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. 2019 Jan 29;8:e39123. doi: 10.7554/eLife.39123

Figure 5. Clemastine efficacy requires cytosolic access and inflammasome signaling.

(A) Quantification of bacterial burden of Mm:Wasabi and MmΔRD1:Wasabi in wildtype larvae treated with 0.5% DMSO or 5 µM clemastine, five dpi. Representative of three independent experiments. Each dot represents an individual animal’s bacterial burden by fluorescence. Red dots denote the animals represented in Figure 5—figure supplement 1A. (B) Quantification of bacterial burden of Mm:tdTomato in pycard (asc) mutants and wildtype/heterozygous siblings after 0.5% DMSO or 5 µM clemastine treatment, 5dpi. Each dot represents an individual animal’s bacterial burden by fluorescence. Representative of three independent experiments. Blue (WT/het) and purple (asc mutants) dots denote representative larvae in Figure 5—figure supplement 2B. Fold change over DMSO for each genotype is presented in Figure 5—figure supplement 2C. (A) Ordinary one-way ANOVA with Tukey’s multiple comparison test. All error bars are s.d. ns1 > 0.9999, ns2 = 0.9452, ns3 = 0.9430. (B) One-way ANOVA with Tukey’s multiple comparison test. All error bars are s.d. ns1 = 0.9998, ns2 = 0.5798, ns3 = 0.3723. p Values from statistical tests on untransformed data are provided in Supplementary file 2.

Figure 5.

Figure 5—figure supplement 1. Clemastine does not enhance microbicidal macrophage activities in MmΔRD1 infections.

Figure 5—figure supplement 1.

(A) Representative animals from the red dots in Figure 5A. Animals were infected with Mm:Wasabi (~50 CFU) and Mm:ΔRD1:Wasabi (~ 50 CFU) and treated with 0.5% DMSO or 5 μM clemastine, 5 dpi. Scale bar is 500 μm. (B) Bacterial burden per animal assessed by fluorescence 5 days post-infection after treatment with 0.5% DMSO or 5 µM clemastine. Infections were performed with Mm:tdTomato (RD1+ and cmaA2+), Tn01901:mCerulean (cmaA2-) or Mm:ΔRD1:Wasabi (RD1-). Each dot represents a single animal bacterial burden by fluorescence, represented as fold chance over DMSO in Figure 5—figure supplement 1C. Representative of two independent experiments. (C) Fold change in bacterial burden per experiment assessed by fluorescence of bacteria (left y-axis) or 16S rRNA qPCR (right y-axis, gray error bars). The experiment in Figure 5—figure supplement 1B is represented with green dots. Each bacterial strain is normalized to the DMSO-treated control within each bacterial strain. (D) Number of Mm:Wasabi or Mm:ΔRD1:Wasabi bacteria per macrophage during treatment with 0.5% DMSO or 5 μM clemastine, 1 dpi. Each dot represents the average number of Mm:Wasabi bacteria inside the macrophages of Tg(mfap4:tdTomato)xt12 of a zebrafish larva. Representative of three experiments. (B) Ordinary one-way ANOVA with Sidak’s multiple comparison test within bacterial strains, ns1 = 0.8757. Error bars are s.d. (C) Paired t-test on left y-axis. Error bars are s.e.m. No statistics were performed on the right y-axis, represents one biological replicate performed in triplicate. (D) Ordinary one-way ANOVA with Holm-Sidak’s multiple comparison test, ns1 = 0.4243, ns2 =0.1495. All error bars are s.e.m., p values from all transformed and untransformed data are provided in Supplementary file 3.
Figure 5—figure supplement 2. Clemastine requires inflammasome components to reduce bacterial burden.

Figure 5—figure supplement 2.

(A) TALEN-mediated lesions in pycard, the zebrafish orthologue of asc, to generate a 14 base pair deletion in exon 1, known as ascw216. (B) Representative infected animals 5 dpi from purple and blue dots in Figure 5B. Blue bar represents wildtype animals and purple bar represents ascw216 infected with Mm:tdTomato. Scale bar is 500 μm. (C) Fold change in bacterial burden per experiment assessed by fluorescence of bacteria. The experiment in Figure 5B is represented with blue dots. Each genotype is normalized to the DMSO-treated control within that genotype. (D) Percent of bacteria decorated with GFP:LC-3 puncta, 3 dpi. Each dot represents percent of bacteria with GFP:Lc-3 puncta from a single animal. Representative of two experiments. (C) Unpaired t-test, error bars are s.e.m. (D) Unpaired t-test, error bars are s.d., ns1 = 0.4585. p Values from all transformed and untransformed data are provided in Supplementary file 3.