CRISPR-Cas systems constitute an adaptive immune system that provides acquired resistance against phages and plasmids in prokaryotes. Upon invasion of foreign nucleic acids, some cells integrate short fragments of foreign DNA as spacers into the CRISPR locus to memorize the invaders and acquire resistance in the subsequent round of infection. This immunization step called adaptation is the least understood part of the CRISPR-Cas immunity. We have focused here on the adaptation stage of DGCC7710 type I-E CRISPR4-Cas (St4) system. Cas1 and Cas2 proteins conserved in nearly all CRISPR-Cas systems are required for spacer acquisition. The St4 CRISPR-Cas system is unique because the Cas2 protein is fused to an additional DnaQ exonuclease domain. Here, we demonstrate that St4 Cas1 and Cas2-DnaQ form a multimeric complex, which is capable of integrating DNA duplexes with 3'-overhangs (protospacers) We further show that the DnaQ domain of Cas2 functions as a 3'-5'-exonuclease that processes 3'-overhangs of the protospacer to promote integration.
Highlights d Cryo-EM structures of three different assemblies of the Cas1, Cas2, and Csn2 proteins d One form, with the stoichiometry Cas1 8-Cas2 4-Csn2 8 , contains 30 bp duplex DNA d Another comprises two of these units with an additional Cas1 8-Cas2 4 interface d Filaments comprise Cas1 8-Cas2 4-Csn2 8 units around a continuous DNA duplex
Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as giant pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which coincided with genes important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and showed that glucosylceramide is important, demonstrating that membrane organization plays a pivotal role in ILY activity. We also cross-validated genes discovered in the ILY screen with vaginolysin and pneumolysin and found that pneumolysin’s cytolytic activity strongly depends on the asymmetric distribution of the membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify the genes involved in biological membrane composition and metabolism.
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