citations

STING mediates immune responses in the closest living relatives of animals

Authors:

Arielle Woznica , Ashwani Kumar , Carolyn R Sturge , Chao Xing , Nicole King , Julie K Pfeiffer

In:

Source: eLife
Publication Date: (2021)
Issue: 10: 1-28

Research Area:

Basic Research

Platform:

4D-Nucleofector® X-Unit

Experiment

Nucleofection
Each transfection reaction was prepared by adding 2 µl of “primed” cells resuspended in SG buffer (Lonza) to a mixture of: 16 µl of SG buffer, 2 µl of 20 µg/µl pUC19, 1 µl of 250 mM ATP (pH 7.5), 1 µl of 100 mg/ml sodium heparin, and =7 µl of reporter DNA (volume is dependent on the number of constructs transfected). Each transfection reaction was transferred to one well in 16-well nucleofection strip (Lonza; Cat. No. V4XC-2032). The nucleofection strip was placed in the X-unit (Lonza; Cat. No. AAF-1002F) connected to a Nucleofector 4D core unit (Lonza; Cat. No. AAF-1002B), and the EO100 pulse was applied to each well.
Recovery
A total of 100 µl of cold recovery buffer (10 mM HEPES-KOH, pH 7.5; 0.9 M sorbitol; 8 % [wt/vol] PEG 8000) was added to the cells immediately after pulsation. After 5 min, the whole volume of the transfection reaction plus the recovery buffer was transferred to 2 ml of Low Nutrient Medium in a 12-well plate. The cells were grown for 24–48 hr before being assayed for luminescence or fluorescence.

Abstract

Animals have evolved unique repertoires of innate immune genes and pathways that provide their first line of defense against pathogens. To reconstruct the ancestry of animal innate immunity, we have developed the choanoflagellate Monosiga brevicollis, one of the closest living relatives of animals, as a model for studying mechanisms underlying pathogen recognition and immune response. We found that M. brevicollis is killed by exposure to Pseudomonas aeruginosa bacteria. Moreover, M. brevicollis expresses STING, which, in animals, activates innate immune pathways in response to cyclic dinucleotides during pathogen sensing. M. brevicollis STING increases the susceptibility of M. brevicollis to P. aeruginosa-induced cell death and is required for responding to the cyclic dinucleotide 2'3' cGAMP. Furthermore, similar to animals, autophagic signaling in M. brevicollis is induced by 2'3' cGAMP in a STING-dependent manner. This study provides evidence for a pre-animal role for STING in antibacterial immunity and establishes M. brevicollis as a model system for the study of immune responses.

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