Filipe, SérgioMartins, Jorge Miguel Alves2021-01-262024-01-012021-01-122021http://hdl.handle.net/10362/110755Bacterial antibiotic resistance constitutes a major and current threat worldwide and the development of new antibacterial strategies is considered a priority for organisms such as Streptococcus pneumoniae. The murMN operon in S. pneumoniae has long been associated with an elevated β-lactam resistance. Its expression is responsible for the presence of branched muropeptides on the peptidoglycan produced by multiple strains resistant to penicillin. Recent work from our lab demonstrated that it was possible to restore penicillin resistance to mutants without the murMN operon. Three mutants, called suppressors, were selected through several rounds of population analysis profiles in increasing penicillin concentrations, in two separate occasions. The suppressor mutants had a decreased susceptibility to penicillin and contained several mutations in regions of the bacterial chromosome, which did not include the gene groups usually associated to β-lactam resistance. Some of these mutations could be associated with the TA synthesis pathway or the cell wall synthesis regulating factors. The purpose of this work was to understand how the suppressor mutants, which express PBP with low affinity to penicillin, but lack the murMN operon, managed to decrease their susceptibility to penicillin. To characterize the phenotypical effects of each mutation, the effects of two mutations present in Pen6ΔmurMN suppressor 1.1, mutations lytR(R76G) e htrA(A361P), were first analyzed. It was observed that the combination of both lytR(R76G) and htrA(A361P) is necessary and sufficient to decrease penicillin susceptibility in the strain Pen6ΔmurMN, but not each mutation individually. In fact, the mutant that received htrA(A361P) does not show any changes in susceptibility, whereas the one that received lytR(R76G) showed only a slight decrease in susceptibility to this antibiotic. To characterize the effect of mutations in LytR protein, and to produce specific antibodies for this protein, derivatives of LytR were expressed, in Escherichia coli, whose purification was successful.engS. pneumoniaemurMN operonLCPLytRβ-lactamHtrAmaster thesis