Design of a de novo aggregating antimicrobial peptide and bacterial conjugation delivery system

Abstract

Traditional antibiotics are reaching obsolescence as a consequence of antibiotic resistance; therefore novel antibiotic approaches are needed. A recent non-traditional approach involves formation of protein aggregates as antimicrobials to disrupt bacterial homeostasis. Previous work on protein aggregates has focused on genome mining for aggregation-prone sequences in bacterial genomes rather than on rational design of aggregating antimicrobial peptides. Here, we use a synthetic biology approach to design an artificial gene encoding the first de novo aggregating antimicrobial peptide. This artificial gene, opaL (overexpressed protein aggregator Lipophilic), disrupts bacterial homeostasis by expressing extremely hydrophobic peptides. When this hydrophobic sequence is disrupted by acidic residues, consequent aggregation and antimicrobial effect decreases. Further, to deliver this artificial gene, we developed a probiotic approach using RK2, a broad host range conjugative plasmid, to transfer opaL from donor to recipient bacteria. We utilize RK2 to mobilize a shuttle plasmid carrying the opaL gene by adding the RK2 origin of transfer. We show that opaL is non-toxic to the donor, allowing for maintenance and transfer since its expression is under control of a promoter with a recipient-specific T7 RNA polymerase. Upon mating of donor and recipient Escherichia coli, we observe selective growth repression in T7 polymerase expressing recipients. This technique could be used to target desired pathogens by selecting pathogen-specific promoters to control opaL expression. This system provides a basis for the design and delivery of novel antimicrobial peptides.\n\nImportanceThe growing threat of antibiotic resistance necessitates new treatment options for bacterial infections that are recalcitrant to traditional antimicrobials. Existing methods usually involve small-molecule compounds which interfere with essential processes in bacterial cells. By contrast, protein aggregates operate by causing widespread disruption of bacterial homeostasis and may provide a new method for combating infections. We used rational design to create and test an aggregating de novo antimicrobial peptide, OpaL. In addition, we employed bacterial conjugation to deliver the opaL gene from donor bacteria to recipient bacteria while using a strain-specific promoter to ensure that OpaL was only expressed in targeted recipients. To the best of our knowledge, this represents the first design for a de novo peptide with aggregation-mediated antimicrobial activity. We envision that OpaLs design parameters could be used in developing a new class of antimicrobial peptides to help treat antibiotic resistant infections.