Date of Degree
PhD (Doctor of Philosophy)
Staphylococcus aureus encodes two extracellular nuclease enzymes, Nuc and Nuc2. Nuc is a secreted enzyme that is cut by signal peptidase (SpsB) at the cell membrane and is further processed into two active forms, NucA and NucB, by an unknown protease. Nuc2 is predicted to be a second extracellular nuclease based on sequence homology to the staphylococcal nuclease (SNase) and is tethered to the membrane with a N-terminal anchor. At the beginning of these studies, little was understood about the biological and physiological roles of Nuc and Nuc2 in S. aureus. The goal of this dissertation was to characterize the extracellular nuclease activity of S. aureus in order to better understand the contributions of Nuc and Nuc2 to the S. aureus life cycle.
The studies presented in Chapter II focus on the role of Nuc in regulating S. aureus biofilm growth. The secreted forms of Nuc, called NucA and NucB, were first identified as anti-biofilm agents present in spent media from a S. aureus alternative sigma factor B (sigB) mutant. Regulation studies identified the major repressors and activators of nuc expression and showed that nuc is repressed under biofilm-forming conditions. By bypassing the native regulatory mechanisms using a nuc inducible plasmid, biofilm growth could be inhibited in a dose-dependent manner. Biofilm testing of nuc mutant strains across genetic backgrounds led to the observation that biofilm thickness increased two-fold in the absence of Nuc. More high molecular weight extracellular DNA (eDNA) accumulated in the nuc mutant compared to wild-type cells, indicating a direct link between Nuc and the availability of eDNA to contribute to the biofilm matrix. These studies showed that nuc expression is tightly regulated in S. aureus biofilms, and Nuc activity can greatly impact biofilm formation and maturation.
In Chapter III, studies were performed to determine whether Nuc2 is an active nuclease in S. aureus and where the protein is localized in the cell. Upon initial comparison to Nuc, Nuc2 has 42% amino acid identity in the proposed SNase domain, and 7 of 9 residues known to be required for Nuc activity are conserved in Nuc2. Fluorescence microscopy of a Nuc2-sGFP translational fusion demonstrated the protein is localized to the cell membrane, and alkaline phosphatase fusion studies showed that the C-terminus of Nuc2 faces out of the cell. Fluorescence resonance energy transfer (FRET) assays facilitated the detection of low levels of Nuc2 activity on the S. aureus cell surface, demonstrating for the first time the enzyme is a functional nuclease, and mutations in the nuc2 gene eliminated this activity. Purification of recombinant Nuc2 also showed that enzyme has DNase activity that is calcium-dependent. Through the construction of Nuc/Nuc2 chimeric proteins, it was determined that localization to the cell membrane does not impair nuclease activity, and the low levels measured for Nuc2 on S. aureus cells is likely due instead to weak expression. The knowledge that Nuc2 is an active nuclease, localized to the cell surface, provides insight into the potential roles Nuc2 may play in a biofilm environment and during S. aureus infection.
Copyright 2012 Megan Rae Kiedrowski