Enhancing bacteriocin antimicrobial activity : from directed evolution to structural insights
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- With the increase in antibiotic resistance, the search for therapeutic alternatives has become a global priority. Bacteriocins are a promising alternative to antibiotics. Bacteriocins are antimicrobial peptides naturally secreted by bacteria, which offer several benefits compared to antibiotics. In this master’s thesis, we have chosen to focus our work on the bacteriocin BlpK, synthesized by the HSISS4 strain of Streptococcus salivarius. This strain is a commensal of the human digestive tract that produces a cocktail of six class II bacteriocins: BlpK, SlvV, SlvW, SlvX, SlvY and SlvZ. Among these, BlpK is responsible for most of the inhibitory ability of S. salivarius HSISS4. This thesis has two main objectives: firstly, to enhance the inhibitory activity of the bacteriocin BlpK using a directed evolution approach, and secondly, to characterize in depth its structure-function relationship. These two goals, both applied and fundamental, were pursued in parallel to optimize BlpK's inhibitory activity against problematic pathogens, in anticipation of future applications, and to improve our understanding of the molecular mechanisms underlying its activity. Concerning the improvement of BlpK activity, we were able to select more active variants from mutant libraries generated by random mutagenesis against Lactococcus lactis used as a model indicator species. Then, we validated the directed evolution strategy for BlpK optimization against the pathogens Enterococcus faecalis and Streptococcus pneumoniae. Two main categories of modifications increased BlpK activity: substitutions in asparagine (N) and substitutions in negatively charged residues (i.e., glutamate (E) or aspartate (D)). We also identified several variants with enhanced activity against these two pathogens, some of which even have a broader spectrum of action: S3N, G6D, F7I, K9N and V33A. Some of these improved variants have substitutions also found in natural BlpK variants from other species. This suggests that experimentally selected mutations reproduce natural adaptations conferring a competitive advantage to the producing strains. Concerning the characterization of the structure-function of BlpK, the analysis of attenuated or inactivated mutants has strengthened our understanding of the essential role of certain residues in the inhibitory activity of BlpK. Analysis of variants with attenuated activity has highlighted the importance of specific residues: cysteines, positively charged residues, and glycines, which contribute to the structural stability and activity of BlpK. Specifically, the positively charged residues and charge polarity within the BlpK structure appear to be an essential feature for its insertion, as well as for its anchoring, in the targeted bacterial membrane. Finally, the data obtained reinforce the proposed model for the mechanism of action of BlpK, based on its oligomerization and permeabilization of the bacterial membrane. In summary, this work has contributed to the enrichment of existing data on the structure-function relationship of BlpK and has updated pre-existing data on the mode of action of BlpK. It also demonstrates the relevance of directed evolution for optimizing the activity of antimicrobial peptides such as BlpK. The integration of high-throughput mutagenesis and automated screening technologies could further accelerate these advances.