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 Mycobacterium marinum 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 Salmonella 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 S. Typhimurium infections. In contrast, overexpression of dram1 by mRNA injection curtailed Salmonella replication and reduced mortality of the infected host. During the early response to infection, GFP-Lc3 levels in transgenic zebrafish larvae correlated with the dram1 expression level, showing over two-fold reduction of GFP-Lc3-Salmonella association in dram1 knockdown or mutant embryos and an approximately 30% increase by dram1 overexpression. Since LAP is known to require the activity of the phagosomal NADPH oxidase, we used a Salmonella biosensor strain to detect bacterial exposure to reactive oxygen species (ROS) and found that the ROS response was largely abolished in the absence of dram1. Together, these results demonstrate the host protective role of Dram1 during S. Typhimurium infection and suggest a functional link between Dram1 and the induction of LAP.

1Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to 3 2 lysozyme, a key effector of the innate immune system. This high level of resistance requires 3 3 several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic 3 4 and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes 3 5 modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in 3 6 the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent 3 7insertions clustered on the chromosome. These mutations were located in a locus referred to as 3 8 the enterococcal polysaccharide antigen (EPA) variable region located downstream of the 3 9 highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa 4 0 variable region was previously proposed to be responsible for EPA decorations, but the role of 4 1 this locus remains largely unknown. Here, we show that EPA decoration contributes to 4 2 resistance towards charged antimicrobials and underpins virulence in the zebrafish model of 4 3 infection by conferring resistance to phagocytosis. Collectively, our results indicate that the 4 4