Abstract
<h3>Abstract</h3> Dram1 is a stress and infection inducible autophagy modulator that functions downstream of transcription factors p53 and NFκB. Using a zebrafish embryo infection model, we have previously shown that Dram1 provides protection against the intracellular pathogen <i>Mycobacterium marinum</i> by promoting the p62-dependent xenophagy of bacteria that have escaped into the cytosol. However, the possible interplay between Dram1 and other anti-bacterial autophagic mechanisms remains unknown. Recently, LC3-associated phagocytosis (LAP) has emerged as an important host defense mechanism that requires components of the autophagy machinery and targets bacteria directly in phagosomes. Our previous work established LAP as the main autophagic mechanism by which macrophages restrict growth of <i>Salmonella</i> Typhimurium in a systemically infected zebrafish host. We therefore employed this infection model to investigate the possible role of Dram1 in LAP. Morpholino knockdown or CRISPR/Cas9-mediated mutation of Dram1 led to reduced host survival and increased bacterial burden during <i>S</i>. Typhimurium infections. In contrast, overexpression of <i>dram1</i> by mRNA injection curtailed <i>Salmonella</i> replication and reduced mortality of the infected host. During the early response to infection, GFP-Lc3 levels in transgenic zebrafish larvae correlated with the <i>dram1</i> expression level, showing over two-fold reduction of GFP-Lc3-<i>Salmonella</i> association in <i>dram1</i> knockdown or mutant embryos and an approximately 30% increase by <i>dram1</i> overexpression. Since LAP is known to require the activity of the phagosomal NADPH oxidase, we used a <i>Salmonella</i> biosensor strain to detect bacterial exposure to reactive oxygen species (ROS) and found that the ROS response was largely abolished in the absence of <i>dram1</i>. Together, these results demonstrate the host protective role of Dram1 during <i>S</i>. Typhimurium infection and suggest a functional link between Dram1 and the induction of LAP.
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