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Metapopulation Structure of CRISPR-Cas Immunity in Pseudomonas aeruginosa and Its Viruses.

ABSTRACT: Viruses that infect the widespread opportunistic pathogen Pseudomonas aeruginosa have been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution of P. aeruginosa and its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark (R. L. Marvig, L. M. Sommer, S. Molin, and H. K. Johansen, Nat Genet 47:57-64, 2015, https://doi.org/10.1038/ng.3148). We show that new spacers are not added to or deleted from CRISPR arrays over time within a single patient but do vary among patients in this data set. We compared assembled CRISPR arrays from this data set to CRISPR arrays extracted from 726 additional publicly available P. aeruginosa sequences to show that local diversity in this population encompasses global diversity and that there is no evidence for population structure associated with location or environment sampled. We compare over 3,000 spacers from our global data set to 98 lytic and temperate viruses and proviruses and find a subset of related temperate virus clusters frequently targeted by CRISPR spacers. Highly targeted viruses are matched by different spacers in different arrays, resulting in a pattern of distributed immunity within the global population. Understanding the multiple immune contexts that P. aeruginosa viruses face can be applied to study of P. aeruginosa gene transfer, the spread of epidemic strains in cystic fibrosis patients, and viral control of P. aeruginosa infection. IMPORTANCE Pseudomonas aeruginosa is a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating how P. aeruginosa uses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicate P. aeruginosa and its viruses evolve in both a broad global population and in isolated human "islands." These data set the stage for exploring metapopulation dynamics occurring within and between isolated "island" populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains.

SUBMITTER: England WE

PROVIDER: S-EPMC6199469 | biostudies-literature | 2018 Sep-Oct

REPOSITORIES: biostudies-literature

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Metapopulation Structure of CRISPR-Cas Immunity in <i>Pseudomonas aeruginosa</i> and Its Viruses.

England Whitney E WE Kim Ted T Whitaker Rachel J RJ

mSystems 20180901 5

Viruses that infect the widespread opportunistic pathogen Pseudomonas aeruginosa have been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution of P. aeruginosa and its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark (R. L. Marvig, L. ...[more]

PMID: 30374457

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Project description:UnlabelledPseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates.ImportanceP. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Project description:CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. IMPORTANCE The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts.

Project description:CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of cas genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for Pseudomonas aeruginosa strains with new spacer acquisition. We used this assay to demonstrate that P. aeruginosa rapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the P. aeruginosa type I-F system, the cas1 gene is required for CRISPR adaptation, recG contributes to (but is not required for) primed CRISPR adaptation, recD is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer.ImportanceOur understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants of Escherichia coli, or cas1-cas2 overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allows P. aeruginosa to rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system of P. aeruginosa Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that many P. aeruginosa strains maintain a CRISPR-Cas system.

Dataset Information

Metapopulation Structure of CRISPR-Cas Immunity in Pseudomonas aeruginosa and Its Viruses.

Publications

Metapopulation Structure of CRISPR-Cas Immunity in <i>Pseudomonas aeruginosa</i> and Its Viruses.

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