Cytolysin A (ClyA) in E. coli: a tool for Outer Membrane Vesicles (OMVs) surface decoration with chimeric proteins

One of the emerging trends in therapeutics is the development of novel drug delivery systems, allowing to convey drugs to a specific cellular target and minimizing adverse effects. Outer Membrane Vesicles (OMVs), small bacterial proteo-liposomes (20-250 nm), hold a great potential to be used as engineerable carriers. In this work we characterized the potential of the recombinant protein Cytolysin A (ClyA) for the recombinant expression of proteins and antigens on bacterial outer membranes and for their inclusions in OMVs. In E. coli pathogenic strains different ClyA monomers assembles in the outer membrane and OMVs forming a cytolytic proteic toxin. We here implemented a truncated isoform of ClyA monomer which pre vented cytolysin complex assembly and that was previously described to be targeted in the outer membrane exposing on the surface recombinant antigens fused to its C-terminus [1]. To this purpose, ClyA sequence with a C-terminal linker and a His-tag was cloned and recombinantly expressed in E. coli BL21(DE3) cells, a commercial strain suitable for optimized recombinant protein production. ClyA enrichment in OMVs was verified by western blotting. ClyA C-terminal exposure on OMVs surface was then investigated by proteolytic degradation of ClyA C-terminal His-tag on intact OMVs, and immunofluorescence experiments using BL21(DE3) cells expressing ClyA. As a proof of concept, we produced two chimeric proteins, composed by two polypeptides of different length fused to ClyA C-terminus. The first (63 kDa) bearing the light variable chain of the AntiCD19 scFV (variable regions of the light and heavy chain) and the second (88 kDa) consisting of both chains of the scFV portion. This latter hybridoma (FMC63-28Z) is currently used in immune-checkpoint inhibition therapy for B cell leukaemia. Both chimeras were recombinantly expressed and OMVs were purified. Our data showed the presence of the partial construct in OMVs. However, the whole scFV fusion protein failed to be encapsulated in the outer membranes. A cell fraction experiment highlighted a lowered OMVs encapsulation efficiency with increasing mass of the fusion protein. Further studies will foresee the addition of a N-terminal leader sequence to improve OMVs encapsulation of longer chimeric constructs and the evaluation of ClyA-AntiCD19 scFV chimera binding affinity to its epitope on CD19 proteins.