Antibody recognition of different staphylococcus aureus wall teichoic acid glycoforms

Wall 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.Bio-organic Synthesi

Impact of glycan linkage to staphylococcus aureus wall teichoic acid on langerin recognition and langerhans cell activation

Staphylococcus aureus is the leading cause of skin and soft tissue infections. It remains incompletely understood how skin-resident immune cells respond to invading S. aureus and contribute to an effective immune response. Langerhans cells (LCs), the only professional antigen-presenting cell type in the epidermis, sense S. aureus through their pattern-recognition receptor langerin, triggering a proinflammatory response. Langerin recognizes the β-1,4-linked N-acetylglucosamine (β1,4-GlcNAc) but not α-1,4-linked GlcNAc (α1,4-GlcNAc) modifications, which are added by dedicated glycosyltransferases TarS and TarM, respectively, on the cell wall glycopolymer wall teichoic acid (WTA). Recently, an alternative WTA glycosyltransferase, TarP, was identified, which also modifies WTA with β-GlcNAc but at the C-3 position (β1,3-GlcNAc) of the WTA ribitol phosphate (RboP) subunit. Here, we aimed to unravel the impact of β-GlcNAc linkage position for langerin binding and LC activation. Using genetically modified S. aureus strains, we observed that langerin similarly recognized bacteria that produce either TarS- or TarP-modified WTA, yet tarP-expressing S. aureus induced increased cytokine production and maturation of in vitro-generated LCs compared to tarS-expressing S. aureus. Chemically synthesized WTA molecules, representative of the different S. aureus WTA glycosylation patterns, were used to identify langerin-WTA binding requirements. We established that β-GlcNAc is sufficient to confer langerin binding, thereby presenting synthetic WTA molecules as a novel glycobiology tool for structure-binding studies and for elucidating S. aureus molecular pathogenesis. Overall, our data suggest that LCs are able to sense all β-GlcNAc-WTA producing S. aureus strains, likely performing an important role as first responders upon S. aureus skin invasion.Bio-organic Synthesi

Leukocyte and complement activation by GM1-specific antibodies is associated with acute motor axonal neuropathy in rabbits

Acute motor axonal neuropathy (AMAN) in humans is associated with the presence of GM1-specific antibodies. Immunization of rabbits with GM1-containing ganglioside mixtures, purified GM1, or Campylobacter jejuni lipo-oligosaccharide exhibiting a GM1-like structure elicits GM1-specific antibodies, but axonal polyneuropathy only occurs in a subset of animals. This study aimed to dissect the molecular basis for the variable induction of AMAN in rabbits. Therefore, we analyzed the pro-inflammatory characteristics of GM1-specific antibodies in plasma samples from ganglioside-immunized rabbits with and without neurological deficits. GM1-specific plasma samples from all rabbits with AMAN were capable of activating both complement and leukocytes, in contrast to none of the plasma samples from rabbits without paralysis. Furthermore, GM1-specific IgG-mediated activation of leukocytes was detected before the onset of clinical signs. These data suggest that AMAN only occurs in rabbits that develop GM1-specific antibodies with pro-inflammatory properties

(Automated) synthesis of well-defined staphylococcus aureus wall teichoic acid fragments

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) 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.NWO91713303 and 09150181910001Bio-organic Synthesi

Do not discard Staphylococcus aureus WTA as a vaccine antigen

NWO91713303Bio-organic Synthesi

Signal inhibitory receptor on leukocytes-1 recognizes bacterial and endogenous amphipathic alpha-helical peptides

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is a negative regulator of myeloid cell function and dampens antimicrobial responses. We here show that different species of the genus Staphylococcus secrete SIRL-1-engaging factors. By screening a library of single-gene transposon mutants in Staphylococcus aureus, we identified these factors as phenol-soluble modulins (PSMs). PSMs are amphipathic alpha-helical peptides involved in multiple aspects of staphylococcal virulence and physiology. They are cytotoxic and activate the chemotactic formyl peptide receptor 2 (FPR2) on immune cells. Human cathelicidin LL-37 is also an amphipathic alpha-helical peptide with antimicrobial and chemotactic activities, structurally and functionally similar to alpha-type PSMs. We demonstrate that alpha-type PSMs from multiple staphylococcal species as well as human cathelicidin LL-37 activate SIRL-1, suggesting that SIRL-1 recognizes alpha-helical peptides with an amphipathic arrangement of hydrophobicity, although we were not able to show direct binding to SIRL-1. Upon rational peptide design, we identified artificial peptides in which the capacity to ligate SIRL-1 is segregated from cytotoxic and FPR2-activating properties, allowing specific engagement of SIRL-1. In conclusion, we propose staphylococcal PSMs and human LL-37 as a potential new class of natural ligands for SIRL-1.Chemical Immunolog

Ganglioside-specific IgG and IgA recruit leukocyte effector functions in Guillain-Barre syndrome

The capacity of ganglioside-specific autoantibodies to recruit leukocyte effector functions was studied. Serum samples from 87 patients with Guillain–Barré (GBS) or Miller Fisher syndrome (MFS), containing GM1-, GQ1b-, or GD1b-specific IgG or IgA, were tested for leukocyte activating capacity. Ganglioside-specific IgG antibodies generally induced leukocyte activation, irrespective of specificity. The magnitude of leukocyte degranulation correlated with GM1- and GQ1b-specific IgG titers, but not with disease severity. Finally, GM1- specific IgA activated leukocytes through the IgA receptor, FcαRI (CD89). Therefore, both ganglioside-specific IgG and IgA can recruit leukocyte effector functions, which may be relevant in the pathogenesis of GBS and MFS. © 2006 Elsevier B.V. All rights reserved. Keywords: Guillain–Barré syndrome; Autoantibodies; Pathogenicity; Gangliosides; Fcγ

The group A streptococcal vaccine candidate VAX-A1 protects against group B Streptococcusinfection via cross-reactive IgG targeting virulence factor C5a peptidase

Bio-organic Synthesi

Ganglioside-specific IgG and IgA recruit leukocyte effector functions in Guillain-Barre syndrome

The capacity of ganglioside-specific autoantibodies to recruit leukocyte effector functions was studied. Serum samples from 87 patients with Guillain–Barré (GBS) or Miller Fisher syndrome (MFS), containing GM1-, GQ1b-, or GD1b-specific IgG or IgA, were tested for leukocyte activating capacity. Ganglioside-specific IgG antibodies generally induced leukocyte activation, irrespective of specificity. The magnitude of leukocyte degranulation correlated with GM1- and GQ1b-specific IgG titers, but not with disease severity. Finally, GM1- specific IgA activated leukocytes through the IgA receptor, FcαRI (CD89). Therefore, both ganglioside-specific IgG and IgA can recruit leukocyte effector functions, which may be relevant in the pathogenesis of GBS and MFS. © 2006 Elsevier B.V. All rights reserved. Keywords: Guillain–Barré syndrome; Autoantibodies; Pathogenicity; Gangliosides; Fcγ