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Browsing by Subject "Enterococcus faecalis"

Now showing 1 - 6 of 6
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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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    A bistable genetic switch controls antibiotic resistance transfer in Enterococcus faecalis.
    (2011-08) Chatterjee, Anushree
    The recent rise in microbial drug resistance is a growing challenge for future therapy of bacterial infections. Increased prevalence of antibiotic resistance in bacteria is an outcome of evolution via natural selection. However, the built-in design feature of bacteria to transfer DNA containing antibiotic resistance both within the same species and across species is the main culprit for the spread of drug resistance. One of the main factors driving the rise of drug resistant microbes is the transfer of antibiotic resistance genes present on mobile plasmids between donor and recipient cells via the mechanism of conjugation. In order to combat microbial drug resistance, novel strategies need to be developed to block such transmission of antibiotic resistance. In this work, the gene regulatory components involved in transfer of tetracycline resistance confers plasmid pCF10 between plasmid-carrying donor cells and plasmid-deficient recipient cells in bacterium Enterococcus faecalis is investigated. In the native state the donor cell exists in an OFF or conjugation-incompetent state. A pheromone released by the recipient cells serves as the chemical trigger for switching the donor cell from OFF to an ON or conjugation-competent state. The onset of conjugation is tightly regulated via multi-layered regulation offered by two-key genes prgQ and prgX present on pCF10 in response to the pheromone secreted by recipient cells. Using mathematical modeling and experimentation, we describe a novel mechanism of gene-regulation due to transcriptional interference and sense-antisense RNA interaction as a result of convergent transcription in the prgX/prgQ operon. We demonstrate that such a multi-layered gene-regulatory mechanism confers the system a bistable genetic switch controlling conjugative gene transfer between donor and recipient cells. A similar regulatory advantage offered by convergent transcription in attributing a bistable switch-like behavior in the scbA-scbR operon controlling antibiotic production in S.coelicolor is also investigated. Both mathematical model and experiments demonstrate that donor cells also control response to pheromone by changing the number of copies of pCF10 plasmid inside the cell. Cells with higher copies show increased robustness of the bistable switch and lower sensitivity to pheromone. Once bistable genetic-switch is ON, expression of genes encoding various proteins involved in the transfer of the plasmid are induced, however, this also causes production of an inhibitor of conjugation, thus giving rise to negative feedback loop which causes the donor to return to OFF state. Modeling and experimental analysis of dynamic response to induction indicate that this negative feed-back loop causes a brief surge of expression of the entire operon. We show that the inhibitor signaling peptide for pCF10 based system, acts as quorum-sensing signal with the role of turning-OFF conjugation at a population-wide scale. An interplay of positive and negative feedback loops allows the donor cell to quickly transition between ON and OFF states and is critical both for the transfer of plasmid and survival of the cell. Studying both the turning-ON and turning-OFF mechanisms of the switch allows identification of potential drug targets for blocking transmission of antibiotic resistance for use in future therapy.
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    Enterococcus faecalis aggregation substance (Asc10) as liaison between bacterium and heart valve in endocarditis.
    (2009-08) Chuang-Smith, Olivia Newton
    Aggregation Substance proteins encoded by sex pheromone plasmids increase virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation Substance Asc10, encoded by the plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the vegetation was the best indicator of virulence. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative where glycine residues in two RGD motifs were changed to alanines showed the greatest reduction in virulence. Remarkably this strain, and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. In addition, mutants carrying Tn917 insertions in the prgB gene demonstrated that secreted N-terminal Asc10 fragments possess activity promoting endocarditis virulence. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis, and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo. Since Asc10 is important as a virulence factor in E. faecalis endocarditis pathogenesis, developing immunization approaches against this surface protein will be useful in combating endocarditis disease. Use of Fab fragment antibodies against Asc10 was found to decrease vegetation size and bacterial load in the rabbit endocarditis model. In addition, microarray and histological studies revealed two routes of infection in vegetation formation; one in the absence of Asc10, characterized by a robust inflammatory response, and the second in which the presence of Asc10 dampens this response, possibly impeding the influx of immune cells into the vegetation. We also employed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10+ E. faecalis and valve tissue, and to examine formation of biofilms. We found that the aggregation domains contribute most to Asc10-mediated E. faecalis valve adherence, whereas the RGD motifs have importance in later stages of valve colonization. Again, an N-terminal Asc10 fragment expressed from a prgB Tn917 insertion mutant mediated adherence of E. faecalis cells, emphasizing the importance of the aggregation domains in valve attachment. Most of the Asc10 mutants examined showed some defects in valve adherence at 4 h, corroborating results from our rabbit endocarditis model, and implying that Asc10 contributes mainly to persistence of E. faecalis during endocarditis infection. Extracellular matrix (ECM) protein studies to determine the eukaryotic Asc10 ligand in valve tissue found that fulllength Asc10 protein did not mediate E. faecalis binding to vitronectin, fibronectin, fibrinogen, von Willebrand factor, heparan sulfate, or chondroitin sulfate. In scanning electron microscopy analysis of the infected valve tissue, we found evidence of biofilm formation, including growing aggregates of bacteria, and the increasing presence of exopolymeric matrix over time. Additionally, E. faecalis cells preferentially bound to damaged tissue, though it was difficult to determine whether the bacteria caused the damage, or if it was due to deterioration of the tissue over time. This porcine heart valve tissue colonization model will serve as a useful tool in future studies of biofilm formation.
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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.
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    Ultrastructural characterization of matrix development and the role of extracellular DNA in early Enterococcus faecalis biofilms
    (2012-11) Barnes, Aaron Michael Tolo
    Enterococcus faecalis is a highly adaptable, gram-positive bacterium that occupies a diverse range of ecological niches. A common soil-dwelling organism, it is also inhabits the metazoan gastrointestinal tract—from insects to humans. E. faecalis is remarkably resistant to a wide range of clinically-relevant antibiotics and readily forms biofilms on both abiotic and biotic surfaces. These latter factors underlie the medical relevance of E. faecalis. This thesis explores the ramifications of early developmental events in E. faecalis biofilm formation. Using correlative microscopy techniques, we investigated a series of mutants with ultrastructural changes in the extracellular matrix that elucidate the roles these genes play in matrix architecture. We also report that extracellular DNA plays a substantial role in stabilizing early (< 8 hr post-inoculation) E. faecalis biofilms, and that the source of this DNA is not via bulk cell lysis, but rather appears to be secreted from metabolically active cells. A putative model for this non-canonical source of DNA in the matrix is also proposed.