Novel signalling pathways in type III CRISPR defence systems

Abstract

CRISPR-Cas systems offer prokaryotes an adaptive defence mechanism, allowing them to respond to the invading nucleic acids. Type III CRISPR systems feature the capacity of synthesising cyclic oligoadenylate (cOA) species, which serve as second messengers to activate ancillary effectors, enhancing immune response. A diverse array of ancillary proteins is predicted to participate in cOA-mediated signalling for immunity enhancement. Nevertheless, the specific functions of many of these ancillary effectors have remained elusive. Here we have unravelled the workings of two novel type III-B CRISPR systems. The first system, from the human gut bacteria Bacteroides fragilis (BfrCmr), associates with an uncharacterised CorA family membrane protein and a NrN family phosphodiesterase. BfrCmr provides defence against mobile genetic elements when expressed in the heterologous host E. coli. A remarkable discovery was the identification of a novel signal molecule, S-adenosyl methionine (SAM)-AMP by conjugating ATP to SAM through a phosphodiester bond, when the BfrCmr system was activated. SAM-AMP in turn binds to the membrane protein CorA, presumably leading to membrane disruption and ultimately cell death. The cognate phosphodiesterase NrN or SAM lyase from Clostridium botulinum degrades SAM-AMP, offering two different means of regulating the signalling pathway.

The second type III CRISPR system investigated is associated with three ancillary proteins, including a Lon protease CalpL, extracytoplasmic function sigma factor CalpS and a toxin MazF homologue CalpT. CalpL consist of a SAVED sensor domain fused with a Lon protease effector domain. CalpL forms a tripartite complex with CalpS and CalpT. When SAVED domain bound to activator cA4, CalpL oligomerises and specifically cleaves CalpT, resulting in the release of the sigma factor CalpS from the complex. This identification of a SAVED domain-containing protease that responses to cOA and triggers the transcriptional regulation provides insights into the sophisticated multi-layered defence mechanisms characterised in type III CRISPR signal-mediated immunity.