64, 4 independent transfections); or ShTRPA (n = 72, 4 independent transfections). *, P < 0.05, ***, P < 0.0001, unpaired two-tailed t-test vs. empty vector data. C. S. haematobium adult females do not exhibit hyperactivity in response to 60 μM AITC (n = 20). Activity was measured as described in Fig 2 and [39]. Data are normalized to Control response for each individual worm. As a positive control, we also tested 40 μM 5-HT, which does produce significant hyperactivity in S. haematobium females (n = 26). *, P < 0.05, paired, two-tailed t-test vs. Control, prior to normalization.
Fig 4.
4-HNE, a host-derived, inflammatory activator of mammalian TRPA1, evokes hyperactivity in adult S. mansoni and activates SmTRPA and ShTRPA.
A. Response of male (left panel) and female (right panel) S. mansoni adults to 0.1% DMSO (n = 12) or 10 μM 4-HNE (n = 47, males; n = 24, females), measured as in Fig 2. **, P < 0.01, paired, two-tailed t-test vs. Control. B. 4-HNE (50 μM) triggers an increase in intracellular GCaMP6f fluorescence in cells expressing either SmTRPA or ShTRPA. Traces of averaged GCaMP6f fluorescence intensity change in cells transfected with empty vector (pcDNA3.1/zeo(+); orange), SmTRPA (purple), or ShTRPA (blue). C. Normalized maximal GCaMP6f fluorescence intensity in response to 50 μM 4-HNE in cells transfected with empty vector (Vector, n = 21, 3 independent transfections); SmTRPA (n = 18, 3 independent transfections); or ShTRPA (n = 95, 4 independent transfections). ***, P < 0.0001, unpaired, two-tailed t-test vs. empty vector data.