Invasive Staphylococcus epidermidis uses a unique processive wall teichoic acid glycosyltransferase to evade immune recognition.

Staphylococcus epidermidis expresses glycerol phosphate wall teichoic acid (WTA), but some health care-associated methicillin-resistant S. epidermidis (HA-MRSE) clones produce a second, ribitol phosphate (RboP) WTA, resembling that of the aggressive pathogen Staphylococcus aureus. RboP-WTA promotes HA-MRSE persistence and virulence in bloodstream infections. We report here that the TarM enzyme of HA-MRSE [TarM(Se)] glycosylates RboP-WTA with glucose, instead of N-acetylglucosamine (GlcNAc) by TarM(Sa) in S. aureus. Replacement of GlcNAc with glucose in RboP-WTA impairs HA-MRSE detection by human immunoglobulin G, which may contribute to the immune-evasion capacities of many invasive S. epidermidis. Crystal structures of complexes with uridine diphosphate glucose (UDP-glucose), and with UDP and glycosylated poly(RboP), reveal the binding mode and glycosylation mechanism of this enzyme and explain why TarM(Se) and TarM(Sa) link different sugars to poly(RboP). These structural data provide evidence that TarM(Se) is a processive WTA glycosyltransferase. Our study will support the targeted inhibition of TarM enzymes, and the development of RboP-WTA targeting vaccines and phage therapies.

N-acetylmuramic acid recognition by MurK kinase from the MurNAc auxotrophic oral pathogen Tannerella forsythia.

The bacterial cell wall consists of a three-dimensional peptidoglycan layer, composed of peptides linked to the sugars N-acetylmuramic acid (MurNAc) and GlcNAc. Unlike other bacteria, the pathogenic Tannerella forsythia, a member of the red complex group of bacteria associated with the late stages of periodontitis, lacks biosynthetic pathways for MurNAc production and therefore obtains MurNAc from the environment. Sugar kinases play a crucial role in the MurNAc recycling process, activating the sugar molecules by phosphorylation. In this study, we present the first crystal structures of a MurNAc kinase, called murein sugar kinase (MurK), in its unbound state as well as in complexes with the ATP analog ß-γ-methylene adenosine triphosphate (AMP-PCP) and with MurNAc. We also determined the crystal structures of K1058, a paralogous MurNAc kinase of T. forsythia, in its unbound state and in complex with MurNAc. We identified the active site and residues crucial for MurNAc specificity as the less bulky side chains of S133, P134, and L135, which enlarge the binding cavity for the lactyl ether group, unlike the glutamate or histidine residues present in structural homologs. In establishing the apparent kinetic parameters for both enzymes, we showed a comparable affinity for MurNAc (Km 180 µM and 30 µM for MurK and K1058, respectively), with MurK being over two hundred times faster than K1058 (Vmax 80 and 0.34 µmol min-1 mg-1, respectively). These data might support a structure-guided approach to development of inhibitory MurNAc analogs for pathogen MurK enzymes.

Principles and Applications of CF2X Moieties as Unconventional Halogen Bond Donors in Medicinal Chemistry, Chemical Biology, and Drug Discovery.

As an orthogonal principle to the established (hetero)aryl halides, we herein highlight the usefulness of CF2X (X = Cl, Br, or I) moieties. Using tool compounds bearing CF2X moieties, we study their chemical/metabolic stability and their logP/solubility, as well as the role of XB in their small molecular crystal structures. Employing QM techniques, we analyze the observed interactions, provide insights into the conformational flexibilities and preferences in the potential interaction space. For their application in molecular design, we characterize their XB donor capacities and its interaction strength dependent on geometric parameters. Implementation of CF2X acetamides into our HEFLibs and biophysical evaluation (STD-NMR/ITC), followed by X-ray analysis, reveals a highly interesting binding mode for fragment 23 in JNK3, featuring an XB of CF2Br toward the P-loop, as well as chalcogen bonds. We suggest that underexplored chemical space combined with unconventional binding modes provides excellent opportunities for patentable chemotypes for therapeutic intervention.

NgR1 binding to reovirus reveals an unusual bivalent interaction and a new viral attachment protein.

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

Structural and functional analysis of natural capsid variants suggests sialic acid-independent entry of BK polyomavirus.

BK polyomavirus (BKPyV) is an opportunistic pathogen that uses the b-series gangliosides GD1b and GT1b as entry receptors. Here, we characterize the impact of naturally occurring VP1 mutations on ganglioside binding, VP1 protein structure, and virus tropism. Infectious entry of single mutants E73Q and E73A and the triple mutant A72V-E73Q-E82Q (VQQ) remains sialic acid dependent, and all three variants acquire binding to a-series gangliosides, including GD1a. However, the E73A and VQQ variants lose the ability to infect ganglioside-complemented cells, and this correlates with a clear shift of the BC2 loop in the crystal structures of E73A and VQQ. On the other hand, the K69N mutation in the K69N-E82Q variant leads to a steric clash that precludes sialic acid binding. Nevertheless, this mutant retains significant infectivity in 293TT cells, which is not dependent on heparan sulfate proteoglycans, implying that an unknown sialic acid-independent entry receptor for BKPyV exists.

Synthesis, Biochemical Characterization, and Genetic Encoding of a 1,2,4-Triazole Amino Acid as an Acetyllysine Mimic for Bromodomains of the BET Family.

Lysine acetylation is a charge-neutralizing post-translational modification of proteins bound by bromodomains (Brds). A 1,2,4-triazole amino acid (ApmTri) was established as acetyllysine (Kac) mimic recruiting Brds of the BET family in contrast to glutamine commonly used for simulating this modification. Optimization of triazole substituents and side chain spacing allowed BET Brd recruitment to ApmTri-containing peptides with affinities similar to native substrates. Crystal structures of ApmTri-containing peptides in complex with two BET Brds revealed the binding mode which mirrored that of Kac ligands. ApmTri was genetically encoded and recombinant ApmTri-containing proteins co-enriched BRD3(2) from cellular lysates. This interaction was blocked by BET inhibitor JQ1. With genetically encoded ApmTri, biochemistry is now provided with a stable Kac mimic reflecting charge neutralization and Brd recruitment, allowing new investigations into BET proteins in vitro and in vivo.

Optimization of the precursor supply for an enhanced FK506 production in Streptomyces tsukubaensis.

Tacrolimus (FK506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK506 is not efficient and costly, whereas the biosynthesis of FK506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis, is very low. We therefore engineered FK506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK506 yield. To increase FK506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis NRRL 18488 by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase AskSt* of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of AskSt* resulted only in a 17% increase in FK506 yield, heterologous overexpression of a feedback deregulated AskCg* from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of AskCg* and DapASt, showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a ∆fkbL mutant led to a complete abolishment of the FK506 production, confirming the indispensability of this enzyme for FK506 production. Chemical complementation of the ∆fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes (pipAf, pipASt, originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis, respectively) completely restored FK506 production. Subsequently, FK506 production was enchanced by heterologous overexpression of PipAf and PipASp in S. tsukubaensis. This resulted in a yield increase by 65% compared to the WT in the presence of PipAf from A. friuliensis. For further rational yield improvement, the crystal structure of PipAf from A. friuliensis was determined at 1.3 Å resolution with the cofactor NADH bound and at 1.4 Å with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in PipAf, which resulted in an overall increase of FK506 production by approx. 100% compared to the WT.

Revisiting a challenging p53 binding site: a diversity-optimized HEFLib reveals diverse binding modes in T-p53C-Y220C.

The cellular tumor antigen p53 is a key component in cell cycle control. The mutation Y220C heavily destabilizes the protein thermally but yields a druggable crevice. We have screened the diversity-optimized halogen-enriched fragment library against T-p53C-Y220C with STD-NMR and DSF to identify hits, which we validated by 1H,15N-HSQC NMR. We could identify four hits binding in the Y220C cleft, one hit binding covalently and four hits binding to an uncharacterized binding site. Compound 1151 could be crystallized showing a flip of C220 and thus opening subsite 3. Additionally, 4482 was identified to alkylate cysteines. Data shows that the diversity-optimized HEFLib leads to multiple diverse hits. The identified scaffolds can be used to further optimize interactions with T-p53C-Y220C and increase thermal stability.

Synthesis of Homo- and Heteromultivalent Fucosylated and Sialylated Oligosaccharide Conjugates via Preactivated N-Methyloxyamine Precision Macromolecules and Their Binding to Polyomavirus Capsid Proteins.

Glycoconjugates are a versatile class of bioactive molecules that have found application as vaccines and antivirals and in cancer therapy. Their synthesis typically involves elaborate functionalization and use of protecting groups on the carbohydrate component in order to ensure efficient and selective conjugation. Alternatively, non-functionalized, non-protected carbohydrates isolated from biological sources or derived through biotechnological methods can be directly conjugated via N-methyloxyamine groups. In this study, we introduce such N-methyloxyamine groups into a variety of multivalent scaffolds─from small to oligomeric to polymeric scaffolds─making use of solid-phase polymer synthesis to assemble monodisperse sequence-defined macromolecules. These scaffolds are then successfully functionalized with different types of human milk oligosaccharides deriving a library of homo- and heteromultivalent glycoconjugates. Glycomacromolecules presenting oligosaccharide side chains with either α2,3- or α2,6-linked terminal sialic acid are used in a binding study with two types of polyomavirus capsid proteins showing that the multivalent presentation through the N-methyloxyamine-derived scaffolds increases the number of contacts with the protein. Overall, a straightforward route to derive glycoconjugates from complex oligosaccharides with high variability yet control in the multivalent scaffold is presented, and applicability of the derived structures is demonstrated.

Screening of a Halogen-Enriched Fragment Library Leads to Unconventional Binding Modes.

We conceived the Halogen-Enriched Fragment Library (HEFLib) to investigate the potential of halogen bonds in the early stages of drug discovery. As the number of competitive interactions increases with ligand size, we reasoned that a binding mode relying on halogen bonding is more likely for fragments than highly decorated molecules. Thus, fragments could feature unexplored binding modes. We screened the HEFLib against the human kinase DYRK1a and verified micromolar binding fragments via isothermal titration calorimetry (ITC). The crystal structure of one fragment revealed a noncanonical binding mode, despite the fragment's classical hinge binding motif. In addition, the fragment occupies a secondary binding site. Both binding modes feature a halogen bond, which we evaluated by ab initio calculations. Structure-affinity relationship (SAR) from a set of analogues improves the affinity, provides a promising fragment-growth vector, and highlights the benefits and applicability of halogen bonds in early lead development.

Homophilic Interaction Between Transmembrane-JAM-A and Soluble JAM-A Regulates Thrombo-Inflammation: Implications for Coronary Artery Disease.

Genetic predisposition through F11R-single-nucleotide variation (SNV) influences circulatory soluble junctional adhesion molecule-A (sJAM-A) levels in coronary artery disease (CAD) patients. Homozygous carriers of the minor alleles (F11R-SNVs rs2774276, rs790056) show enhanced levels of thrombo-inflammatory sJAM-A. Both F11R-SNVs and sJAM-A are associated with worse prognosis for recurrent myocardial infarction in CAD patients. Platelet surface-associated JAM-A correlate with platelet activation markers in CAD patients. Activated platelets shed transmembrane-JAM-A, generating proinflammatory sJAM-A and JAM-A-bearing microparticles. Platelet transmembrane-JAM-A and sJAM-A as homophilic interaction partners exaggerate thrombotic and thrombo-inflammatory platelet monocyte interactions. Therapeutic strategies interfering with this homophilic interface may regulate thrombotic and thrombo-inflammatory platelet response in cardiovascular pathologies where circulatory sJAM-A levels are elevated.

Exploring divalent conjugates of 5-N-acetyl-neuraminic acid as inhibitors of coxsackievirus A24 variant (CVA24v) transduction.

Coxsackievirus A24 variant (CVA24v) is responsible for several outbreaks and two pandemics of the highly contagious eye infection acute hemorrhagic conjunctivitis (AHC). Currently, neither prevention (vaccines) nor treatments (antivirals) are available for combating this disease. CVA24v attaches to cells by binding Neu5Ac-containing glycans on the surface of cells which facilitates entry. Previously, we have demonstrated that pentavalent Neu5Ac conjugates attenuate CVA24v infection of human corneal epithelial (HCE) cells. In this study, we report on the structure-based design of three classes of divalent Neu5Ac conjugates, with varying spacer lengths, and their effect on CVA24v transduction in HCE cells. In relative terms, the most efficient class of divalent Neu5Ac conjugates are more efficient than the pentavalent Neu5Ac conjugates previously reported.

Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26).

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.

Genetic diversity of Staphylococcus aureus wall teichoic acid glycosyltransferases affects immune recognition.

Staphylococcus aureus is a leading cause of skin and soft tissue infections and systemic infections. Wall teichoic acids (WTAs) are cell wall-anchored glycopolymers that are important for S. aureus nasal colonization, phage-mediated horizontal gene transfer, and antibiotic resistance. WTAs consist of a polymerized ribitol phosphate (RboP) chain that can be glycosylated with N-acetylglucosamine (GlcNAc) by three glycosyltransferases: TarS, TarM, and TarP. TarS and TarP modify WTA with ß-linked GlcNAc at the C-4 (ß1,4-GlcNAc) and the C-3 position (ß1,3-GlcNAc) of the RboP subunit, respectively, whereas TarM modifies WTA with α-linked GlcNAc at the C-4 position (α1,4-GlcNAc). Importantly, these WTA glycosylation patterns impact immune recognition and clearance of S. aureus. Previous studies suggest that tarS is near-universally present within the S. aureus population, whereas a smaller proportion co-contain either tarM or tarP. To gain more insight into the presence and genetic variation of tarS, tarM and tarP in the S. aureus population, we analysed a collection of 25 652 S. aureus genomes within the PubMLST database. Over 99 % of isolates contained tarS. Co-presence of tarS/tarM or tarS/tarP occurred in 37 and 7 % of isolates, respectively, and was associated with specific S. aureus clonal complexes. We also identified 26 isolates (0.1 %) that contained all three glycosyltransferase genes. At sequence level, we identified tar alleles with amino acid substitutions in critical enzymatic residues or with premature stop codons. Several tar variants were expressed in a S. aureus tar-negative strain. Analysis using specific monoclonal antibodies and human langerin showed that WTA glycosylation was severely attenuated or absent. Overall, our data provide a broad overview of the genetic diversity of the three WTA glycosyltransferases in the S. aureus population and the functional consequences for immune recognition.

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

Exploring the Effect of Structure-Based Scaffold Hopping on the Inhibition of Coxsackievirus A24v Transduction by Pentavalent N-Acetylneuraminic Acid Conjugates.

Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.

The polyfunctional polysialic acid: A structural view.

Polysialic acid (polySia), a homopolymer of α2,8-linked sialic acid residues, modifies a small number of proteins and has central functions in vertebrate signalling. Here, we review the regulatory functions of polySia in signalling processes and the immune system of adult humans, as well as functions based on their chemical properties. The main focus will be on the structure-function relationship of polySia with its interaction partners in humans. Recent studies have indicated that the degree of polymerisation is an important parameter that can guide the regulatory effect of polySia in addition to its binding to target proteins. Therefore, the structures of polySia in solution and bound to interaction partners are compared in order to identify the key factors that define binding specificity.

Structural Insight into Non-Enveloped Virus Binding to Glycosaminoglycan Receptors: A Review.

Viruses are infectious agents that hijack the host cell machinery in order to replicate and generate progeny. Viral infection is initiated by attachment to host cell receptors, and typical viral receptors are cell-surface-borne molecules such as proteins or glycan structures. Sialylated glycans (glycans bearing sialic acids) and glycosaminoglycans (GAGs) represent major classes of carbohydrate receptors and have been implicated in facilitating viral entry for many viruses. As interactions between viruses and sialic acids have been extensively reviewed in the past, this review provides an overview of the current state of structural knowledge about interactions between non-enveloped human viruses and GAGs. We focus here on adeno-associated viruses, human papilloma viruses (HPVs), and polyomaviruses, as at least some structural information about the interactions of these viruses with GAGs is available. We also discuss the multivalent potential for GAG binding, highlighting the importance of charged interactions and positively charged amino acids at the binding sites, and point out challenges that remain in the field.

NeutrobodyPlex-monitoring SARS-CoV-2 neutralizing immune responses using nanobodies.

In light of the COVID-19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.

Cell wall glycosylation in Staphylococcus aureus: targeting the tar glycosyltransferases.

Peptidoglycan (PG) is the major structural polymer of the bacterial cell wall. The PG layer of gram-positive bacterial pathogens such as Staphylococcus aureus (S. aureus) is permeated with anionic glycopolymers known as wall teichoic acids (WTAs) and lipoteichoic acids (LTAs). In S. aureus, the WTA backbone typically consists of repeating ribitol-5-phosphate units, which are modified by enzymes that introduce glycosylation as well as amino acids at different locations. These modifications are key determinants of phage adhesion, bacterial biofilm formation and virulence of S. aureus. In this review, we examine differences in WTA structures in gram-positive bacteria, focusing in particular on three enzymes, TarM, TarS, and TarP that glycosylate the WTA of S. aureus at different locations. Infections with S. aureus pose an increasing threat to human health, particularly through the emergence of multidrug-resistant strains. Recently obtained structural information on TarM, TarS and TarP has helped to better understand the strategies used by S. aureus to establish resistance and to evade host defense mechanisms. Moreover, structures of complexes with poly-RboP and its analogs can serve as a platform for the development of new inhibitors that could form a basis for the development of antibiotic agents.