Impact of Glycan Linkage to Staphylococcus aureus Wall Teichoic Acid on Langerin Recognition and Langerhans Cell Activation

Abstract

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.

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

“…The enzymatic activity of the tar- variants was assessed by analyzing WTA glycosylation using WTA-specific Fab fragments 11 . Bacteria were grown to mid-exponential growth phase, collected by centrifugation (4,000 rpm, 8 min, 4°C) and resuspended at an OD 600 of 0.4 (∼1× 10 8 colony forming units (CFU)/mL) in phosphate-buffered saline (PBS; pH 7) with 0.1% bovine serum albumin (BSA; Sigma).…”

“…Bacteria were grown to mid-exponential growth phase, collected by centrifugation (4,000 rpm, 8 min, 4°C) and resuspended at an OD 600 of 0.4 (∼1× 10 8 colony forming units (CFU)/mL) in phosphate-buffered saline (PBS; pH 7) with 0.1% bovine serum albumin (BSA; Sigma). Bacteria (1.25 × 10 6 CFU) were incubated with 3.3 μg/mL monoclonal Fab fragments (pre-diluted in PBS 0.1% BSA) specific to β-GlcNAc (clone 4497) or α-1,4-GlcNAc (clone 4461) WTA, respectively 11 . After washing, bacteria were incubated with a Goat F(ab’) 2 anti-human Kappa-Alexa Fluor 647 (pre-diluted in PBS 0.1%BSA; 2.5 μg/mL, Southern Biotech #2062-31) to detect bound Fab fragments.…”

“…The specific WTA GlcNAc modifications greatly influence β-lactam-and phage resistance of S. aureus 2,3 as well as host-pathogen interaction [9][10][11][12] . For instance, the WTA glycan modifications represent dominant antigens in the S. aureus-reactive antibody pool in humans 2,9,12 .…”

“…Most antibodies are directed against β-GlcNAc modified WTA 9 and not every WTA glycoform may be similar in terms of immunogenicity or as target for antibody-mediated clearance 6, 9 . With regard to innate immunity, β-GlcNAc-WTA but not α-GlcNAc-WTA is detected by the innate receptor langerin, which is expressed on skin epidermal Langerhans cells 10, 11 . These findings indicate that different WTA glycosylation profiles, which depend on the presence and activity of the three known glycosyltransferases, impact S. aureus immune recognition and clearance.…”

Epitope Recognition of a Monoclonal Antibody Raised against a Synthetic Glycerol Phosphate Based Teichoic Acid

“…As shown in Figure 2 A, the tested library comprises GroP-based fragments varying in length (hexamers or pentadecamers), nature of the glucosyl substituent (α-glucose, α-glucosamine, or α- N -acetylglucosamine), the position of the carbohydrate on the chain (terminal or in the middle), and the stereochemistry of the glycerol unit ( sn- 1 vs sn -3). Compound 7 , a ribitol phosphate chain resembling the structure of S. aureus WTAs, 24 was included as a negative control. As can be observed from the results shown in Figure 2 B, the WH7.01 mAb recognized all printed GroP-based TA fragments well, indicating that the GroP backbone is the main recognition element for the mAb.…”