Browsing by Author "Huo, Wenwen"
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Item A Type I Restriction-Modification System Associated with Enterococcus Faecium Subspecies Separation(Amer Soc Microbiology, 2019-01-09) Huo, Wenwen; Adams, Hannah M.; Trejo, Cristian; Badia, Rohit; Palmer, Kelli L.; 0000-0002-7343-9271 (Palmer, KL); Huo, Wenwen; Adams, Hannah M.; Trejo, Cristian; Badia, Rohit; Palmer, Kelli L.The gastrointestinal colonizer Enterococcus faecium is a leading cause of hospital-acquired infections. Multidrug-resistant (MDR) E. faecium isolates are particularly concerning for infection treatment. Previous comparative genomic studies revealed that subspecies referred to as clade A and clade B exist within E. faecium. MDR E. faecium isolates belong to clade A, while clade B consists of drug-susceptible fecal commensal E. faecium isolates. Isolates from clade A are further grouped into two subclades, clades A1 and A2. In general, clade A1 isolates are hospital-epidemic isolates, whereas clade A2 isolates are isolates from animals and sporadic human infections. Such phylogenetic separation indicates that reduced gene exchange occurs between the clades. We hypothesize that endogenous barriers to gene exchange exist between E. faecium clades. Restriction-modification (R-M) systems are such barriers in other microbes. We utilized a bioinformatics analysis coupled with second-generation and third-generation deep-sequencing platforms to characterize the methylomes of two representative E. faecium strains, one from clade A1 and one from clade B. We identified a type I R-M system that is clade A1 specific, is active for DNA methylation, and significantly reduces the transformability of clade A1 E. faecium. Based on our results, we conclude that R-M systems act as barriers to horizontal gene exchange in E. faecium and propose that R-M systems contribute to E. faecium subspecies separation. IMPORTANCE Enterococcus faecium is a leading cause of hospital-acquired infections around the world. Rising antibiotic resistance in certain E. faecium lineages leaves fewer treatment options. The overarching aim of this work was to determine whether restriction-modification (R-M) systems contribute to the structure of the E. faecium species, wherein hospital-epidemic and non-hospital-epidemic isolates have distinct evolutionary histories and highly resolved clade structures. R-M provides bacteria with a type of innate immunity to horizontal gene transfer (HGT). We identified a type I R-M system that is enriched in the hospital-epidemic clade and determined that it is active for DNA modification activity and significantly impacts HGT. Overall, this work is important because it provides a mechanism for the observed clade structure of E. faecium as well as a mechanism for facilitated gene exchange among hospital-epidemic E. faecium isolates.Item Enterococcus Faecalis Genome Defense Systems and Their Impact on Conjugative Antibiotic Resistance Plasmid Transfer(2017-05) Huo, Wenwen; Zhang, Michael Q; Palmer, Kelli LEnterococcus 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.