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Staphylococcus aureus peptidoglycan (PG) is densely functionalized with anionic polymers called wall teichoic acids (WTAs). These polymers contain three tailoring modifications: d -alanylation, α- O -GlcNAcylation, and β- O -GlcNAcylation. Here we describe the discovery and biochemical characterization of a unique glycosyltransferase, TarS, that attaches β- O -GlcNAc (β- O - N -acetyl- d -glucosamine) residues to S. aureus WTAs. We report that methicillin resistant S. aureus (MRSA) is sensitized to β-lactams upon tarS deletion. Unlike strains completely lacking WTAs, which are also sensitive to β-lactams, Δ tarS strains have no growth or cell division defects. Because neither α- O -GlcNAc nor β- O -Glucose modifications can confer resistance, the resistance phenotype requires a highly specific chemical modification of the WTA backbone, β- O -GlcNAc residues. These data suggest β- O -GlcNAcylated WTAs scaffold factors required for MRSA resistance. The β- O -GlcNAc transferase identified here, TarS, is a unique target for antimicrobials that sensitize MRSA to β-lactams.

The growing prevalence of multidrug-resistant Gram-negative pathogens highlights the urgent need for therapeutic strategies that complement traditional antibiotics by targeting essential virulence pathways. Glycosyltransferase LgtC, a key enzyme in lipooligosaccharide (LOS) biosynthesis, represents an attractive target for antivirulence approaches because of its essential role in bacterial immune evasion and pathogenicity. In this work, we employed an integrated in silico pipeline to identify thioglycoside analogs structurally related to the metabolic decoys FucSBn and BacSBn, evaluating their potential to hit the UDP-galactose donor pocket of LgtC. A similarity-based screening in PubChem, followed by ADME–Tox filtering yield 18 candidate analogs. Molecular docking using AutoDock-GPU revealed several candidates, most notably C-5 (− 8.36 kcal/mol) and C-18 (− 8.13 kcal/mol), to bind favorably within the donor site, showing more negative mean scoring values than natural donor UDP-α-D-galactose (− 6.74 kcal/mol). Redocking of the natural ligand reproduced the crystallographic pose, supporting the reliability of the docking protocol. To assess dynamic behavior, 100 ns molecular dynamics simulations (AMBER14) were performed for each complex. The top-scoring analogs maintained stable binding poses, with RMSD values of ~ 2.0–3.0 Å and preserved donor-like hydrogen-bond networks complemented by π-stacking and sulfur-mediated contacts. These interaction patterns suggest that the thioglycoside analogs may occupy the donor site in a manner compatible with competitive binding. While docking and MD describe different aspects of ligand recognition, several trends observed in docking, such as the favorable binding scores of C-5 and C-18, are broadly consistent with their ability to maintain stable poses during MD. Based on this consistency and scoring, the thioglycoside scaffolds C-5, C-14, and C-18 emerge as computationally prioritized candidates for subsequent biochemical testing against LgtC. Furthermore, these scaffolds offer a mechanistic basis and putative starting points for future structure-based optimization of thioglycoside analogs aimed at disrupting LOS biosynthesis in multidrug-resistant Gram-negative bacteria.

The Glycine max xyloglucan endotransglycosylase/hydrolase (EC 2.4.1.207), GmXTH43, has been identified through RNA sequencing of RNA isolated through laser microdissection of Heterodera glycines -parasitized root cells (syncytia) undergoing the process of defense. Experiments reveal that genetically increasing XTH43 transcript abundance in the H . glycines -susceptible genotype G . max [Williams 82/PI 518671] decreases parasitism. Experiments presented here show decreasing XTH43 transcript abundance through RNA interference (RNAi) in the H . glycines -resistant G . max [Peking/PI 548402] increases susceptibility, but it is unclear what role XTH43 performs. The experiments presented here show XTH43 overexpression decreases the relative length of xyloglucan (XyG) chains, however, there is an increase in the amount of those shorter chains. In contrast, XTH43 RNAi increases XyG chain length. The experiments show that XTH43 has the capability to function, when increased in its expression, to limit XyG chain extension. This outcome would likely impair the ability of the cell wall to expand. Consequently, XTH43 could provide an enzymatically-driven capability to the cell that would allow it to limit the ability of parasitic nematodes like H . glycines to develop a feeding structure that, otherwise, would facilitate parasitism. The experiments presented here provide experimentally-based proof that XTHs can function in ways that could be viewed as being able to limit the expansion of the cell wall.

Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections. Biosynthesis of glycosylphosphatidylinositol (GPI) is essential for the integrity of the fungal cell wall. Here, the authors show that the natural product jawsamycin inhibits GPI biosynthesis by targeting a subunit of the fungal UDP-glycosyltransferase, and displays pronounced activity against pathogenic fungi of the order Mucorales.

Most bacterial cell envelopes contain a cell wall layer made of peptidoglycan. The synthesis of new peptidoglycan is critical for cell growth, division, and morphogenesis and is also coordinated with peptidoglycan hydrolysis to accommodate the new material. However, the enzymes that cleave peptidoglycan must be carefully controlled to avoid autolysis. In recent years, some control mechanisms have begun to emerge, although there are many more questions than answers for how most cell wall hydrolases are regulated. Here, we report a novel cell wall hydrolase control mechanism in Pseudomonas aeruginosa , which we discovered during our characterization of a mutant sensitive to the overproduction of a secretin protein. The mutation affected an uncharacterized Sel1-like repeat protein encoded by the PA3978 locus. In addition to the secretin-sensitivity phenotype, PA3978 disruption also increased resistance to a β-lactam antibiotic used in the clinic. In vivo and in vitro analyses revealed that PA3978 binds to the catalytic domain of the lytic transglycosylase MltF and inhibits its activity. ∆PA3978 mutant phenotypes were suppressed by deleting mltF , consistent with them having been caused by elevated MltF activity. We also discovered another interaction partner of PA3978 encoded by the PA5502 locus. The phenotypes of a ∆PA5502 mutant suggested that PA5502 interferes with the inhibitory function of PA3978 toward MltF, and we confirmed that activity for PA5502 in vitro . Therefore, PA3978 and PA5502 form an inhibitor/anti-inhibitor system that controls MltF activity. We propose to name these proteins IltA ( i nhibitor A of l ytic t ransglycosylase) and LiiA ( l ytic transglycosylase i nhibitor A’s i nhibitor). A peptidoglycan cell wall is an essential component of almost all bacterial cell envelopes, which determines cell shape and prevents osmotic rupture. Antibiotics that interfere with peptidoglycan synthesis have been one of the most important treatments for bacterial infections. Peptidoglycan must also be hydrolyzed to incorporate new material for cell growth and division and to help accommodate important envelope-spanning systems. However, the enzymes that hydrolyze peptidoglycan must be carefully controlled to prevent autolysis. Exactly how this control is achieved is poorly understood in most cases but is a highly active area of current research. Identifying hydrolase control mechanisms has the potential to provide new targets for therapeutic intervention. The work here reports the important discovery of a novel inhibitor/anti-inhibitor system that controls the activity of a cell wall hydrolase in the human pathogen Pseudomonas aeruginosa , which also affects resistance to an antibiotic used in the clinic.

Iminosugars are sugar analogues endowed with a high pharmacological potential. The wide range of biological activities exhibited by these glycomimetics associated with their excellent drug profile make them attractive therapeutic candidates for several medical interventions. The ability of iminosugars to act as inhibitors or enhancers of carbohydrate-processing enzymes suggests their potential use as therapeutics for the treatment of cystic fibrosis (CF). Herein we review the most relevant advances in the field, paying attention to both the chemical synthesis of the iminosugars and their biological evaluations, resulting from in vitro and in vivo assays. Starting from the example of the marketed drug NBDNJ (N-butyl deoxynojirimycin), a variety of iminosugars have exhibited the capacity to rescue the trafficking of F508del-CFTR (deletion of F508 residue in the CF transmembrane conductance regulator), either alone or in combination with other correctors. Interesting results have also been obtained when iminosugars were considered as anti-inflammatory agents in CF lung disease. The data herein reported demonstrate that iminosugars hold considerable potential to be applied for both therapeutic purposes.

Chemical tools have proven indispensable for studies in glycobiology. Synthetic oligosaccharides and glycoconjugates provide materials for correlating structure with function. Synthetic mimics of the complex assemblies found on cell surfaces can modulate cellular interactions and are under development as therapeutic agents. Small molecule inhibitors of carbohydrate biosynthetic and processing enzymes can block the assembly of specific oligosaccharide structures. Inhibitors of carbohydrate recognition and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. Carbohydrate biosynthetic pathways are often amenable to interception with synthetic unnatural substrates. Such metabolic interference can block the expression of oligosaccharides or alter the structures of the sugars presented on cells. Collectively, these chemical approaches are contributing great insight into the myriad biological functions of oligosaccharides.

Drug-resistant bacteria have caused serious medical problems in recent years, and the need for new antibacterial agents is undisputed. Transglycosylase, a multidomain membrane protein essential for cell wall synthesis, is an excellent target for the development of new antibiotics. Here, we determined the X-ray crystal structure of the bifunctional transglycosylase penicillin-binding protein 1b (PBP1b) from Escherichia coli in complex with its inhibitor moenomycin to 2.16-Å resolution. In addition to the transglycosylase and transpeptidase domains, our structure provides a complete visualization of this important antibacterial target, and reveals a domain for protein-protein interaction and a transmembrane helix domain essential for substrate binding, enzymatic activity, and membrane orientation.

The rapid rise of multi-drug-resistant bacteria is a global healthcare crisis, and new antibiotics are urgently required, especially those with modes of action that have low-resistance potential. One promising lead is the liposaccharide antibiotic moenomycin that inhibits bacterial glycosyltransferases, which are essential for peptidoglycan polymerization, while displaying a low rate of resistance. Unfortunately, the lipophilicity of moenomycin leads to unfavourable pharmacokinetic properties that render it unsuitable for systemic administration. In this study, we show that using moenomycin and other glycosyltransferase inhibitors as templates, we were able to synthesize compound libraries based on novel pyranose scaffold chemistry, with moenomycin-like activity, but with improved drug-like properties. The novel compounds exhibit in vitro inhibition comparable to moenomycin, with low toxicity and good efficacy in several in vivo models of infection. This approach based on non-planar carbohydrate scaffolds provides a new opportunity to develop new antibiotics with low propensity for resistance induction. The inhibition of bacterial glycosyltransferase has the potential to be an effective therapeutic target against drug resistance bacteria. Here, the authors present a novel class of inhibitor compounds based on a monosaccharide scaffold, which are able to eliminate bacterial infections in mice.

Bacteria surround themselves with peptidoglycan, an adaptable enclosure that contributes to cell shape and stability. Peptidoglycan assembly relies on penicillin-binding proteins (PBPs) acting in concert with SEDS-family transglycosylases RodA and FtsW, which support cell elongation and division respectively. In Bacillus subtilis , cells lacking all four PBPs with transglycosylase activity (aPBPs) are viable. Here, we show that the alternative sigma factor σ I is essential in the absence of aPBPs. Defects in aPBP-dependent wall synthesis are compensated by σ I -dependent upregulation of an MreB homolog, MreBH, which localizes the LytE autolysin to the RodA-containing elongasome complex. Suppressor analysis reveals that cells unable to activate this σ I stress response acquire gain-of-function mutations in the essential histidine kinase WalK, which also elevates expression of sigI , mreBH and lytE . These results reveal compensatory mechanisms that balance the directional peptidoglycan synthesis arising from the elongasome complex with the more diffusive action of aPBPs.