Browsing by Subject "Pheromone"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    Characterization of the effects of regulators PrgY and PrgZ on the inducibility of plasmid pCF10 in Enterococcus faecalis
    (2012-09) Barr, Frank T.
    Enterococcus faecalis is an increasingly significant pathogenic bacterium in humans, as well as a commensal member of the intestinal flora. Pheromone-responsive plasmid systems play an important role in the dissemination of antibiotic resistance among this and other species. pCF10 is a well-characterized member of this class of plasmids, and allows for transmission of tetracycline resistance in E. faecalis. The experiments described in this thesis were designed to assess the effects of three genes thought to be important for the response of plasmid pCF10 to the peptide mating pheromone cCF10 in E. faecalis: 1) the chromosomal locus responsible for pheromone production- ccfA, 2) the plasmid-encoded negative regulator prgY thought to be responsible for sequestration of endogenous pheromone at the cell surface, and 3) the plasmid-encoded positive regulator prgZ, an oligopeptide permease subunit A homolog responsible for specific import of pheromone. In this study transcription emanating from regulatory regions of this plasmid was characterized over time using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results from these assays were confirmed using phenotypic analysis and Northern blotting. Expression of ccfA was shown to be important to moderate the pheromone response. This moderation effect is believed to be the result of basal induction of donor cells, resulting in production of the peptide inhibitor iCF10 from the plasmid. As the first study to probe PrgY function at the level of transcription, RNA quantitation revealed that levels of endogenous pheromone cCF10 resulting from processing of the CcfA lipoprotein precursor are insufficient to fully induce a conjugation response in the absence of negative regulator PrgY, suggesting secondary negative regulation. Finally, PrgZ was shown to be important for regulated shutdown of the conjugation response in addition to initiation. This lends evidence to previous research suggesting specific import of inhibitor iCF10 by PrgZ. Probing the effects of these regulators over time in strains that differ in their ability to secrete pheromone allows us to paint a more complete regulatory picture of this complex and dynamic system.
  • Thumbnail Image
    Item
    Intramolecular Diels–Alder/Tsuji Allylation assembly of the functionalized trans-Decalin of Salvinorin A and sea lamprey migratory pheromone structure activity relationship studies.
    (2010-05) Burns, Aaron C.
    In the first portion of this work, an intramolecular Diels–Alder/Tsuji allylation assembly of functionalized trans-decalin systems will be discussed (Chapter I). This is presented in the context of synthetic studies toward the psychoactive (κ-opioid agonist) natural product salvinorin A. Synthetic studies of the sea lamprey (Petromyzon marinus) migratory pheromone will be discussed in Chapter II. In the third chapter, a structure activity relationship study of the sea lamprey migratory pheromone will be presented in which results suggest that the sea lamprey olfactory system is not uniquely and specifically recognizing the allo (i.e., 5α-H) configuration in bile acid derivatives.
  • Thumbnail Image
    Item
    Sensory and chemical basis of off-host aggregation behavior by bed bugs, Cimex lectularius
    (2015-02) Olson, Joelle F.
    After feeding on hosts, bed bugs, Cimex lectularius L., aggregate in cracks and crevices near their hosts. Off-host aggregation is mediated by sensory organs on the bed bug antennae and chemical stimuli associated with bug feces. This dissertation examined the sensory bases of bed bug off-host aggregation behavior, and results are presented in four chapters. Chapter one provides a basic overview of existing literature on the sensory structures located on adult antennae and the chemical stimuli that influence bed bug behavior. The chapter concludes with a discussion of practical applications for bed bug control. In chapter two, behavioral assays and microscopy were used to study sensilla on the bed bug antenna. A multi-choice behavioral assay using fecal stained filter papers determined which antennal segments mediate off-host aggregation. Both scanning and transmission electron microscopy techniques were used to determine the type and function of sensilla on the pedicel of adults and nymphs. In addition to an abundance of serrated hairs, several smooth hairs with gustatory function were sparsely distributed throughout the segment and a distal patch of sensilla with olfactory function was also described. The identification of sensilla with olfactory and gustatory function on the pedicel suggests off-host aggregation by bed bugs may be mediated by a volatile or semi-volatile compound or compounds. In chapter three, the chemical stimulus associated with bed bug feces was analyzed, including stimulus volatility, extraction, isolation, and separation of component molecules. Solid phase microextration (SPME) techniques were used to assess the presence of known bed bug pheromones, (E)-2-hexenal (E2H) and (E)-2-octenal (E2O) on fecal stained papers that were heat treated for several days. In addition, multi-choice behavioral assays were used to assess aggregation response to fecal stained papers that were heated, to papers washed in various solvents, and to concentrated methanol extracts and extracts separated by solid phase extraction (SPE) techniques. Results demonstrated that E2H and E2O decrease significantly with heat exposure; however, aggregation response to fecal stained disks remained relatively constant, suggesting that the chemical stimulus is less volatile compared to previous reports. The chemical stimulus was soluble in methanol and water, with bed bug response greatest to concentrations of fecal extracts above 30 mg/ml methanol. Separation of the active components was possible using a C18 SPE cartridge and gas chromatography techniques, which prepared the chemical stimulus for further identification. In chapter four, crude extracts from bed bug feces were analyzed by a gas chromatograph coupled with an electro-antennogram detector (GC-EAD) and mass spectrometer (GC-MS) to identify essential components of the off-host aggregation pheromone. Adult antennae responded to compounds associated with three elution regions of the crude extract. Several chemical compounds were identified in each of the active regions, and selected groups of compounds were evaluated in multiple choice assays to assess aggregation response. A combination of two compounds, dimethyl trisulfide (DMTS) and methyldiethanolamine (MDEA) resulted in an aggregation response that was equivalent to original extract. This final chapter concludes with a discussion of potential applications of a synthetic aggregation pheromone for surveillance and bed bug control.