Enhancing Bacteriocin Antimicrobial Activity: From Directed Evolution to Structural Insights

Details

Supervisors

Hols, Pascal

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

enIn response to the rising threat of antibiotic resistance, the exploration for alternative therapeutic strategies has become a global priority. Among the potential solutions, bacteriocins, peptides naturally secreted by bacteria, represent a promising alternative. This work focuses on the bacteriocin BlpK, produced by Streptococcus salivarius HSISS4, which contributes the majority of this strain’s inhibitory activity. The objective of this Master’s thesis was to characterize BlpK and identify the structural determinants of its antimicrobial activity. Wild-type and mutant BlpK variants were analyzed to establish structure-function relationships, while a directed-evolution approach was used to refine its mechanism of action and assess changes in potency and activity spectrum. BlpK structure-function relationships were investigated through the systematic characterization of mutant variants generated by random mutagenesis. Screening identified 38 attenuated and 7 fully inactivated single-amino acid mutants, revealing residues critical for activity. Structural mapping showed that reduced activity mainly resulted from altered charge polarization, disrupted α-helix packing (notably within conserved GxxxG motifs), impaired oligomerization, or changes affecting nanopore properties. Complete loss of activity was associated with disruption of the disulfide bridge, introduction of helix-breaking proline residues, or severe charge mispolarization, supporting a pore-forming mechanism that requires precise structural organization and charge distribution. Directed evolution revealed that BlpK activity can be enhanced through specific amino acid substitutions strengthening charge polarization, hydrogen bonding, flexibility, and helix stabilization. The variants S3N and G6D showed improved activity and lower MICs against Lactococcus lactis and Enterococcus faecalis. A second directed-evolution round based on the S3N variant identified enhanced double mutants, most notably S3N-G6D, whereas an independent evolution performed on the attenuated T14I background selected compensatory, charge-modulating mutations that restored WT-like activity, highlighting the major role of electrostatic tuning in BlpK function. The S3N-G6D double mutant emerged as one of the most potent BlpK variants, combining two naturally frequent substitutions. MIC assays using synthetic peptides showed that S3N-G6D remains active at significantly lower concentrations than wild-type BlpK, with a species-dependent improvement that was most pronounced against E. faecalis. Although inhibition zone sizes were similar to those of the single mutants, the double mutant displayed enhanced potency and retained activity across a broad range of Gram-positive bacteria. Single-cell microscopy further revealed faster membrane disruption and increased killing efficacy compared to wild-type BlpK, demonstrating that the S3N-G6D combination enhances the robustness of bactericidal action. These findings establish BlpK as a tunable, powerful bacteriocin, where precise structural features shape activity and spectrum. This work shows that bacteriocins can be engineered as next-generation antimicrobials, offering a promising alternative to traditional antibiotics in the fight against resistance.