L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking

Abstract

Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand the internal pressure of bacteria. Bacteria are known to heavily remodel their peptidoglycan stem peptides, yet little is known about the physiological impact of these chemical variations on peptidoglycan cross-linking. Furthermore, there are limited tools to study these structural variations, which can also have important implications on cell wall integrity and host immunity. Cross-linking of peptide chains within PG is an essential process, and its disruption thereof underpins the potency of several classes of antibiotics. Two primary cross-linking 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 cross-linking 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 tetrapeptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG cross-linking. Ldt probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results confirm that our Ldt probes are specific and suggest that the primary sequence of the stem peptide can control Ldt cross-linking levels. We anticipate that unraveling the interplay between Ldts and other cross-linking modalities may reveal the organization of the PG structure in relation to the spatial localization of cross-linking machineries.

Packed Like Sardines– How Surface Crowdedness Impacts Accessibility to Peptidoglycan ofStaphylococcus aureus

“…A wide range of single D-amino acid PG probes have been developed to elucidate fundamental steps in bacterial cell wall biology. 13,[22][23][24][25][26][27][28][29][30][31][32] We also included oxidized D-cystine (2) and D-cysteine amidated in the C-terminus (3) in an attempt to maximize PG tagging. 26,30 Cell treatment with the dipeptide D-Cys-D-Ala (4) was expected to result in the incorporation of D-cysteine at the 4 th position of the stem peptide.…”

“…Lastly, cysteine was placed at the N-terminus of a tetrapeptide synthetic analog (5) of the PG stem peptide. We 32,35 , and others [36][37][38][39] , recently showed that structural analogs of PG stem peptides can be crosslinked into the growing PG scaffold of live cells. We proceeded to investigate the role of surface biopolymers on PG accessibility.…”

Building peptidoglycan inside eukaryotic cells: A view from symbiotic and pathogenic bacteria

“…I would like to speculate on a putative role of l , d ‐transpeptidases to restrain active growth. These enzymes catalyze the formation of cross‐links between amino acids in third position of adjacent stem peptides, l , d ‐(3‐3) bridges of structure m‐ Dap‐ m‐ Dap (Pidgeon et al, ). Unlike the d , d ‐(4‐3) bridge catalyzed by PBPs, the l , d ‐(3‐3) bridge does not depend on an intact pentapeptide side chain and, as consequence, can be formed in structured mature PG (Figure a).…”

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms

“…Because bacteria can recycle more than d ‐Ala, dipeptide reporters have also been developed to monitor the de novo biosynthesis and turnover of peptidoglycan (Figure C) . In addition, dipeptide reporters have also been employed to monitor l,d ‐transpeptidase‐mediated peptidoglycan crosslinking as well as photo‐crosslinking of lipid II‐binding proteins (Figure D) …”