Enterococcus Faecalis Genome Defense Systems and Their Impact on Conjugative Antibiotic Resistance Plasmid Transfer

Enterococcus faecalis is a Gram-positive bacterium that naturally colonizes humans and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Considering that bacteria commonly possess two genome defense mechanisms to prevent MGE acquisition, restriction-modification (R-M, analogous to an innate immune system) and CRISPR-Cas (adaptive immune system), we hypothesize that these barriers may have been compromised in MDR E. faecalis strains. However, little was known about the activities of E. faecalis R-M and CRISPR-Cas systems. In my dissertation, a functional E. faecalis OG1RF encoded R-M system was identified and its activity against MGEs was confirmed using both conjugation and transformation assays. This work was the first to demonstrate that R-M provides E. faecalis with significant defense capability against antibiotic resistance plasmids. Subsequently, the distribution of R-M systems in a larger collection of E. faecalis strains was studied. To predict the novel R-M systems, I developed an R-M prediction algorithm based on amino acid sequence homology, and successfully predicted new R-M systems in 75 E. faecalis genomes. Remarkably, some lineage-specific R-M systems were detected. Especially, hospital-adapted lineages were found to be enriched for certain R-M systems, suggesting that these bacteria can readily exchange DNA with each other. Another active form of genome defense in E. faecalis, namely CRISPR-Cas, has also been investigated. In experimental in vitro evolution studies, we observed that chromosomally-encoded CRISPR-Cas systems tend to be compromised upon enforced maintenance of antibiotic resistance plasmids possessing sequences targeted by CRISPR-Cas. Using deep sequencing, we found that CRISPR array alleles are naturally heterogeneous, which provides an evolutionary basis for compromised CRISPR-Cas under selection pressure. This work demonstrates that antibiotic use can inadvertently select for E. faecalis with enhanced abilities to acquire mobile genetic elements. Finally, I studied lytic enterococcal phages for their interactions with E. faecalis hosts. This work was undertaken because phage therapy is increasingly of interest as an alternative to antibiotics for infection treatment. The genome modification status of one novel enterococcal phage was characterized, and the phage was found to be modified at most cytosine residues. This phage evades E. faecalis R-M defense, most likely due to this ubiquitous genome modification. That the phage encodes an anti-R-M strategy is beneficial for phage therapy applications.