Identification and quantification of peptidoglycan cytoplasmic fragments by LC-MS

In Bacillus licheniformis 749/I, the induction of BlaP beta-lactamase relies on a complex regulation system. During this process, the intracellular repressor BlaI is inactivated by a dipeptide coming from the peptidoglycan (PG) degradation via an “AND Gate” regulation. This regulation involves the cellular stress induced by the beta-lactam, the membrane receptor BlaR1 and the PG turnover.
Briefly, the induction occurs when the extracellular domain of BlaR1 is acylated by the antibiotic which leads to a reorganization of the transmembrane segments and the receptor autocleavage. Simultaneously, the beta-lactam partially inhibits the penicillin-binding protein 1 (PBP1), triggering increased PG turnover and accumulation of PG fragments. Some of these fragments could enter in the cytoplasm and undergo enzymatic degradations which lead to the formation of the pro co-activator (tripeptide L-Ala-D-Glu-m-A2pm). This pro co-activator generates the co-activator, the dipeptide D-Glu-m-A2pm.
Nowadays the nature and the concentration of PG fragments inside the cytoplasm are unknown. Therefore, the development and the validation of a zwitterionic hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry method (ZIC-HILIC-MS) are required in order to identify and quantify those cytoplasmic fragments.