Abstract :
[en] Considering the increasing emergence of antibiotic resistant bacterial
strains, the main objective of this thesis is to increase our knowledge on
these resistance systems and, more particularly, that of Bacillus
licheniformis in which a fragment from its cell wall – the peptidoglycan –
triggers the induction of the β-lactamase BlaP.
The first objective of this thesis was to work on the development of analytical
methods to analyze all types of soluble peptidoglycan fragments. In a first
step, capillary zone electrophoresis (CZE) coupled with mass spectrometry
was considered for the characterization of the mixture of muropeptides
derived from the peptidoglycan of B. licheniformis and compared to the
“gold standard" represented by reverse phase liquid chromatography
followed by an off-line analysis by mass spectrometry. Secondly, given the
polar nature of peptides triggering the β-lactamase induction, their
separation in a complex mixture by chromatography is not easy: they are
usually eluted in the dead volume in reverse phase chromatography,
whereas their separation by ion exchange chromatography is often
incompatible with mass spectrometry coupling. Two alternatives for their
separation and analysis were therefore proposed: capillary zone
electrophoresis and hydrophilic interaction liquid chromatography (HILIC).
As with the analysis of muropeptides, the first was directly coupled to the
mass spectrometer while the second is followed by an off-line mass
spectrometry analysis. In addition, the quantification of peptides by these
analytical methods has been achieved by the spiking of an isotopically
labelled equivalent, allowing the direct dosage of these molecules in the
cytoplasm.
The second objective of this work was to test these peptide quantification
methods in order to characterize a biological system, namely the activity of
the L3 cytoplasmic loop of the BlaR1 receptor of B. licheniformis. Indeed, in
the presence of an antibiotic in the extracellular medium, the presumed
activity of this loop would be an L-Ala-aminopeptidase enabling the
tripeptide L-Ala-D-iGlu-m-A2pm derived from peptidoglycan to be cleaved
into the dipeptide D-iGlu-m-A2pm, the co-activator of -lactamase BlaP
induction. However, in the absence of antibiotics, this loop would show no
activity. The L3 loop activity was therefore studied by assaying the
respective cytoplasmic concentrations of di- and tripeptide in different
mutants of BlaR1, in the presence and absence of antibiotics, in order to
confirm its activity.
