Rapid response reagents for the detection of Burkholderia pseudomallei

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Biochemistry, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Northern Australia and Southeast Asia. Clinical infection can progress rapidly, in serious cases resulting in septicaemia that leads to death within 48 hours if left untreated (up to 50% of all diagnosed cases). Therefore rapid diagnosis of melioidosis is critical. Current culture[-]based identification techniques are time-and-labour-intensive, while new molecular detection techniques achieved limited success. Therefore, this study aims to develop novel rapid response reagents towards B. pseudomallei for detection and diagnosis applications. This project investigates the development of an integrated process to generate novel high-affinity protein reagents towards B. pseudomallei that incorporates recombinant B. pseudomallei antigen expression and subsequent biopanning of the shark antibody variable domain (VNAR) and 20-mer peptide chains phage display libraries. Six B. pseudomallei surface proteins were identified as potential target antigens of which four proteins, flagellin, pilin, Outer membrane protein A (OmpA) and a hemagglutinin-like protein, were successfully produced recombinantly using Escherichia coli cells. These four recombinant proteins were subjected to biopanning using the phage display libraries to generate the novel binding reagents. Novel VNAR and peptide binders were success[s]fully isolated towards B. pseudomallei OmpA and the purification and extensive characterisation of the novel binding reagents using various techniques are presented. The ability of the respective VNAR and peptide binders to bind to B. pseudomallei OmpA were examined by ELISA, and the stability and kinetics of binding interactions were determined using column shift binding assay and surface plasmon resonance. Additionally, efforts were made to elucidate the structural basis for the binding interactions by subjecting the B. pseudomallei antigens and the respective binders to crystallization and co-crystallisation trials. The results achieved in this study demonstrate the feasibility of generating novel binding reagents towards B. pseudomallei antigens in an integrated process and form the foundation for further development of novel binders using alternative phage display libraries.