Abstract Title of dissertation: ENGINEERING ENHANCED STRUCTURAL STABILITY TO THE STREPTOCOCCAL BACTERIOPHAGE ENDOLYSIN PLYC

Title of dissertation: ENGINEERING ENHANCED STRUCTURAL STABILITY TO THE STREPTOCOCCAL BACTERIOPHAGE ENDOLYSIN PLYC Ryan Daniel Heselpoth, Doctor of Philosophy, 2014 Dissertation Directed by: Associate Professor Daniel C. Nelson Institute for Bioscience and Biotechnology Research and Department of Veterinary Medicine Antibiotic misuse and overuse has prompted bacteria to rapidly develop resistance, thereby hindering the efficacy of these chemotherapeutics. Due to antibiotic resistant strains expeditiously disseminating, antimicrobial resistance has been labeled as one of the greatest threats to human health globally. An emerging alternative antimicrobial strategy involves using bacteriophage-derived enzymes, termed endolysins. Endolysins are peptidoglycan hydrolases that liberate lytic bacteriophage virions late in the infection cycle by cleaving critical covalent bonds in the bacterial cell wall. As a result, the high intracellular osmotic pressure induces cell lysis. Antimicrobial strategies have been devised involving the extrinsic application of recombinant endolysins to susceptible Gram-positive pathogens. The efficacy of these enzymes has been validated in vitro and in vivo, with no resistance observed to date. One such example is the streptococcal-specific endolysin PlyC. This endolysin is currently the most bacteriolytically-active and possesses the ability to lyse human and animal pathogens known to cause serious health complications. Unfortunately, like numerous other endolysins, PlyC is relatively unstable and accordingly has short shelf life expectancy. With a long-term goal of using endolysins for industrial applications, furthering the development of a thermolabile translational antimicrobial with a short shelf life is ambitious. The main objective of this dissertation is to develop and validate bioengineering strategies for thermostabilizing bacteriolytic enzymes. Using PlyC as the model enzyme, we first used a rationale-based computational screening methodology to identify stabilizing mutations to a thermosusceptible region of the catalytic subunit, PlyCA. One mutation, T406R, caused a 2.27°C increase in thermodynamic stability and a 16 fold improvement in kinetic stability. Next, we developed a substantiated novel directed evolution protocol that involves randomly incorporating mutations into a bacteriolytic enzyme followed by a screening process that effectively identifies mutations that are stabilizing. Finally, applying multiple rounds of directed evolution to PlyC allowed for the identification of a thermostabilizing mutation, N211H, which increased the thermodynamic stability by 4.10°C and kinetic stability 18.8 fold. Combining the N211H and T406R mutations was additive in terms of thermal stability, with thermodynamic and kinetic stability enhancements of 7.46°C and 28.72 kcal/mol activation energy (EA) of PlyCA unfolding, respectively. ENGINEERING ENHANCED STRUCTURAL STABILITY TO THE STREPTOCOCCAL BACTERIOPHAGE ENDOLYSIN PLYC