Browsing by Author "Rodrigues, Marinelle"
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Item An Attenuated CRISPR-Cas System In Enterococcus faecalis Permits DNA Acquisition(Amer Soc Microbiology) Hullahalli, Karthik; Rodrigues, Marinelle; Uyen Thy Nguyen; Palmer, Kelli L.; 0000-0002-7343-9271 (Palmer, KL); Hullahalli, Karthik; Rodrigues, Marinelle; Uyen Thy Nguyen; Palmer, Kelli L.Antibiotic-resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Fnterococcus faecolis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) genome defense systems. CRISPRCas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in F. faecolis is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this "CRISPR tolerance." We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly. DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR- assisted genome-editing scheme. Our results suggest that E. faecatis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense. IMPORTANCE CRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently nonlethal. We uncover novel phenotypes associated with this "CRISPR tolerance" and, after determining its genetic basis, develop a genome-editing platform in E. faecafis with negligible off target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR- induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.Item Comparative Analysis of the Orphan Crispr2 Locus in 242 Enterococcus Faecalis Strains(Public Library of Science, 2015-09-23) Hullahalli, Karthik; Rodrigues, Marinelle; Schmidt, Brendan D.; Li, Xiang; Bhardwaj, Pooja; Palmer, Kelli L.; Hullahalli, Karthik; Rodrigues, Marinelle; Schmidt, Brendan D.; Li, Xiang; Bhardwaj, Pooja; Palmer, Kelli L.Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs.;Item CRISPR-Cas systems in Enterococcus faecalis and their application in probiotics for the removal of antibiotic resistance(2018-05) Rodrigues, Marinelle; 0000-0002-5509-605X (Rodrigues, M); Palmer, Kelli L.The gut microbiome is composed of diverse bacterial, fungal and archaeal species which function dynamically and reside commensally within a host, proving health benefits such as immune stimulation, nutrient supplementation, and resistance to pathogen colonization. Enterococcus faecalis is a native, commensal inhabitant of the GI tract of most terrestrial animals. It is typically an underrepresented member of the healthy microbiome with its prevalence impeded by competing organisms. However, during antibiotic-mediated dysbiosis, its population blooms. They are then able to reach the bloodstream and cause infections such as endocarditis and bacteremia. E. faecalis infections are challenging to treat due to their intrinsic resistance to certain antibiotics and propensity for acquiring antibiotic resistance genes through horizontal gene transfer. Sequence analysis of clinical isolates has revealed that these strains possess expanded genomes of which >25% is derived from mobile genetic elements. It has previously been observed that MDR strains of E. faecalis lack complete CRISPR-Cas systems, an adaptive immune system which protects bacteria from invading DNA. These systems are able to recognize and cleave specific sequences of DNA by using RNA guides and have found many applications as genetic tools in the manipulation of DNA or the rational design of sequence-specific antimicrobials. In this dissertation, I inserted the interference machinery of a CRISPR-Cas system in E. faecalis into an MDR strain and restored activity for genome defense using conjugation assays. I noted that CRISPR-mediated defense was not entirely effective in this species and a significant number of transconjugants were obtained even when the plasmid was targeted. Further examination of these transconjugants showed that they were unstable, and depending on the presence or absence of selection for the plasmid, the cells were able to either compromise their CRISPR system or lose the targeted plasmid. More importantly, this instability conferred a growth defect which could then be exploited in composite populations to selectively eliminate undesirable traits. Using this, we were able to target an antibiotic resistance gene and abolish resistance from heterogeneous populations of E. faecalis. Following this discovery, I improved the system by incorporating the entire CRISPR-Cas targeting system on a pheromone-responsive plasmid (PRP) encoding a bacteriocin which enforced its selection. PRPs have notoriously high conjugation frequencies and are known to efficiently disseminate in E. faecalis populations in both in vivo and in vitro conditions. Using these plasmids, I was able to significantly decrease antibiotic resistance from in vitro populations and in an in vivo model of mouse gut colonization. The work presented here provides evidence supporting the use of CRISPR-targeting constructs in probiotics to reduce the circulation of undesirable traits among E. faecalis strains colonizing patients in hospitals with the aim of mitigating the occurrence of MDR infections.Item Exploiting CRISPR-Cas to Manipulate Enterococcus Faecalis Populations(eLife Sciences Publications Ltd, 2018-08-20) Hullahalli, Karthik; Rodrigues, Marinelle; Palmer, Kelli L.; 0000-0002-5509-605X (Rodrigues, M); 0000-0002-7343-9271 (Palmer, KL); Rodrigues, Marinelle; Palmer, Kelli L.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.Item Modulators of Enterococcus Faecalis Cell Envelope Integrity and Antimicrobial Resistance Influence Stable Colonization of the Mammalian Gastrointestinal Tract(Amer Soc Microbiology, 2018-10-22) Banla, Ismael L.; Kommineni, Sushma; Hayward, Michael; Rodrigues, Marinelle; Palmer, Kelli L.; Salzman, Nita H.; Kristich, Christopher J.; 0000-0002-7343-9271 (Palmer, KL); Rodrigues, Marinelle; Palmer, Kelli L.The Gram-positive bacterium Enterococcus faecalis is both a colonizer of the gastrointestinal tract (GIT) and an agent of serious nosocomial infections. Although it is typically required for pathogenesis, GIT colonization by E. faecalis is poorly understood. E. faecalis tolerates high concentrations of GIT antimicrobials, like cholate and lysozyme, leading us to hypothesize that resistance to intestinal antimicrobials is essential for long-term GIT colonization. Analyses of E. faecalis mutants exhibiting defects in antimicrobial resistance revealed that IreK, a determinant of envelope integrity and antimicrobial resistance, is required for long-term GIT colonization. IreK is a member of the PASTA kinase protein family, bacterial transmembrane signaling proteins implicated in the regulation of cell wall homeostasis. Among several determinants of cholate and lysozyme resistance in E. faecalis, IreK was the only one found to be required for intestinal colonization, emphasizing the importance of this protein to enterococcal adaptation to the GIT. By studying.ireK suppressor mutants that recovered the ability to colonize the GIT, we identified two conserved enterococcal proteins (OG1RF_11271 and OG1RF_11272) that function antagonistically to IreK and interfere with cell envelope integrity, antimicrobial resistance, and GIT colonization. Our data suggest that IreK, through its kinase activity, inhibits the actions of these proteins. IreK, OG1RF_11271, and OG1RF_11272 are found in all enterococci, suggesting that their effect on GIT colonization is universal across enterococci. Thus, we have defined conserved genes in the enterococcal core genome that influence GIT colonization through their effect on enterococcal envelope integrity and antimicrobial resistance.