UCLA

Pathogenic Gram-positive bacteria cause a range of serious infections in humans and represent a significant threat to global health. The rising emergence of virulent strains which are resistant to our current arsenal of antibiotics escalates these dangers. Many Gram-positive bacteria display an array of proteins on their cell surface that enable them to interact with their environment during infections. Among the most important extracellular virulence factors are bacterial pili, which are adhesive filaments that are constructed by specialized cysteine transpeptidases through isopeptide linkages. This dissertation describes my efforts to elucidate the assembly mechanism of the archetypal SpaA-pilus from Corynebacterium diphtheriae. Further, it describes parallel experiments aimed at repurposing the enzymes used in pilus biogenesis for protein engineering applications.

This thesis is divided into two major sections: Pilus Biology (Chapters 2 - 4) and Sortase Bioconjugation (Chapters 5 - 7). The Pilus Biology section describes efforts to elucidate the mechanism of Gram-positive pilus biogenesis using kinetic, structural and cellular experiments. Chapter 2 describes the biochemical reconstitution and characterization of the assembly reaction that builds the C. diphtheriae SpaA-pilus. SpaA-type pili are assembled by a Class C pilin polymerase sortase, CdSrtA. Examination of the structure of this enzyme revealed that CdSrtA is held in an inactive state in vitro by an autoinhibitory “lid” structure. We discovered that amino acid substitutions introduced into the “lid” activate the enzyme and permit biochemical characterization of the polymerization reaction. Chapter 3 characterizes the mechanism and in vitro kinetics of the lysine-isopeptide transpeptidation reaction that builds the SpaA pilus. We identify the rate limiting step in the mechanism and offer a kinetic explanation that explains why “lid” alterations activate CdSrtA. Chapter 4 describes the solution structure of the pilin-pilin linkage which joins successive protomers in the SpaA pilus. Data from NMR dynamics, SAXS and biophysical measurements of protein stability reveal the mechanism by which Gram-positive pili are stabilized at each linkage site throughout the elongated pilus fiber.

The Bioconjugation section of this thesis describes our efforts to develop sortase enzymes into useful bioconjugation tools. Chapter 5 describes a versatile sortase-mediated protein nanocage ligation platform that facilitates enzymatic synergy by enhancing pathway flux between enzymes with complementary activities. As a proof of principle, the nanocage scaffold was functionalized with cellulolytic enzymes, demonstrating a marked enhancement in degradative synergy of cellulose substrate compared to unbound cellulases. Chapter 6 builds upon our understanding of the pilus biogenesis reaction to engineer the CdSrtA sortase into a viable bioconjugation tool that can be used to attach peptide fluorophores to proteins via isopeptide bonds. Chapter 7 describes ongoing efforts to further optimize the CdSrtA bioconjugation tool using a directed evolution approach, with the goal of altering its substrate specificity and increasing its thermostability. Together, the research described in this thesis provides new insight into the biogenesis mechanism that is used by Gram-positive bacteria to produce adhesive pili and describes the development of a promising molecular tool for producing novel bioconjugates.