Enterococcus faecalis strains with compromised CRISPR-Cas defense emerge under antibiotic selection for a CRISPR-targeted plasmid
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Abstract
Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Non-MDR E. faecalis strains frequently possess CRISPR-Cas systems, which reduce the frequency of mobile genetic element (MGE) acquisition. We demonstrated in previous studies that E. faecalis populations can transiently maintain both a functional CRISPR-Cas system and a CRISPR-Cas target. In this study, we used serial passage and deep sequencing to analyze these populations. In the presence of antibiotic selection for the plasmid, mutants with compromised CRISPR-Cas defense and enhanced ability to acquire a second antibiotic resistance plasmid emerged. Conversely, in the absence of selection, the plasmid was lost from wild-type E. faecalis populations, but not E. faecalis populations that lacked the cas9 gene. Our results indicate that E. faecalis CRISPR-Cas can become compromised under antibiotic selection, generating populations with enhanced abilities to undergo horizontal gene transfer.
Importance
Enterococcus faecalis is a leading cause of hospital-acquired infections and disseminator of antibiotic resistance plasmids among Gram-positive bacteria. We have previously shown that E. faecalis strains with an active CRISPR-Cas system can prevent plasmid acquisition and thus limit the transmission of antibiotic resistance determinants. Yet, CRISPR-Cas was not a perfect barrier. In this study, we observed populations of E. faecalis with transient co-existence of CRISPR-Cas and one of its plasmid targets. Our experimental data demonstrate that antibiotic selection results in compromised E. faecalis CRISPR-Cas function, thereby facilitating the acquisition of additional resistance plasmids by E. faecalis .
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The MDR E. faecalis strain KUB3006, for which a completely closed genome is available (44), possesses 4 plasmids, including one that encodes linezolid resistance. Yet, it also encodes CRISPR3-Cas (44). The cas9 gene is frameshifted, and the frameshift occurs within the codon for one of the two Cas9 active sites that we previously experimentally confirmed in E. faecalis
Very cool!
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Overall, we posit that the interplay of CRISPR-Cas, plasmids, and antibiotic selection should be further investigated to understand the role of CRISPR-Cas in the antibiotic resistance crisis.
It would be fascinating to see how the presence of phage might shift this balance!
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No mutations were identified in the S6 protospacer or the PAM region of the repB gene in pAM714.
Curious that no protospacer mutations were found! I'm assuming that the plasmid replicates to a high copy number than the host chromsome, meaning there would be more opportunity for mutation to happen in the plasmid relative to the chromosome. I wonder if you would see the same patterns if the adaptation machinery were absent from the strain. Can you comment on why you think chromosomal mutations are favored over plasmid mutations ?
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We sequenced the cas9 coding region of the WT4 population from Day 0 and identified a mutation resulting in an Ala749Thr substitution. Ala749 occurs within the RuvC nuclease domain and is conserved in the well-studied Cas9 of Streptococcus pyogenes (17). The Ala749Thr substitution may result in loss in Cas9 function, causing WT4 to phenocopy the Δ1-Δ6 populations
Interesting that this this mutation was present in day 0 - do you think this Cas9 deactivation mutation was pre-existing in the WT population, and the conjugation experiment directly selected for it? Do you think that there is any low level toxicity of the CRISPR system in WT strains (even in the absence of selection) that might be generating CRISPR-null mutants at some rate?
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