Sortase-mediated modification and intracellular delivery of cholera toxin analogues

Macromolecule therapeutics, such as oligonucleotides, are playing an increasingly important role in the fight against diseases. Unfortunately, the majority of macromolecules are unable to cross the cell membrane barrier, which severely reduces the number of potential intracellular targets this class of therapeutic can act on. The AB5 complexes, cholera toxin and heat-labile enterotoxin have evolved the ability to enter mammalian cells effortlessly by using a retrograde transport mechanism. It has been previously reported that non-toxic analogues of these proteins can be used to transport macromolecular cargo into cells. However, the majority of these strategies utilise recombinant technologies and as a result are limited to protein based cargo.
Sortase A (SrtA) is a class of transpeptidase enzyme that attaches virulence factors to the cell wall of gram Gram-positive bacteria. The enzyme recognises proteins carrying a C-terminal LPXTG motif and ligates them to peptidoglycans at the N-terminus of an oligoglycine sequence. Since its discovery, SrtA has been viewed as a potential new tool for protein modification. However, the enzyme-mediated ligation reaction is relatively inefficient in vitro.
In this thesis, I will describe the development of depsipeptide substrates for SrtA that allow the quantitative N-terminal labelling of proteins/peptides. In addition, a simple and robust strategy to chemically-modified AB5 complexes that are able to enter mammalian cells will be reported. Finally, a novel approach to generate oligonucleotide-conjugated AB5 complexes using a combination of enzymatic and bioorthogonal reactions will also be outlined.