Uncovering New Mechanisms of Type III CRISPR-Cas System Inhibition and Effector Function
Bacteria and their viruses, called bacteriophages (phages), are in a constant arms race for survival. Phages are one of the most abundant biological entities in the world, with an estimated 1031on earth, outnumbering bacteria ten to one. To defend against phages, bacteria have evolved numerous defense mechanisms. One such defense system is the bacterial adaptive immune system called CRISPR-Cas, which consists of clustered, regularly interspaced short palindromic repeats (CRISPRs) and a set of genes that encode CRISPR-associated (Cas) proteins. While there are seven distinct types of CRISPR-Cas systems identified thus far, we focus on specifically the type III CRISPR system which contains a multi-protein effector complex that functions together with its crRNA in target binding and cleavage. During type III CRISPR-Cas immunity in prokaryotes, RNA-guided recognition of viral (phage) transcripts stimulates the Cas10 complex to convert ATP into cyclic oligoadenylates. These act as signaling molecules that bind to CRISPR-associated Rossmann Fold (CARF) proteins and activate their effector domains. The effector part unleashes various activities that are toxic to the host cell, inhibiting cell growth and leading to growth arrest. We are now beginning to uncover that CARF effectors use a wide array of mechanisms of action. In the first part of this study, we report the structure and function of the Cap1 effector, composed of a pair of transmembrane helices (TM1/2), a CARF-like (CARFL) domain and a domain of unknown function (DUF4579). Cryo-EM studies on apo-and ligand-bound states of Cap1 in glyco-diosgenin detergent revealed the formation of tetrameric complexes in both states, with one cyclic tetra-adenylate (cA4) molecule bound in a pocket composed by the four CARFL domains. Binding of cA4 triggers conformational changes that widen an otherwise narrow pore formed by the four TM1/2 domains. In vivo, Cap1 activation results in membrane depolarization, a growth arrest of the bacterial host and the abrogation of the viral lytic cycle. Our findings reveal the mechanistic basis of membrane depolarization mediated by cyclic nucleotide signaling during the type III CRISPR-Cas response. On the other side of the bacteria-phage arms race, phages have evolved mechanisms to counteract CRISPR-Cas; many viruses express anti-CRISPR (Acr) proteins that interact directly with Cas proteins and inactivate them. To date, there are over 40 Acrs of the type II CRISPR-Cas systems, but in comparison, there are only 4 confirmed Acrs of the type III systems. To identify novel inhibitors of the type III-A system, we performed a transformation screen with a type III-A CRISPR system in E.coli and environmental DNA (eDNA). In the second part of this study, we identified a single open reading frame, ORF6, from the MA1 eDNA library. We show that ORF6 inhibits CRISPR immunity against plasmids and phages in vivo and has high sequence and structural homology to known oligoribonucleases. As we continue to elucidate ORF6’s mechanism of action, we hypothesize that, while it may not be phage encoded, ORF6 could reveal an interesting incompatibility with the type III CRISPR-Cas system that will deepen our understanding of the function of type III CRISPR in bacteria.
CRISPR-Cas, CARF effector, anti-CRISPRs, Type III, CRISPR-Cas system, Life Sciences
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