CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23

Area of Science:

• Molecular Biology
• Microbiology
• Immunology

Background:

• CRISPR-Cas systems confer adaptive immunity in prokaryotes.
• Type III CRISPR systems utilize effector proteins activated by cyclic nucleotide second messengers to combat viral infections.
• Csx23 is an uncharacterized effector protein prevalent in Vibrio cholerae.

Purpose of the Study:

• To characterize the uncharacterized effector Csx23 from Vibrio cholerae.
• To elucidate the mechanism of action of Csx23 in CRISPR-Cas mediated immunity.
• To investigate the structural and functional properties of Csx23.

Main Methods:

• Expression of Csx23 in Escherichia coli with its cognate type III CRISPR system.
• Structural studies to determine the protein fold and ligand binding.
• Pulse Electron Paramagnetic Resonance (EPR) spectroscopy to study conformational changes.

Main Results:

• Csx23 localizes to the membrane via an N-terminal transmembrane domain.
• The C-terminal domain binds cyclic tetra-adenylate (cA4), activating defense function.
• Structural analysis revealed a novel tetrameric fold specific for cA4 binding.
• Binding of cA4 induces significant perturbation of the transmembrane domain, suggesting pore formation or membrane disruption.

Conclusions:

• Csx23 represents a new class of cyclic nucleotide-binding proteins.
• Csx23 functions as a membrane-associated effector in Type III CRISPR-Cas systems.
• The mechanism involves cA4-mediated disruption of membrane integrity for prokaryotic defense.