Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

Abstract

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, and only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in the absence of Dram1. The phagocytic ability of macrophages in 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 increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (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 the absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic 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.

DRAM1 requires PI(3,5)P2generation by PIKfyve to deliver vesicles and their cargo to endolysosomes

“…When host cells detect pathogenic mycobacteria that cause tuberculosis, DRAM1 expression is activated downstream of the immunity regulating transcription factor NFκB (van der Vaart et al, 2014). Knockdown or knockout of dram1 increased susceptibility to mycobacterial infection in zebrafish, identifying Dram1 as a host resistance factor (van der Vaart et al, 2014;Zhang et al, 2020). In support of this finding, overexpression of dram1 was protective against mycobacterial infection by enhancing autophagic defences against intracellular bacteria and stimulating vesicle fusion events with lysosomes (van der Vaart et al, 2014).…”

“…This role of Dram1 is important in defence against mycobacterial infection in the zebrafish model for tuberculosis, and mammalian DRAM1 was found to associate with Mycobacterium tuberculosis phagocytosed by primary human macrophages (van der Vaart et al, 2014). Furthermore, we recently described how zebrafish macrophages lacking Dram1 failed to deliver pathogenic mycobacteria to acidic endolysosomal compartments, ultimately resulting in an inflammatory type of cell death -called pyroptosis -which disseminates the infection (Zhang et al, 2020). Besides its function in vesicle trafficking, DRAM1 is required for apoptosis mediated by the tumour suppressor p53 in relation to cancer and in HIV infected CD4(+) T cells (Crighton et al, 2006;Laforge et al, 2013).…”

Development of a seven-gene tumor immune microenvironment prognostic signature for high-risk grade III endometrial cancer

“…DRAM1 is a stress-induced regulator of autophagy and cell death associated with cancer, myocardial infarction, and infectious disease. 42 Its acts as a tumor inhibitor in non-small-cell lung carcinoma (NSCLC) by promoting epidermal growth factor receptor (EGFR) lysosome degradation and regulates autophagy as well as cell proliferation by inhibiting the phosphatidylinositol 3-kinase-Akt-mechanistic target of rapamycin (mTOR)-ribosomal protein S6 pathway. 43 , 44 It has been reported that DNER promotes the epithelial-to-mesenchymal transition in breast cancer through the Wnt/β-catenin pathway.…”

Autophagy and Host Defense in Nontuberculous Mycobacterial Infection

“…Indeed, DRAM1 functions through autophagic targeting and phagosomal maturation of M. marinum , thereby restricting bacteria during the early phase of infection. Dissemination of M. marinum infection is associated with defective autophagy and gasdermin Eb-mediated pyroptotic cell death in dram1 mutant zebrafish larvae ( 82 ). However, it remains to be characterized whether DRAM1 plays a crucial role in host defense to other NTM strains through activation of autophagy and prevention of cell death.…”