Wall teichoic acids (WTAs) are glycopolymers decorating the surface of Gram-positive bacteria and potential targets for antibody-mediated treatments against Staphylococcus aureus, including... Show moreWall teichoic acids (WTAs) are glycopolymers decorating the surface of Gram-positive bacteria and potential targets for antibody-mediated treatments against Staphylococcus aureus, including methicillin-resistant (MRSA) strains. Through a combination of glycan microarray, synthetic chemistry, crystallography, NMR, and computational studies, we unraveled the molecular and structural details of fully defined synthetic WTA fragments recognized by previously described monoclonal antibod-ies (mAbs 4461 and 4497). Our results unveiled the structural requirements for the discriminatory recognition of alpha- and beta-GlcNAc-modified WTA glycoforms by the complementarity-determining regions (CDRs) of the heavy and light chains of the mAbs. Both mAbs interacted not only with the sugar moiety but also with the phosphate groups as well as residues in the ribitol phosphate (RboP) units of the WTA backbone, highlighting their significant role in ligand specificity. Using elongated WTA fragments, containing two sugar modifications, we also demonstrated that the internal carbohydrate moiety of alpha-GlcNAc-modified WTA is preferentially accommodated in the binding pocket of mAb 4461 with respect to the terminal moiety. Our results also explained the recently documented cross-reactivity of mAb 4497 for beta-1,3/beta-1,4-GlcNAc-modified WTA, revealing that the flexibility of the RboP backbone is crucial to allow positioning of both glycans in the antibody binding pocket. Show less
Berni, F.; Kalfopoulou, E.; Cardells, A.M.G.; Carboni, F.; Es, D. van der; Romero-Saavedra, F.; ... ; Codee, J.D.C. 2021
Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both... Show moreGlycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody. Show less
Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP)... Show moreWall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (alpha-GlcNAc at the RboP C4; beta-GlcNAc at the RboP C4; beta-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains. Show less
Gerlach, D.; Guo, Y.; De Castro, C.; Kim, S.H.; Schlatterer, K.; Xu, F.F.; ... ; Peschel, A. 2018
Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are... Show moreMethicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5–40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences. Show less
Hagen, B.; Ali, S.; Overkleeft, H.S.; Marel, G.A. van der; Codee, J.D.C. 2017
