Producing intracellular and extracellular lipid vesicles in E. coli for vaccine development

Membrane-bound structures such as intracellular compartments and extracellular vesicles appear to play important roles in bacterial biochemistry and survival. Whilst bacterial extracellular vesicles (BEVs) are naturally secreted, intracellular vesicle formation in bacteria has only been observed following the overproduction of some membrane proteins. The proteins of the LemA family have previously been shown to have membrane restructuring capabilities when recombinantly expressed in Escherichia coli. Consequently, this project set out to investigate the potential of utilising LemA proteins together with protein engineering approaches to produce intracellular and extracellular vesicles in E. coli. Initial studies focussed on two distinct LemA proteins from Pseudomonas aeruginosa (Pa) LemA1 and LemA2, which were predicted to be targeted to the inner and the outer membranes, respectively. Transmission electron microscopy (TEM) analysis revealed the formation of intracellular membrane vesicles in both samples, while scanning electron microscopy (SEM) studies showed increased outer membrane vesiculation in the cells overexpressing PalemA2; findings that were consistent with the predicted localisation of these proteins. Moreover, the purification and structural studies of PaLemA1 provided a solid basis for future structural work on this protein. The construction and overproduction of the transmembrane and soluble domains of MamQ, LemA.153, LemA.159, LemA.501 and PaLemA1 proteins individually resulted in inclusion body formation in the majority of samples. However, the expression of the transmembrane domain (TMD) of PalemA1 alone was sufficient to induce intracellular vesicle production. A 'mix-and-match' approach of the different TMDs and soluble domains further yielded a wide range of novel membranous phenotypes in E. coli. Together, these results provide good evidence for the recombinant production of membranous compartments following LemA protein overproduction in E. coli. Furthermore, such membranous structures hold a lot of potential for targeted protein engineering approaches in the generation of a novel vaccine delivery platform