Identification of DEAD-Box RNA Helicase DDX41 as a Trafficking Protein That Involves in Multiple Innate Immune Signaling Pathways in a Zebrafish Model

Abstract

DDX41 is an important sensor for host recognition of DNA viruses and initiation of nuclear factor-κB (NF-κB) and IFN signaling pathways in mammals. However, its occurrence and functions in other vertebrates remain poorly defined. Here, a DDX41 ortholog [Danio rerio DDX41 (DrDDX41)] with various conserved structural features to its mammalian counterparts was identified from a zebrafish model. This DrDDX41 was found to be a trafficking protein distributed in the nucleus of resting cells but transported into the cytoplasm under DNA stimulation. Two nuclear localization signal motifs were localized beside the coiled-coil domain, whereas one nuclear export signal motif existed in the DEADc domain. DrDDX41 acts as an initiator for the activation of NF-κB and IFN signaling pathways in a Danio rerio STING (DrSTING)-dependent manner through its DEADc domain, which is a typical performance of mammalian DDX41. These observations suggested the conservation of DDX41 proteins throughout the vertebrate evolution, making zebrafish an alternative model in understanding DDX41-mediated immunology. With this model system, we found that DrDDX41 contributes to DrSTING–Danio rerio STAT6 (DrSTAT6)-mediated chemokine (Danio rerio CCL20) production through its DEADc domain. To the best of our knowledge, this work is the first report showing that DDX41 is an upstream initiator in this newly identified signaling pathway. The DrDDX41-mediated signaling pathways play important roles in innate antibacterial immunity because knockdown of either DrDDX41 or DrSTING/DrSTAT6 significantly reduced the survival of zebrafish under Aeromonas hydrophilia or Edwardsiella tarda infection. Our findings would enrich the current knowledge of DDX41-mediated immunology and the evolutionary history of the DDX41 family.

Characterization of cGAS homologs in innate and adaptive mucosal immunities in zebrafish gives evolutionary insights into cGAS‐STING pathway

“…pRL‐TK renilla luciferase reporter vector was purchased from Promega. Zebrafish IFNφ1/3 reporter plasmids were produced in our laboratory 34 . The primers used for construct generation are listed in Table S1.…”

“…Embryos were collected 24 hours after microinjection and GFP fluorescence was visualized via fluorescence microscopy (Axiovert 40 CFL; Carl Zeiss, Oberkochen, Baden‐Württemberg, Germany). MOs against Dr STING mRNA ( Dr STINGMO) was used, and its efficiency was tested in a previous study 34 …”

“…HEK293T cells were transfected with Dr cGASa/b/ Dr STING expression plasmids and IFN‐β or NF‐κB luciferase reporter construct (200 ng/mL; Clontech, San Francisco, CA, USA) and pGL‐TK Renilla luciferase internal control (20 ng/mL; Clontech, San Francisco, CA, USA). After 24 hours, the relative luciferase activity unit was determined using a Dual‐Luciferase reporter assay system (Promega, Madison, Wis., USA) as previously described 34 . Meanwhile, the mRNA level of CCL20 was examined by real‐time PCR.…”

“…For antibacterial assay, the Dr cGASa/b/ Dr STING morphornts and wild‐type control embryos at 24 hours post fertilization (hpf) were exposed to A hydrophila or Edwardsiella tarda ( E tarda , 1 × 10 8 CFU/mL) 34,39 . After immersion in the bacterial suspension for 5 hours, the mortality of each group was recorded at 12‐72 hours post infection (hpi).…”

“…For 2′3′‐cGAMP antibacterial assay, adult zebrafish were stimulated with 2′3′‐cGAMP or PBS (control) for 12 hours, followed by i.p challenge with A hydrophila (2.1 × 10 5 CFU). Mortality in each group was monitored during the 5 day‐period at 24 hours of interval and the relative survival rate (RSR) was calculated as previously described 34 …”