Drug-resistant bacterial infections threaten to overburden our healthcare system and disrupt modern medicine. A large class of potent antibiotics, including vancomycin, operate by interfering with bacterial cell wall biosynthesis. Vancomycin-resistant enterococci (VRE) evade the blockage of cell wall biosynthesis by altering cell wall precursors, rendering them drug insensitive. Herein, we reveal the phenotypic plasticity and cell wall remodeling of VRE in response to vancomycin in live bacterial cells via a metabolic probe. A synthetic cell wall analog was designed and constructed to monitor cell wall structural alterations. Our results demonstrate that the biosynthetic pathway for vancomycin-resistant precursors can be hijacked by synthetic analogs to track the kinetics of phenotype induction. In addition, we leveraged this probe to interrogate the response of VRE cells to vancomycin analogs and a series of cell wall-targeted antibiotics. Finally, we describe a proof-of-principle strategy to visually inspect drug resistance induction. Based on our findings, we anticipate that our metabolic probe will play an important role in further elucidating the interplay among the enzymes involved in the VRE biosynthetic rewiring.

The surge in drug-resistant bacterial infections threatens to overburden healthcare systems worldwide. Bacterial cell walls are essential to bacteria, thus making them unique targets for the development of antibiotics. We describe a cellular reporter to directly monitor the phenotypic switch in drug-resistant bacteria with temporal resolution. Vancomycin-resistant enterococci (VRE) escape the bactericidal action of vancomycin by chemically modifying their cell-wall precursors. A synthetic cell-wall analogue was developed to hijack the biosynthetic rewiring of drug-resistant cells in response to antibiotics. Our study provides the first in vivo VanX reporter agent that responds to cell-wall alteration in drug-resistant bacteria. Cellular reporters that reveal mechanisms related to antibiotic resistance can potentially have a significant impact on the fundamental understanding of cellular adaption to antibiotics.

β-Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin-binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP-Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d-amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.

The number of antibiotic-resistant bacterial infections has increased dramatically over the past decade. To combat these pathogens, novel antimicrobial strategies must be explored and developed. We previously reported a strategy based on hapten-modified cell wall analogues to induce recruitment of endogenous antibodies to bacterial cell surfaces. Cell surface remodeling using unnatural single d-amino acid cell wall analogues led to modification at the C-terminus of the peptidoglycan stem peptide. During peptidoglycan processing, installed hapten-displaying amino acids can be subsequently removed by cell wall enzymes. Herein, we disclose a two-step dipeptide peptidoglycan remodeling strategy aimed at introducing haptens at an alternative site within the stem peptide to improve retention and diminish removal by cell wall enzymes. Through this redesigned strategy, we determined size constraints of peptidoglycan remodeling and applied these constraints to attain hapten-linker conjugates that produced high levels of antibody recruitment to bacterial cell surfaces.

A strategy has been devised for increasing the cellular selectivity of membrane-disrupting antibiotics based on the attachment of a facially amphiphilic sterol. Using Amphotericin B (AmB) as a prototype, covalent attachment of cholic acid bound to a series of α,ω-diamines has led to a dramatic reduction in hemolytic activity, a significant reduction in toxicity toward HEK293T cells, and significant retention of antifungal activity.

Bacteria can either exist in the planktonic (free floating) state or in the biofilm (encased within an organic framework) state. Bacteria biofilms cause industrial concerns and medical complications and there has been a great deal of interest in the discovery of small molecule agents that can inhibit the formation of biofilms or disperse existing structures. Herein we show that, contrary to previously published reports, d-amino acids do not inhibit biofilm formation of Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Staphylococcus epidermis (S. epidermis) at millimolar concentrations. We evaluated a diverse set of natural and unnatural d-amino acids and observed no activity from these compounds in inhibiting biofilm formation.

Bacterial peptidoglycan is a mesh-like network comprised of sugars and oligopeptides. Transpeptidases cross-link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous D-amino acids onto bacterial cell surfaces with vast promiscuity for the side-chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C-terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C-terminus amidated D-amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species.

Bioorthogonal click ligations are extensively used for the introduction of functional groups in biological systems. Tetrazine ligations are attractive in that they are catalyst-free and display favorable kinetics. We describe the efficient remodeling of bacterial cell surfaces using unnatural d-amino acids derivatized with tetrazine ligation handles. The metabolic incorporation of these unnatural d-amino acids onto bacterial cell surfaces resulted in a site-selective installation of fluorophores.

The post-transitional modification of peptidyl arginine to citrulline by PAD2 can affect the inherent biophysical properties of the citrullinated protein. Furthermore, dysregulation of PAD2 activity has been implicated in a number of human diseases. Inhibition of these enzymes by small molecules can serve as essential probes in establishing a link to pathogenesis. Herein, we describe a profluorescent substrate analog that reports on the activity and the inhibition of PAD2 in a robust assay. Most noteworthy, we expect future drug discovery efforts based on PAD2 inhibition can be pursued via this assay.

Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape, and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organism. Intriguingly, bacteria can metabolically incorporate unnatural D-amino acids into the peptidoglycan stem peptide directly from the surrounding medium, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural D-amino acids has provided unique insights into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural side chains. However, the chemical space and physical features of this side chain promiscuity have not been determined systematically. In this report, we designed and synthesized a library of variants displaying diverse side chains to comprehensively establish the tolerability of unnatural D-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural D-amino acid incorporation and gaining insights into the temporal control of PBP activity. We empirically established the scope of physical parameters that govern the metabolic incorporation of unnatural D-amino acids into bacterial peptidoglycan.