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Browsing by Subject "Conjugation"

Now showing 1 - 4 of 4
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    Biochemical Analysis Of Modulation Of Sex Pheromone Production By Prgy, A Structural Homolog Of A Metalloprotease (Tiki) That Modulates Wnt Signaling In Eukaryotes
    (2017-05) Le, Thinh
    Enterococcus faecalis contains mobile genetic elements that can rapidly spread antibiotic resistance and virulence genes through its population by conjugation. The chromosomally encoded pheromone cCF10 (LVTLVFV) induces conjugative transfer of E. faecalis plasmid pCF10. Pheromone inducible plasmids have evolved a highly specific and sensitive response to pheromone to allow their host (donor) cell to sense recipients while minimizing expression of the conjugation genes in the absence of recipients. The pCF10 PrgY protein reduces production of endogenous pheromone by donor cells to prevent self-induction. Recent data suggested that PrgY shares significant homology to the eukaryotic metalloprotease TIKI that has been shown to cleave the amino terminus of mature Wnt proteins, thereby regulates the Wnt signaling. Comparative modeling of PrgY active site revealed that PrgY and TIKI share key conserved residues in the metal-binding catalytic core as well as overall secondary structure. Based on the structural similarity between PrgY and TIKI, we hypothesized that PrgY reduces endogenous pheromone production in donor cells by specifically binding and degrading cCF10 as it is secreted across the cytoplasmic membrane. To test the working model, affinity chromatography and Surface Plasmon Resonance were used to demonstrate the direct interaction between PrgY and cCF10, and their binding affinities. The results of this work revealed that PrgY directly interacts with cCF10. Strong binding between cCF10 and PrgY was observed, and the binding can be saturated at a level comparable to the calculated theoretical maximum assuming 1:1 binding. Mass spectrometry was used to identify possible degradation products of cCF10 in the culture supernatant from a strain expressing PrgY. Peptide LVTL was uniquely identified in the donor culture supernatant expressing PrgY. This suggests that PrgY cleaved cCF10 after the second leucine, and released the degraded peptide fragments LVTL and VFV that do not have any pheromone activity. The cumulative results of this research provide important insights into the molecular mechanism of PrgY, and advance our understanding on the function of each of the PrgY family members found in a diverse range of species.
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    The role of Enterococcus faecalis biofilm formation in the regulation of conjugation.
    (2012-06) Cook, Laura Carol Case
    Enterococcus faecalis has recently emerged as an important nosocomial pathogen. Pathogenicity of these organisms depends greatly on a few important aspect of enterococcal physiology. The ability of enterococci to form biofilms greatly enhances their virulence. Their innate resistance to many antibiotics and their ability to transfer these resistance genes through conjugation heightens their threat to human health. The work described in this thesis attempts to explain the roles of biofilm growth, conjugation, and cell communication in E. faecalis. To examine the role of biofilm growth on the E. faecalis transcriptome, RNAseq analysis was undertaken. We found that over 100 genes were measurably upregulated during biofilm growth while approximately 26 genes were downregulated. These data gives us important insights into the biology of enterococcal biofilms. In clinical settings, biofilms are likely locations for antibiotic resistance transfer events involving nosocomial pathogens such as E. faecalis. Conjugation is an important mode of horizontal gene transfer in bacteria, enhancing the spread of antibiotic resistance. In this work, I demonstrate that growth in biofilms alters the induction of conjugation by a sex pheromone in E. faecalis. Mathematical modeling suggested that a higher plasmid copy number in biofilm cells would enhance a switch-like behavior in the pheromone response of donor cells with a delayed, but increased response to the mating signal. Alterations in plasmid copy number and a bimodal response to induction of conjugation in populations of plasmid-containing donor cells were both observed in biofilms, consistent with the predictions of the model. The pheromone system may have evolved such that donor cells in biofilms are only induced to transfer when they are in extremely close proximity to potential recipients in the biofilm community. These results have important implications for development of chemotherapeutic agents to block resistance transfer and treat biofilm-related clinical infections.
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    Single cell analysis of bacterial communication and gene transfer by Enterococcus faecalis
    (2019-02) Erickson, Rebecca
    Enterococcus faecalis is a commensal member of the gastrointestinal tract of animals including humans but is also an opportunistic pathogen and a major cause of healthcare-associated infections. Its pathogenicity is thought to arise in immunocompromised people and after infection, treatment is difficult due to antibiotic resistance. E. faecalis is particularly good at transferring antibiotic resistance by mechanisms like conjugation and conjugative transfer of plasmids can occur at a high frequency without antibiotic selection. Conjugative plasmid pCF10 encodes tetracycline resistance and transfer between E. faecalis cells is facilitated by cell-to-cell communication. This signaling triggers expression of genes from pCF10 that encode for transfer machinery. The response to signaling is robust and has been extensively studied at the population level. However, it has recently become apparent that there is response variation. Understanding the mechanisms that underlie variation in response initiation is important to preventing transfer. Studies presented in this dissertation adapt fluorescence in situ Hybridization Chain Reaction (HCR) for single cell analysis of transcripts and explore questions about the pCF10 conjugation system that would not have otherwise been possible. In chapter 3, variation in the signaling response was assessed and the response was shown to be very heterogenous. When the level of signal is low, (like what might occur naturally), a minority of cells respond. Although stochasticity in the system may give rise to such heterogeneity, work in chapter 4 investigates the response impact of a few specific mechanistic players (PrgX, C, and I). Changing the levels of these components was shown to change the outcome. Lastly, single cell analysis was used in chapter 5 to assess the expression of genes required for conjugative transfer. These results show that the few responding cells commit to expression of all the genes encoding for production of the conjugation machinery. Overall, these results suggest that the pCF10 system is evolutionarily tuned for specific levels of each component and poised to have response variation for a population of cells. Thus, a small percent of cells can respond and since the majority of responding cells are able to conjugate, plasmid transfer is highly efficient. These results also exemplify how small differences in two cells can precipitate different responses in otherwise identical cells exposed to very similar conditions. Information about variation in the initiation of the signaling response required for pCF10 transfer is important to understanding the general biology of gene transfer among bacteria. In the future, this information will be important for successful design of effective interventions to the transfer of genes conferring antibiotic resistance.
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    Systems analysis of pheromone signaling and antibiotic resistance transfer in Enterococcus faecalis
    (2018-01) Bandyopadhyay, Arpan Anup
    Antibiotics have been an extremely important weapon in the fight against bacterial infections for over half a century. However, excessive use of antibiotics has led to increased frequencies of resistance among bacteria. Antibiotic resistance is an inevitable outcome of natural selection as organisms undergo random mutations to escape lethal selective pressure. Many of these resistant bacteria can also transfer their genetic material to other bacteria through direct cell-cell contact via conjugation, further facilitating the spread of resistance. The human gastrointestinal tract, replete with a high density of bacteria and often exposed to antibiotics, provides an ideal environment for antibiotic resistance genes to arise and propagate through bacterial populations. Enterococcus faecalis, a commensal bacterium of the human intestinal tract, has emerged as a major cause of healthcare-associated infections. Treatment of these infections has become increasingly difficult with the emergence of E. faecalis strains that are resistant to multiple major classes of antibiotics. The organism’s ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious clinical concern. Here we present our study on conjugation of a tetracycline-resistance plasmid pCF10 which is regulated by intercellular communication using two antagonistic signaling peptides. An inducer peptide produced by the plasmid-free recipient cells functions as a “mate-sensing” signal and triggers the conjugative plasmid transfer in donors. The donors encode an inhibitor peptide on the plasmid which represses conjugation and functions as a "self-sensing" signal, reducing the response to the inducer in a density-dependent fashion. This form of dual signaling-controlled conjugation was also found to be prevalent across other pheromone-responsive plasmids, including pAD1 and pAM373. Though the donors calibrate their conjugation response in accordance with the relative abundance of donors and recipients, plasmid transfer can occur under otherwise unfavorable conditions, such as low inducing pheromone and high inhibitor concentrations. To better understand this apparent inconsistency, we formulated a stochastic mathematical model that integrates intracellular molecular regulation of conjugation and interactions between donors and recipients through the signaling peptides. Kinetic parameters for the model were estimated from literature and augmented by experimental RNA-Seq data and binding constant measurements. Simulations of the stochastic model and single-cell analysis using transcript quantification by HCR-FISH and GFP reporter fusions revealed distinct subpopulations of rapid responders under unfavorable conditions for plasmid transfer. We developed a series of fluorescent reporters to track the uninduced/induced donors, recipients, and uninduced/induced transconjugants in real-time using confocal microscopy and flow cytometry. We are further developing a microfluidic gut model which allow for co-culturing of human and bacteria cells in an in vivo-simulated microenvironment. This system will be used to model the in vivo biology of conjugation and gain a better mechanistic understanding of the community balance between the microbial inhabitants of the GI tract. A better understanding of the bacterial signaling mechanisms in vivo and the downstream effects on microbiome community balance may help us identify alternate strategies to prevent the spread of antibiotic resistance.