Balancing of immune activation and suppression during phage infection

Area of Science:

• Molecular Biology
• Bacteriology
• Immunology

Background:

• Signaling-based bacterial immunity systems (e.g., CBASS, Thoeris) use nucleotide signals to activate anti-phage effectors.
• Mechanisms of phage evasion and the specific phage features targeted by these bacterial defenses are not fully understood.

Purpose of the Study:

• Investigate phage evasion strategies against CBASS and Thoeris immunity in *Pseudomonas aeruginosa*.
• Identify phage-encoded factors responsible for evading bacterial signaling immunity.

Main Methods:

• Comparative genomic analysis of *Migulavirinae* and related N4-like phages.
• Functional characterization of phage genes involved in evading bacterial defenses.
• Biochemical assays to study protein interactions and molecular sponge activity.

Main Results:

• Closely related phages exhibit allelic variations in side tail fiber proteins influencing Thoeris sensitivity.
• An 'anti-defense hotspot' with three genes confers resistance to both CBASS and Thoeris defenses.
• A novel Thoeris anti-defense (Tad) protein was identified, functioning as a molecular sponge for NAD-derived molecules (e.g., gcADPR).
• The Tad protein shares sequence and structural similarity with a human ryanodine receptor domain.

Conclusions:

• Phage evasion relies on a balance between immune activation and antagonism, mediated by specific protein variations and defense-counteracting genes.
• The discovery of the Tad protein highlights a novel phage anti-immunity mechanism and reveals an unexpected evolutionary link between phage and human proteins.