The CRISPR/Cas Adaptive Immune System of Pseudomonas aeruginosa Mediates Resistance to Naturally Occurring and Engineered Phages

Abstract

bHere we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/ Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function. C lustered regularly interspaced short palindromic repeats (CRISPR) are found in roughly 50% of sequenced bacterial genomes and 90% of archaeal genomes (2, 24). CRISPR regions are composed of multiple repeated sequences ranging from 21 to 48 bp in length separated by 26-to 72-bp spacers (1, 2). The sequences of spacer regions are variable but are often identical to sequences found within phages, plasmids, or other foreign DNA (24). CRISPR loci are transcribed as one large transcript that is processed within the identical repeat sequence into mature, small CRISPR RNAs (crRNAs) by either CRISPR-associated (Cas) proteins alone or RNase III associated with Cas proteins (5, 11, 15). These mature crRNAs are then complexed with subtype-specific Cas proteins, and this complex specifically interacts with nucleotide target sequences complementary to spacer sequences (5,14,16,26). Complementarity between mature crRNAs and sequences found within phages or plasmids leads to inhibition of their replication through target nucleotide cleavage (12,14,25). The ability of CRISPR/Cas systems to also incorporate DNA sequences from newly encountered foreign DNA and subsequently resist phages or plasmids containing these sequences has led these systems to be referred to as bacterial adaptive immune systems (24).Pseudomonas aeruginosa is an opportunistic pathogen of humans and animals that is capable of becoming highly antibiotic resistant (18); thus, it has become a model for new or previously overlooked antibacterial ...

Quorum sensing controls the Pseudomonas aeruginosa CRISPR-Cas adaptive immune system

“…To do this, we investigated whether QS controls CRISPR-Cas in the pathogen Pseudomonas aeruginosa (21). We used P. aeruginosa UCBPP-PA14 (denoted PA14), which has a type I-F CRISPR-Cas system (22) that provides phage resistance (8,23,24). In PA14, two CRISPR regions flank the cas genes: cas1, cas3, and csy1-4.…”

Immigration of susceptible hosts triggers the evolution of alternative parasite defence strategies

“…The bacterial strains used in this study are P. aeruginosa UCBPP-PA14 (wild-type, WT), UCBPP-PA14 csy3::LacZ (CRISPR-Cas KO) and the CRISPR-Cas KO-derived surface mutant sm1, which have been described previously [10,25]. The CRISPR-Cas KO strain has a non-functional CRISPR-Cas system due to disruption of the essential csy3 gene.…”

“…In addition, BIM-4sp was used, which is a WT-derived strain that acquired four spacers against phage DMS3vir. Phage DMS3vir is a previously described mutant of phage DMS3 that cannot enter the lysogenic life cycle owing to mutation of the repressor gene [10,25].…”

Holding a grudge

“…73 Indeed, almost all CRISPR spacers that matched database sequences in clinical isolates of P. aeruginosa targeted prophages, 74 and CRISPR/Cas has been shown to limit growth of lysogenic phages in this species. 75 Another intriguing finding revealed a role for the Cas protein complex in inhibition of a defective prophage through repeat-directed cleavage of a prophage RNA transcript, independent of spacer targeting, in the haloarchaeon Haloferax mediterranei. 76 Taken together, these findings provide strong impetus to the concept that in some ecological niches, the role of CRISPR/ Cas may be much more multi-faceted than previously appreciated.…”