Selective autophagy receptors Optn and p62 can compensate for each other’s loss-of-function during mycobacterial infection in zebrafish. (A). Schematic diagram showing the current model of the roles of autophagy modulator Dram1 and selective autophagy receptors Optn and p62 in defense against Mm infection in zebrafish. Dram1 is induced after pathogen recognition by Tlr-Myd88-NFκB signaling and localizes predominantly to lysosomes, where it is proposed to facilitate the fusion with autophagosomes. The ubiquitin receptors Optn and p62 mediate the selective autophagy (xenophagy) of cytosolic bacteria. Dram1, Optn and p62 have all been shown to contribute to the defense response of the zebrafish host to mycobacterial infection. (B) Overexpression of optn in p62 wildtype and mutant background. In the experimental workflow, optn mRNA was injected into p62 +/+ and -/- embryos at the one cell stage, followed by injection of 200 CFU of Mm into the blood island at 28 hpf, and assessment of bacterial burden at 4 dpi (representative images shown). Quantification shows that overexpression of optn could decrease bacterial burden independent of p62. (C) Overexpression of p62 in optn wildtype and mutant background. In the experimental workflow, p62 mRNA was injected into optn +/+ and -/- embryos at the one cell stage, followed by infection and bacterial burden assessment as in (C) (representative images shown). Quantification shows that overexpression of p62 could decrease bacterial burden independent of optn. Data are displayed as percentage difference to the control group set at 100% and are accumulated from three independent infection experiments (each data point representing an individual embryo), indicated with different colors. * p<0.05, **p<0.01,***p<0.001.

Double mutation of optn and p62 increases the susceptibility to Mm infection but combined overexpression has no additive effect. (A) Comparison of single and double optn/p62 mutants. Four different genotypes were infected with 200 CFU of Mm following the indicated experimental workflow and assessed for bacterial burden at 4 dpi (representative images shown). Quantification shows that optn/p62 double mutant embryos were hypersusceptible compared to the wild type and single mutants. (B) Comparison of separate and combined overexpression of optn/p62. One cell stage embryos were injected with 100 pg optn mRNA, 100 pg p62 mRNA, a combination of 50 pg optn mRNA and 50 pg of p62 mRNA, or buffer as a control. The subsequent workflow for infection and analysis of bacterial burden (representative images shown) was the same as in (A). Quantification shows that the combined overexpression of optn and p62 was less effective in reducing bacterial burden. Data are displayed as percentage difference to the control group set at 100% and are accumulated from two (A) or three (B) independent infection experiments (each data point representing an individual embryo), indicated with different colors. ns, non-significant, * p<0.05, ***p<0.001.

Optn and p62 can protect against Mm independently of the autophagy modulator Dram1. (A) Overexpression of optn in dram1 wildtype and mutant background. In the experimental workflow, optn mRNA was injected into dram1 +/+ and -/- embryos at the one cell stage, followed by injection of 200 CFU of Mm into the blood island at 28 hpf and assessment of bacterial burden at 4 dpi (representative images shown). Quantification shows that optn overexpression could decrease bacterial burden independent of dram1. (B) Overexpression of p62 in dram1 wildtype and mutant background. Overexpression of p62 was studied by an experimental workflow analogous to that in (A) and found to decrease bacterial burden independent of dram1. Data are displayed as percentage difference to the control group set at 100% and are accumulated from three independent infection experiments (each data point representing an individual embryo), indicated with different colors. ns, non-significant, * p<0.05, **p<0.01, ***p<0.001.

Dram1 can protect against Mm in the absence of Optn and p62. (A-C)dram1 overexpression in single and double mutants of optn and p62. In the experimental workflow, dram1 mRNA was injected at the one cell stage into optn+/+ and -/- embryos (A), p62+/+ and -/- embryos (B), optn+/+p62+/+ and optn-/-p62-/- embryos (C), followed by injection of 200 CFU of Mm into the blood island at 28 hpf and assessment of bacterial burden at 4 dpi (representative images shown). Quantifications show that dram1 could decrease bacterial burden independent of the loss of optn(A), p62(B) or both receptors (C). (D) Overexpression of dram1 in myd88 wild type and mutant background. dram1 mRNA was injected into myd88+/+ and myd88-/- embryos at the one cell stage, followed by infection and bacterial burden assessment as in (A-C) (representative images shown). Quantification showed that dram1 overexpression could not decrease bacterial burden in myd88 mutants. Data are displayed as percentage difference to the control group set at 100% and are accumulated from three (A-C) or two (D) independent infection experiments (each data point representing an individual embryo), indicated with different colors. ns, non-significant, * p<0.05, ***p<0.001.

Dram1 increases the colocalization between Lc3 and Mm in optn/p62 double mutant lines. Effect of dram1 overexpression on the colocalization between Mm and GFP-Lc3 in optn/p62 double mutant and wildtype background. In the experimental workflow (A), dram1 mRNA was injected into optn+/+p62+/+ and optn-/-p62-/- embryos at the one cell stage, followed by injection of 200 CFU of Mm into the blood island at 28 hpf and imaging of a region of interest (ROI) in the caudal hematopoietic region at 2 dpi (representative confocal microscopy images shown). (B) Quantification shows that dram1 overexpression increased Mm/GFP-Lc3 colocalization independent of optn and p62. Complete or partial overlap of GFP-Lc3 with Mm was considered as co-localizing events. Data are displayed as percentage of GFP-Lc3-positive Mm clusters relative to the total number of Mm clusters and are accumulated from three independent infection experiments, of which the data points (each representing a single ROI from an individual embryo) are indicated with different colors. (C) Quantification shows that dram1 overexpression increased Mm and LysoTracker colocalization independent of optn and p62. Complete or partial overlap of LysoTracker with Mm was considered as co-localizing events. Data are accumulated from three independent experiments (each data point representing a single ROI from an individual embryo)* p<0.05, ** p<0.01.