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.
New therapeutics to augment current approaches and shorten treatment duration are of critical importance for combating tuberculosis (TB), especially those with novel mechanisms of action to counter the emergence of drug-resistant TB. Host-directed therapy (HDT) offers a novel strategy with mechanisms that include activating immune defense mechanisms or ameliorating tissue damage. These and related concepts will be discussed along with issues that emerged from the workshop organized by the Stop TB Working Group on New Drugs, held at the Gordon Research Conference for Tuberculosis Drug Development in Lucca, Italy in June 2017, titled "Strategic Discussion on Repurposing Drugs & Host Directed Therapies for TB. " In this review, we will highlight recent data regarding drugs, pathways, and concepts that are important for successful development of HDTs for TB.
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