Stella, G. | Search bioRxiv

bioRxiv2026

A lipid fenestration-gated membrane depolarization mechanism expands the repertoire of CRISPR-mediated anti-phage defense strategies

Prokaryotic Type III CRISPR-Cas systems synthesize cyclic oligoadenylate (cOA) second messengers that activate CRISPR-associated Rossmann fold (CARF) immune effectors during viral infection. Here we characterize Chp1, a membrane-embedded CARF effector that assembles as a tetrameric pore harboring autoinhibitory lipid fenestrations that occlude the pore in the inactive state. cOA binding triggers a structural rearrangement that (i) closes the fenestrations, (ii) eliminates the lipid obstruction of the pore to open it, and (iii) remodels the pore entrance from hydrophobic to polar to allow ion permeation. This conformational switch triggers depolarization of the membrane of the infected cell, which enters growth arrest and becomes inhospitable for viral replication. Our results uncover a lipid-gated mechanism that expands the repertoire of CRISPR-mediated defense strategies.

microbiology↗

bioRxiv2025

A CARF-HAD phosphatase effector provides immunity during the type III-A CRISPR-Cas response

CRISPR-Cas systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, Type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain, which we named Chp. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.

microbiology↗