The Kinetic and Structural Investigation of Pilus Assembly and the Development of Sortase Inhibitors for Gram-Positive Bacteria
Pathogenic multidrug resistant bacteria cause a range of serious infections in humans. These bacteria have developed mechanisms to counteract the lethal effects of currently used antibiotics, creating a need for novel therapeutics. Gram-positive bacteria display a wide assortment of cell surface proteins that are important for bacterial survival and host-pathogen interactions. These key surface structures included pili, proteinaceous fibers that assist in microbial survival by mediating adhesion to host tissues and aid in the formation of biofilm. A large number of gram-positive bacterial species assemble pili and append surface proteins to the cell wall using sortase cysteine transpeptidase enzymes. These enzymes link the components of the pilus together via covalent lysine isopeptide bonds which confer enormous tensile strength. This dissertation describes my investigation of the assembly mechanism of the archetypal SpaA-pilus from Corynebacterium diphtheriae. It also describes my contributions to develop small molecule sortase inhibitors that could function as anti-infective agents and my work towards exploiting the activity of sortase enzymes as a protein engineering tool.
This thesis focuses on sortase enzymes and can be divided into two major sections: studies to determine how sortases construct pili (Chapters 2-4) and work designed to discover a small molecule inhibitor for the Staphylococcus aureus Sortase A enzyme (Chapter 5). All of the studies have been published in peer reviewed papers, with the exception of work detailed in Chapter 4. Chapter 2 describes the NMR solution structure of the lysine isopeptide bond interface that connects the pilin components of the pilus. This structural information combined with biophysical and cellular analyses led to the formulation of the “latch” mechanism of pilus assembly. Chapter 3 describes an enzyme kinetic study of the sortase enzyme from C. diphtheriae (CdSrtA) which catalyzes the formation of lysine isopeptide bonds between components of the SpaA pilus. In this study, the rate-limiting step of catalysis was determined and variants of CdSrtA with improved activity were discovered. Chapter 4 describes research that employed biochemical and structural approaches to investigate how the incorporation of the SpaB pilin subunit terminates pilus assembly. Key differences were observed between the reaction that terminates assembly and the process of polymerization that builds the shaft of the pilus. Chapter 5 describes efforts to discover a small molecule inhibitor of the Staphylococcus aureus Sortase A enzyme that has the potential to be a therapeutically useful anti-infective agent. The first half of Chapter 5 describes the work done to improve the activity of previously discovered pyridazinone-based molecules using computational and synthetic chemistry methods. The second half describes the implementation of a novel cell-based screen to identify sortase inhibitors. This work leveraged the unique sortase-dependent growth phenotype of Actinomyces oris to screen for sortase inhibitors. Over 200,000 small molecules were screened for their ability to impair A.oris’s growth, which led to the identification of three molecular scaffolds that inhibit sortase activity in vitro. In totality, my thesis research has shed light on how sortase enzymes assemble pili in gram-positive bacteria, led to improved variants of CdSrtA sortase that can be used in protein engineering, and helped identify new small molecule sortase inhibitors with potential therapeutic applications.