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Design of a De Novo Aggregating Antimicrobial Peptide and a Bacterial Conjugation-Based Delivery System
journal contribution
posted on 2018-11-07, 00:00 authored by Logan
T. Collins, Peter B. Otoupal, Jocelyn K. Campos, Colleen M. Courtney, Anushree ChatterjeeAntibacterial
resistance necessitates the development of novel
treatment methods for infections. Protein aggregates have recently
been applied as antimicrobials to disrupt bacterial homeostasis. Past
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 a 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 decrease. Further, we developed a probiotic delivery system
using the broad-host range conjugative plasmid RK2 to transfer the
gene from donor to recipient bacteria. We utilize RK2 to mobilize
a shuttle plasmid carrying opaL by adding the RK2
origin of transfer. We show that opaL is nontoxic
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 T7 RNA polymerase
expression and provides a basis for the design and delivery of aggregating
antimicrobial peptides.
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Keywords
pathogen-specific promotersgene encodingaggregating antimicrobial peptidescontrol T 7 RNA polymerase expressionacidic residuesprobiotic delivery systemdonorgenome mininghomeostasigrowth repressionopaLPast workProtein aggregatesRK 2 originaggregating antimicrobial peptidetransferrecipient-specific T 7 RNA polymeraseEscherichia coliaggregation-prone sequencesbiology approachRK 2Bacterial Conjugation-Based Delivery System Antibacterial resistanceprotein aggregatesantimicrobial effect decreasenovel treatment methodsbroad-host range conjugative plasmid RK 2T 7 polymerase-expressing recipientsshuttle plasmidDe Novo Aggregating Antimicrobial Peptide
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