Dram1 deficiency enhances mycobacterial disease progression due to pyroptotic cell death of infected macrophages

DNA Damage Regulated Autophagy Modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish dram1 mutant and investigated its loss-of-function effects during Mycobacterium marinum (Mm) infection, a widely used model in TB research. In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, whereas only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, while autophagic targeting of Mm was reduced in absence of Dram1, despite overall increased Lc3-II accumulation. The phagocytic ability of dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting enhanced bacterial burden phenotype could be rescued by knockdown of inflammatory caspase (caspa) and gasdermin (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic cell death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death. Overall design: Mutant embryos and their controls were manually dechorionated at 24 hours post fertilization (hpf) and at 28 hpf they were infected by injecting 150 or 300 colony forming units of M. marinum strain M into the blood island, or mock-injected with PBS/2%PVP. After injections embryos were transferred into fresh egg water containing 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) to prevent melanisation and incubated for 4 days at 28,5°C. After the incubation period, infected and uninfected mutants and their controls were imaged and groups of 20 embryos were snap-frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.

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Zhang R, Varela M, Forn-Cuní G, Torraca V, van der Vaart M, Meijer AH