Monday 9 September at 1:00pm
Biochemistry Seminar Room BIG13
Hazel Sisson
PhD Swansong
Department of Microbiology and Immunology
Developing novel phage-derived antimicrobials against the phytopathogen Pseudomonas syringae pv. actinidiae
Plant pathogens cause considerable damage to crop production resulting in global food shortages and severe economic losses. There is growing restriction for the use of agrichemicals, such as copper and antibiotics, in response to escalating resistance and environmental accumulation. Therefore, there is a desperate need for new, alternative, environmentally friendly antimicrobial agents. Bacteriophages (phages) naturally predate bacteria and thus can be harnessed as biocontrol agents. An alternative strategy is the use of phage-derived enzymes, including endolysins. These enzymes degrade the peptidoglycan of the cell wall and natively act from the “inside-out”. Applying them to the outside of bacteria as antimicrobials has shown efficacy against gram-positive bacteria, however the additional outer membrane of gram-negative bacteria impedes the endolysin from accessing the peptidoglycan. Therefore, current research majorly focuses on approaches to enhance the ability of endolysins to overcome the gram-negative outer membrane. These strategies include the use of chemical membrane permeabilisers and the engineering of fusion proteins incorporating endolysins, antimicrobial peptides and phage-derived receptor binding proteins. Here, we explore the use of phage-derived proteins against the causal agent of bacterial canker on kiwifruit; the gram-negative bacterium Pseudomonas syringae pv. actinidiae (Psa). We describe and investigate the mechanism of an antimicrobial synergy between citric acid and the endolysin from the phage ⏀Psa374 that is effective at reducing copper resistant Psa in vitro. We then demonstrate the development of novel chimeric phage-derived proteins incorporating additional phage proteins such as tail fibers, baseplates, tail spikes, holins and virion-associated lysozymes. By generating and screening large numbers of variant fusion proteins we produced a lead variant, L4E10, that utilises a modular endolysin and a lipase, which displays moderate exogenous antibacterial activity that is specific to Pseudomonas syringae pathovars. Finally, we developed a vector, containing Escherichia coli SlyD, which can be co-expressed to enhance the soluble expression and purification of aggregation-prone proteins; including endolysin - tail fiber fusions. Overall, this research evidences the potential of endolysin fusion proteins as alternative antimicrobials against gram-negative phytopathogens and explores the prospects of further phage proteins as an untapped antimicrobial reservoir.
