Engineering an antimicrobial chimeric endolysin that targets the phytopathogen Pseudomonas syringae pv. actinidiae

Global food shortages and rising antimicrobial resistance require alternatives to antibiotics and agrichemicals for the management of agricultural bacterial pathogens. The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of kiwifruit canker and is responsible for major agricultural losses. Bacteriophage enzymes present an emerging antimicrobial option. Endolysins possess the ability to cleave peptidoglycan and are effective antimicrobials against gram-positive bacteria. Delivery of endolysins to the peptidoglycan of gram-negatives is impeded by the additional outer membrane. To overcome this barrier, we used VersaTile molecular shuffling to produce Psa-targeting chimeric proteins which were then tested for antimicrobial activity. These chimeras consist of endolysins linked by polypeptides to diverse phage proteins mined from Psa phage genomes. A preferential configuration for antibacterial activity was observed for enzymatic domains at the N terminus and alternative phage proteins at the C terminus. The lead variant possessed an N-terminal modular endolysin and a C-terminal lipase. Antibacterial activity was enhanced with the addition of the chemical permeabilizers such as citric acid or EDTA. Mutagenesis of the lipase active site eliminated exogenous antibacterial activity toward Psa. The endolysin-lipase chimera demonstrated specificity toward Psa, illustrating potential as a targeted biocontrol agent. Overall, we generated a chimeric endolysin with exogenous and specific activity toward Psa, the causative agent of kiwifruit canker.