Research Topic: Leveraging URS-Induced Host Stress Cascades for thePhage-Assisted Evolution of Antimicrobial Peptides
Abstract: ** Lecture will be given in English**
The global escalation of antimicrobial resistance (AMR) demands novel therapeutic strategies, including the development of RNA- and peptide-based antimicrobial agents derived from underrepresented sequences (URSs). URSs are short amino acid triplets (such as CMY) naturally rare in bacterial proteomes that induce irreversible translation arrest within the ribosomal exit tunnel. To optimize and enhance URS peptide potency from a genomic perspective, this thesis aimed to engineer an indirect phage-assisted directed evolution system in Escherichia coli. By coupling the internal toxic effect of URS expression to the indirect transcriptional activation of host stress promoters driving the essential phage coat protein gene III (gIII), this platform seeks to establish a positive selection loop for evolving toxic peptide variants.
Initial optimization of induction parameters demonstrated that low inducer (IPTG) concentrations (0.1–0.25 mM) achieved significant URS-mediated bactericidal activity—reducing cell viability by up to four orders of magnitude—without inducer-related growth interference. Western blot analysis confirmed time-dependent intracellular accumulation of the 32-amino-acid CMYWKx5 peptide (~5 kDa) over two hours post-induction. High-throughput RNA sequencing (RNA-seq) of E. coli BL21 expressing CMYWKx5 revealed distinct transcriptomic alterations, identifying ibpA (ibpAB operon) and pspA (pspACDG operon) as the most strongly upregulated host promoters based on normalized transcript abundance (TPM). Dual-plasmid fluorometric and cell viability assays confirmed that ibpA promoter activation (measured as relative fluorescence units normalized to OD600 and log(CFU/ml)) is specifically triggered by active URS peptides (CMYWx5 and CMYWKx5), whereas non-toxic URS-like control peptides (SMYWKx5) failed to activate the promoter.
To construct the continuous evolution vector, the CMYWKx5 cassette under an arabinose-inducible pBAD promoter was cloned into a gIII-deleted fth1 (Fd) filamentous bacteriophage backbone. Evaluation of this selection phage construct across multiple E. coli strains demonstrated an absence of arabinose-induced toxicity or viability reduction, highlighting key architectural and expression bottlenecks when porting toxic URS sequences into bacteriophage vectors. Overall, while this study successfully identified endogenous host stress promoters capable of linking internal peptide toxicity to indirect gene activation, full operational assembly of the phage-assisted continuous evolution system requires further vector engineering to maintain selection pressure within the phage framework.
