Biochemical Analysis Of Modulation Of Sex Pheromone Production By Prgy, A Structural Homolog Of A Metalloprotease (Tiki) That Modulates Wnt Signaling In Eukaryotes

University of Minnesota Ph.D. dissertation. May 2017. Major: Oral Biology. Advisor: Gary Dunny. 1 computer file (PDF); iv, 87 pages.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