Peptidoglycan (PG) is a crosslinked, mesh-like scaffold endowed with the strength to withstand the internal turgor pressure of bacteria. Crosslinking of peptide chains within PG is an essential process and its disruption thereof underpins the potency of several classes of antibiotics. Two primary crosslinking modes have been identified that are carried out by D,D-transpeptidases and L,D-transpeptidases (Ldts). The nascent PG from each enzymatic class is structurally unique, which results in different crosslinking configurations. Recent advances in PG cellular probes have been powerful in advancing the understanding of D,D-transpeptidation by Penicillin Binding Proteins (PBPs). In contrast, no cellular probes have been previously described to directly interrogate Ldt function in live cells. Herein, we describe a new class of Ldt-specific probes composed of structural analogs of nascent PG, which are metabolically incorporated into the PG scaffold by Ldts. With a panel of tetra- and penta-peptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG crosslinking. Ldt-probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results suggest that Ldt crosslinking may have distinct functions that work in concert with PBP crosslinking to assemble the PG scaffold. We anticipate that unraveling the interplay between Ldts and PBP crosslinking may guide drug regimen and establish new drug targets.

L,D-transpeptidase specific cell wall probes

2019 Spring National Meeting

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