DRAM1 confers resistance to Salmonella infection

Abstract

DRAM1 is an infection inducible autophagy modulator, previously shown to promote autophagic and lysosomal defense responses against the intracellular pathogen Mycobacterium marinum. However, its possible role in other anti-bacterial autophagic mechanisms remains unknown. Recently, LC3-associated phagocytosis (LAP) has emerged as autophagy-related mechanism that 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 infection. 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-Salmonella associations were reduced in dram1 knockdown or mutant embryos, and increased 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 with deficiency of dram1, while it was increased with dram1 overexpression. Corroborating these results in a mammalian model, the LC3 and ROS responses to Salmonella were similarly reduced or increased by knockdown or overexpression of Dram1, respectively, in murine RAW264.7 macrophages. Together, these results demonstrate the host protective role of Dram1/DRAM1 during S. Typhimurium infection and suggest a functional link between Dram1/DRAM1 and the induction of LAP.Abbreviations: ATG8: Autophagy related protein 8; ATG16: Autophagy related protein 16; CFU: colony-forming unit; DRAM1: DNA damage regulated autophagy modulator gene 1; dpf: days post fertilization; GFP: green fluorescent protein; hpi: hours post infection; LAP: LC3 associated phagocytosis; LC3, microtubule-associated protein 1 light chain 3; NADPH: Nicotinamide dinucleotide phosphate; p53: Tumor suppressor protein 53: ROS; reactive oxygen species; S. Typhimurium: Salmonella enterica serovar Typhimurium; TIPTP: 2(tetrahydroindazolyl) phenoxy-N-(thiadiazolyl)propenamide 2; UVRAG: UV radiation resistance associated protein