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Kmo is required for intracellular control of bacterial burden within macrophages. (A) Treatment with the Kmo inhibitor Ro61-8048 does not change macrophage numbers in either infected or uninfected larvae compared to DMSO control. Macrophage numbers from Tg(mpeg1.1:mCherry) larvae either treated with 50µM Ro61-8048 or DMSO control were quantified by microscopy from both infected (16 HPI) and sibling uninfected larvae (values, mean + /- SEM). Each data point represents an individual larva. Data are representative of 3 experiments. (One-way ANOVA followed by Tukey’s multiple comparisons, ns, non-significant). (B) Kmo inhibition with Ro61-8048 does not impair macrophage engulfment of S. Typhimurium-GFP. Tg(mpeg1.1:mCherry) larvae (3 dpf) were infected with S. Typhimurium-GFP and the percentage of mCherry + /GFP+ macrophages were quantified by flow cytometry at 4 HPI (Unpaired t-test, ns, non-significant). Each data point represents an individual larva. Data are representative of 3 experiments. (C-F) Kmo inhibition (C) or depletion (D-F) impairs macrophage control of bacterial burden. (C) Tg(mpeg1.1:mCherry) larvae (3 dpf) were infected with S. Typhimurium-GFP (CFU/ larvae) and treated with 50µM Ro61-8048 or DMSO control or (D)kmo or control morphant larvae were infected with S. Typhimurium-GFP (CFU/ larvae) and the number of live bacteria per sorted macrophage (mCherry + / GFP+) was quantified by isolating macrophages by FACS at 4 HPI followed by lysis and enumeration of bacteria by plating for CFU (Unpaired t-test; C, ****p < 0.0001, D, ***p < 0.001). Data are representative of 3 experiments. (E, F) Kmo depletion increases numbers of intracellular bacteria per macrophage by transmission electron microscopy (TEM). kmo or control morphant larvae were infected with S. Typhimurium, fixed at 4 HPI and infected phagocytes in the caudal hematopoietic tissue were examined by TEM. (E) Representative transmission electron microscopy images. (F) Quantification of observed bacteria per phagocyte (Values, mean + /- SEM; Unpaired t-test, **p < 0.01). Infected phagocytes were imaged and quantified from 3 biological replicates. Infected phagocytes were identified by the presence of black, electron dense bacteria. Each data point represents a single phagocyte. CFU, colony forming units. HPI, hours post infection. dpf, days post fertilization. Ro61-8048, Kmo inhibitor.
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