Engineering a Recombinant BrkA Autotransporter with an Antimicrobial Colicin E1 Passenger in Escherichia coli

Abstract

Bacteriocins are proteins with bactericidal effects that offer alternatives to antibiotics, and in some cases have anti-cancer properties. Colicin E1 is a bacteriocin from E. coli that hijacks the outer membrane (OM) protein TolC to enter target cells and form pores in the inner membrane, causing cell death. Its cationic, amphiphilic nature also demonstrates cytotoxic activity against anionic tumour cells, and bacteria as delivery vesicles for cancer therapeutics is being increasingly studied. We proposed that gram-negative bacteria’s autotransporters could secrete Colicin E1 for anticancer treatments. Autotransporters are multidomain proteins with an N-terminal passenger domain and a C-terminal translocator domain. The latter forms a ꞵ-barrel in the bacterial OM through which the passenger domain migrates, folding as it exits. The passenger domain can remain embedded in the OM or be cleaved by proteases and secreted. Previous research has replaced the native passenger in the autotransporter Bordetella resistance to killing A (BrkA) with functional recombinant proteins, and insertion of an OmpT protease cut-site between the BrkA passenger and translocator domain may facilitate passenger secretion. Using that framework, we replaced the native BrkA passenger with Colicin E1 to create pColE1 with an OmpT cut-site. pImmE1, encoding the Colicin E1 immunity protein, was also created to prevent self-toxicity, and successful plasmid construction was confirmed with nanopore sequencing. However, culture broths lacked antibacterial activity, and Colicin E1 secretion into cell culture broths could not be confirmed with Western blot. This suggests that more work is needed to make BrkA an efficient recombinant protein secretion system.