Exploiting CRISPR-Cas to manipulate Enterococcus faecalis populations.

Karthik Hullahalli,Kelli L Palmer,Marinelle Rodrigues

doi:10.7554/elife.26664

Karthik Hullahalli, Kelli L Palmer + Show 1 more

Open Access

https://doi.org/10.7554/elife.26664

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Journal: eLife	Publication Date: Jun 23, 2017
Citations: 45	License type: CC BY 4.0

Affiliation: The University of Texas at Dallas

Abstract

CRISPR-Cas provides a barrier to horizontal gene transfer in prokaryotes. It was previously observed that functional CRISPR-Cas systems are absent from multidrug-resistant (MDR) Enterococcus faecalis, which only possess an orphan CRISPR locus, termed CRISPR2, lacking cas genes. Here, we investigate how the interplay between CRISPR-Cas genome defense and antibiotic selection for mobile genetic elements shapes in vitro E. faecalis populations. We demonstrate that CRISPR2 can be reactivated for genome defense in MDR strains. Interestingly, we observe that E. faecalis transiently maintains CRISPR targets despite active CRISPR-Cas systems. Subsequently, if selection for the CRISPR target is present, toxic CRISPR spacers are lost over time, while in the absence of selection, CRISPR targets are lost over time. We find that forced maintenance of CRISPR targets induces a fitness cost that can be exploited to alter heterogeneous E. faecalis populations.

Highlights

Enterococcus faecalis is a Gram-positive opportunistic pathogen that commensally inhabits the gastrointestinal tracts of humans and other mammals (Lebreton et al, 2014)
We have previously reported that CRISPR-Cas lowers the frequency of acquisition of large (50–70 kbp) conjugative pheromone responsive plasmids (PRPs) in E. faecalis T11, a strain closely related to V583 but lacking the HGT-driven genome expansion characteristic of V583 (Price et al, 2016)
We inserted the oriT sequence from the pheromone-responsive plasmid pCF10 into the cloning vector pLZ12 (Perez-Casal et al, 1991), creating pKH12. pKH12 was linearized via PCR using primers with overhangs engineered to introduce different protospacers with the CRISPR1/CRISPR2 NGG protospacer adjacent motif (PAM) sequence (Price et al, 2016) to generate the pKHSX series, where X defines a spacer from our CRISPR dictionary (Hullahalli et al, 2015)

Summary

Introduction

Enterococcus faecalis is a Gram-positive opportunistic pathogen that commensally inhabits the gastrointestinal tracts of humans and other mammals (Lebreton et al, 2014). Sequence analysis of multidrug-resistant (MDR) isolates of E. faecalis reveals that they typically possess expanded genomes relative to more drug-sensitive isolates and have acquired large segments of mobile DNA in the form of prophage, genomic islands, transposons, and plasmids (Raven et al, 2016; Palmer et al, 2012; Paulsen et al, 2003; Bourgogne et al, 2008) These extraneous DNA elements often encode antibiotic resistance determinants and virulence factors that facilitate host infection and colonization, making the horizontal dissemination of DNA one of the prime causative factors for the emergence of MDR E. faecalis (Palmer et al, 2014).

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