New age antibiotics: Bacteriocins

Many phages produce specialized small proteins, which specifically inhibit bacterial RNAP in order to redirect host resources to allow phage replication. We have extensive experience in the area of phage-derived bacterial RNAP inhibitors and have uncovered and characterised two bacterial RNAP inhibitors from the E. coli phage T7, called Gp2 and Gp5.7. Gp2 is a potent inhibitor of the bacterial RNAP responsible for the transcription of 'housekeeping' genes, whilst Gp5.7 is a potent inhibitor of the bacterial RNAP responsible for the transcription of stress response associated genes, including genes required for adapting to NSIGA. We have previously shown that recombinant Gp2 and Gp5.7 effectively inhibit the E. coli RNAP in vitro and that expression of plasmid-borne Gp2 or Gp5.7 in exponentially growing E. coli cells rapidly attenuated growth. However, a major obstacle in using Gp2 and Gp5.7 as antibacterial compounds is their inability to penetrate bacterial cells (and thus inhibit the RNAP) when added directly to a culture of bacteria. To overcome this obstacle, we will collaborate with Syngulon - a Belgian SME operating in the space of AMR research - and leverage our combined strength and experience in phage-derived RNAP inhibitors and antibacterial peptides (bacteriocins) and develop chimeric bacteriocin-Gp2/Gp5.7 proteins that are able to translocate into Gram-negative bacteria and interfere with transcription. Put simply, we will use bacteriocins as a vehicle to deliver phage-derived toxic cargo into bacteria.
We will specifically focus on a class of bacteriocins called colicins, which are antibacterial proteins produced by, and effective against, E. coli and very closely related bacteria. Colicins are categorised based on their mode of bactericidal activity, their membrane receptors, and the mechanism they use for translocation through the outer membrane and across the periplasmic space of Gram-negative bacteria. We will specifically focus on colicin E2 (ColE2), which, like many other colicins, is produced by growth-attenuated E. coli as a weapon of interbacterial competition, and has been previously shown to be effective against foodborne pathogenic strains of E. coli such as EHEC O157:H7. ColE2 producing bacterial cells are protected against self-toxicity by the constitutive expression of an immunity (ImmE2) protein, which forms a tight complex with ColE2 and renders it inactive. The ImmE2 protein dissociates from ColE2 during the translocation process into the target bacterial cell. ColE2 is a modular protein and consists of three domains: the receptor binding domain (R-domain) binds with high affinity to the cell surface receptor, ButB; the translocation domain (T-domain) functions in transferring the colicin from the cell surface to or through the periplasm via the OmpF/Tol system; and the catalytic domain (C-domain) contains the cytotoxic function - a DNA endonuclease. Although the finer details of how ColE2 is translocated into target cells remain elusive, freeing the cytotoxic C-domain from the rest of the molecule is a key step in the killing function of ColE2. This is achieved by the inner-membrane protease, FtsH, which cleaves ColE2 near residue D420 (located between the R and C domain) and thereby frees the cytotoxic C-domain to enter the cytoplasm to carry out its killing function.
As the structures of ColE2, Gp2 and Gp5.7 are available to us, we will use a structure-guided approach to construct chimeric ColE2-Gp2 and ColE2-Gp5.7 proteins using standard molecular biology methods.
An entry plasmid containing a C-domain truncated ColE2, to make the ColE2-Gp2 or ColE2-Gp5.7 proteins, is available to us through Syngulon. For ease of purification, we will introduce a 6His-tag at the carboxyl terminal end of Gp2 and Gp5.7.