Mechanisms of resistance and immunity against bacteriocins in Streptococcus salivarius HSISS4

Details

Supervisors

Hols, Pascal ; Mahillon, Jacques

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

One of the major challenges of our century is the fight against the spread of antibiotic resistance. The WHO predicts that the increase in antibiotic resistance will be responsible for up to 10 million deaths by 2050, so there is an urgent need to find alternatives. One promising solution is based on bacteriocins, i.e. antimicrobial peptides synthesised by bacteria. In this master’s thesis, we focused on a Gram-positive commensal bacterium: Streptococcus salivarius HSISS4. This strain produces several bacteriocins, including BlpK. BlpK inhibits the growth of clinically relevant pathogens such as Staphylococcus aureus and Enterococcus faecium but also of some bacteria involved in more common health problems. Many bacteriocins act by creating pores in the cell wall of their target. This often requires the help of a receptor, which is used as the anchor point and/or as the starting point of the pore. Bacteriocin producers have immunity mechanisms to protect themselves from their weapons. In this work, we sought to identify the receptor used by BlpK as well as the proteins involved in the immunity and resistance mechanisms. To achieve those objectives, we based our work on two approaches. The first was to identify the receptor by purifying the complex (receptor-bacteriocin-immunity protein) after incubation with a cross-linking agent. To this end, we attached a strep-tag to the immunity protein. Then, we successfully detected the immunity protein by western blot and evaluated the best solvent to solubilize it. Finally, we used a strep-Tactin column to purify the protein. The second method was based on the selection of spontaneous mutants of resistance. We performed several tests to discriminate the mutants we obtained. As many bacteriocins target sugar transporters, we measured their growth in presence of different carbon sources to see if some mutants had lost their ability to metabolize some of them. Then, we observed their sensitivity towards different bacteriocins and their ability to spontaneously produce bacteriocins. On the basis of these phenotypic analyses, we selected nine mutants with different phenotypes to sequence their whole genome. We observed that a mutation in the gene coding for a transcriptional regulator, gntR, was present in eight of them. By sequencing this gene in the 45 mutants, we discovered a mutation in gntR for 33 of them. This clearly suggests that gntR is somehow involved in bacteriocin resistance, potentially through the regulation of an ABC transporter. In conclusion, our work has determined the parameters necessary to achieve cross-linking and purification of the receptor. We have also identified a regulator, GntR, which plays an important role in the mechanism of resistance to BlpK. The data collected during this master’s thesis could eventually help to anticipate or slow the emergence and spread of bacterial resistances, and bring a better understanding of the mechanisms of immunity. This is essential for clinical applications, such as the use of HSISS4 as probiotic or the development of drug similar to BlpK.