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Abstract

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 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.

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Data availability

All major data generated or analysed in this study are included in the article or its supplementary information files. The coordinates and structure factors were deposited in the Protein Data Bank under accession numbers 6H1J, 6H21, 6H2N, 6H4F, 6H4M and 6HNQ. Source data for experiments with animals (Fig. 4c, d) are provided. Additionally, a gel image of Extended Data Fig. 1f is supplied as Supplementary Fig. 1. All other data relating to this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank S. Popovich and P. Kühner for technical assistance; E. Weiß for help with phagocytosis experiments; R. Rosenstein and X. Li for discussions; B. Blaum and G. Zocher for assistance with NMR analysis and support for structure phasing and discussion; and the Swiss Lightsource beamline staff of the Paul Scherrer Institute for beam time and technical support. This work was financed by grants from the German Research Foundation to A.P. (TRR34, CRC766, TRR156, RTG1708), T.S. (TRR34, CRC766), C.W. (TRR34, CRC766, TRR156, RTG1708), and G.X. (CRC766); the German Center of Infection Research to A.P. (HAARBI); the Ministry of Science and Technology, Thailand Government to W.S.; the Korean Drug Development Foundation to S.-H.K. and B.L.L. (KDDF-201703-1); and the Max-Planck-Society to P.H.S.

Reviewer information

Nature thanks M. Crispin, F. DeLeo, M. Gilmore and J. Zimmer for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: David Gerlach, Yinglan Guo

  2. These authors jointly supervised this work: Thilo Stehle, Andreas Peschel

Affiliations

  1. Interfaculty Institute of Microbiology and Infection Medicine, Infection Biology, University of Tübingen, Tübingen, Germany

    • David Gerlach
    • , Katja Schlatterer
    •  & Andreas Peschel
  2. German Centre for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany

    • David Gerlach
    • , Katja Schlatterer
    • , Berit Schulte
    • , Christiane Wolz
    •  & Andreas Peschel
  3. Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany

    • Yinglan Guo
    •  & Thilo Stehle
  4. Department of Agricultural Sciences, University of Naples, Naples, Italy

    • Cristina De Castro
  5. National Research Laboratory of Defense Proteins, College of Pharmacy, Pusan National University, Pusan, South Korea

    • Sun-Hwa Kim
    •  & Bok Luel Lee
  6. Max-Planck-Institute for Colloids and Interfaces, Potsdam, Germany

    • Fei-Fei Xu
    • , Claney Pereira
    •  & Peter H. Seeberger
  7. Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

    • Sara Ali
    •  & Jeroen Codée
  8. Lydia Becker Institute of Immunology and Inflammation, Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK

    • Wanchat Sirisarn
    •  & Guoqing Xia
  9. Interfaculty Institute of Microbiology and Infection Medicine, Medical Microbiology, University of Tübingen, Tübingen, Germany

    • Berit Schulte
    •  & Christiane Wolz
  10. Bacteria, Parasites and Fungi, Statens Serum Institut, Copenhagen, Denmark

    • Jesper Larsen
  11. Department of Chemical Sciences, University of Naples, Naples, Italy

    • Antonio Molinaro
  12. Vanderbilt University School of Medicine, Nashville, TN, USA

    • Thilo Stehle

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Contributions

D.G. characterized TarP in vivo and its genomic context, created mutants, designed experiments, purified WTA, and performed experiments with human IgGs. Y.G. designed experiments, purified proteins, crystallized proteins, solved the structures, and performed in vitro analysis of TarP. C.D.C. performed NMR experiments. C.D.C. and A.M. analysed the NMR data and wrote the NMR discussion. S.-H.K. performed and B.L.L. designed and interpreted mouse immunization and infection experiments. K.S. designed IgG deposition experiments. B.S. and C.W. collected and characterized CC5 MRSA strains. J.L. collected and characterized CC398 strains. J.L. and C.W. analysed S. aureus genomes. F.-F.X, C.P., and P.H.S. designed and synthesized 3RboP. S.A. and J.C. designed and synthesized 6RboP-(CH2)6NH2. W.S. performed MIC experiments. G.X. identified tarP, and characterized and interpreted MIC data. D.G., Y.G., A.P., T.S., and G.X. designed the study, analysed results, and wrote the paper.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Thilo Stehle or Andreas Peschel.

Extended data figures and tables

  1. Extended Data Fig. 1 Characterization of TarP, deposition of human IgGs, and presence of tarP in the producer of antigen 336.

    a, Analysis of WTA by PAGE. WTA from RN4220 ΔtarM/S expressing either tarP or tarS was compared with non-glycosylated WTA. Data shown are representative of two experiments. b, MIC values of oxacillin against MW2 wild type, tarS mutant, and tarP-complemented tarS mutant. Data are medians of ten independent experiments. c, Efficiency of plating (EOP) of phage Φ11 against tarS or tarP-expressing RN4420 ΔtarM/S. Values of tarP relative to tarS expression are given as mean ± s.d. (n = 3). Statistical significance was calculated by paired Student’s t-test (two-sided) with significant P values (P ≤ 0.05) indicated. d, The level of WTA glycosylation catalysed by TarP or TarS was determined by analysing the GlcNAc and phosphate content of WTA isolated from a N315 strain panel. Depicted is the ratio of GlcNAc and phosphate as mean with s.d. of three technical replicates. The values are in good agreement with NMR data (Supplementary Table 3). e, Relative deposition of IgG from intravenous immunoglobulins enriched for WTA binding on different CC5 wild-type and tarP mutant cells. Values are given as mean percentage ± s.d. of four independent experiments. Statistical significance was calculated by paired Student’s t-test (two-sided). P values ≤ 0.05 were considered significant and are indicated. f, Presence of tarP and tarS in S. aureus ATCC55804, expressing antigen 336, described as 3-O-GlcNAc-WTA25. Shown is a representative of two independent replicates. g, TarP reduces neutrophil phagocytosis of N315 strains lacking protein A, opsonized with indicated concentrations of IgG enriched for WTA binding. Values are depicted as mean fluorescence intensity (MFI). Shown are two independent experiments with neutrophils from different donors. These data supplement data presented in Fig. 4b: upper panel, mean of three technical replicates of an independent experiment, lower panel, mean of two technical replicates.

  2. Extended Data Fig. 2 NMR analysis of WTA from N315 mutant panel.

    All depicted experiments were repeated twice. y-axes and x-axes show 13C and 1H chemical shifts, respectively. ad, NMR spectra of non-glycosylated WTA (ΔtarSΔtarP mutant). a, HSQC expansion of the region containing the ribitol and glycerol protons shifted by acylation; b, c, HSQC-TOCSY-20 and HSQC-TOCSY-80 spectra, respectively. d, HSQC area of the non-acylated ribitol and glycerol proton. eh, NMR spectra of TarS-WTA (ΔtarP mutant). e, HSQC expansion of the region containing the ribitol and glycerol protons shifted by acylation. f, g, HSQC-TOCSY-20 and HSQC-TOCSY-80, respectively. h, HSQC area of the non-acylated ribitol and glycerol proton. io, NMR spectra of TarP-WTA (ΔtarS mutant). i, HSQC expansion of the region containing the ribitol and glycerol protons shifted by acylation. j, k, HSQC-TOCSY-20 and HSQC-TOCSY-80 spectra, respectively. l, HSQC area of the non-acylated ribitol and glycerol protons. m, Expansion of l with HSQC (black/grey) overlapped with HSQC-TOCSY-20 (cyan). n, Overlap of HSQC-TOCSY-20 (cyan) and HSQC-TOCSY-80 (black). o, HSQC (black) and HMBC (grey) detailing the GlcNAc signals. p, NOESY expansion detailing the correlations of the β-GlcNAc anomeric protons: GlcNAc ‘b*’ differs from unit ‘b’, which has the same anomeric proton chemical shift, but is linked to a different ribitol unit. All densities are labelled with the letters used in Supplementary Table 2. The density marked with an asterisk in m is consistent with ribitol glycosylated at O-4.

  3. Extended Data Fig. 3 Secondary structure of a TarP monomer and interactions with UDP-GlcNAc.

    a, Cartoon representation of a TarP monomer bound to UDP-GlcNAc (yellow) and Mn2+ (lime green). The CTD is coloured red. b, Interactions of TarP with UDP-GlcNAc and Mn2+, coloured as in a. Hydrogen bonds and salt bridges are shown as black dashed lines. c, Interactions of TarP with UDP-GlcNAc (yellow) and Mg2+ (magenta). d, Simulated-annealing (mFo − DFc) omit map of UDP-GlcNAc (grey mesh, contoured at 2.0σ) and Mn2+ (magenta mesh, at 3.0σ) in the TarP–UDP-GlcNAc–Mn2+ complex structure. UDP-GlcNAc and Mn2+ are coloured as in a. e, Simulated-annealing (mFo − DFc) omit map of UDP-GlcNAc (grey mesh, at 2.0σ) and Mg2+ (blue mesh, at 2.0σ) in the TarP–UDP-GlcNAc–Mg2+ complex structure. UDP-GlcNAc and Mg2+ are coloured as in c.

  4. Extended Data Fig. 4 Simulated-annealing (mFo − DFc) omit maps of 3RboP and UDP-GlcNAc, and characterization of TarP mutant proteins.

    a, Chemical structures of synthetic 3RboP and 6RboP-(CH2)6NH2. The unit numbers are indicated. b, Simulated-annealing (mFo − DFc) omit map of 3RboP (lime green) in the binary structure (magenta mesh, contoured at 2.0σ). c, Simulated-annealing (mFo − DFc) omit map of UDP-GlcNAc (yellow), Mg2+ (magenta) and 3RboP (lime green) in the ternary complex structure (red mesh, at 1.8σ, blue mesh, at 2.0σ or magenta mesh, at 1.5σ). d, Circular dichroism spectra of wild-type and mutant TarP proteins (for wild type, R76A and D181A, n = 3; for D92A, Y152A and R259A, n = 2). e, Size-exclusion chromatography elution profiles of wild-type and mutant TarP proteins (for wild type, n = 8; for R76A, D181A and R259A, n = 3; for D92A and Y152A, n = 2, all with similar results). Mutant proteins D94A, E180A, D209A, K255A, R262A, and H263A showed similar circular dichroism spectra and size-exclusion chromatography elution profiles (data not shown).

  5. Extended Data Fig. 5 Gating strategy for flow cytometry experiments.

    a, Gating strategy for IgG deposition experiments. To distinguish bacteria from background signals, pure PBS was measured. Left, bacterial gating occurred at the FSC/SCC density plot omitting PBS-derived signals. Bacterial aggregates of high SSC and FSC values were excluded from the gated population as well. Right, the mean fluorescence of the bacterial population (black) was determined and compared with non-IgG-treated bacteria (grey) to control for nonspecific binding of the secondary FITC-labelled antibody. Subsequently, mean fluorescence values of individual mutants were compared relatively to the corresponding wild-type strain. b, Gating strategy for phagocytosis experiments. Neutrophils were separated by Histopaque/Ficoll gradient and subsequent gating of neutrophils occurred at the FSC/SCC density plot upon size and complexity (left). Histopaque/Ficoll gradient isolations showed a neutrophil purity of more than 80%. Using the CFSE-fluorescence channel, the gated population was subdivided into fluorescence-positive and -negative cells (right). Successful phagocytosis was indicated by uptake of CFSE-labelled bacteria. The phagocytic efficiency was expressed as product of the mean fluorescence of the fluorescence-positive population and their relative abundance (mean fluorescence intensity, MFI).

  6. Extended Data Table 1 tarP presence and podophage susceptibility of CC5 strains, comprising sequence type (ST) 5 and 225, and CC398 isolates
  7. Extended Data Table 2 Enzymatic activities of mutated TarP proteins
  8. Extended Data Table 3 Donor substrate specificity of TarP
  9. Extended Data Table 4 Crystallographic data statistics for TarP and TarP–UDP-GlcNAc–Mg2+
  10. Extended Data Table 5 Crystallographic data statistics for TarP–UDP-GlcNAc–Mn2+, TarP–3RboP, TarP–6RboP-(CH2)6NH2 and TarP–UDP-GlcNAc–3RboP

Supplementary information

  1. Supplementary Figure 1

    Photo of gel electrophoresis for Extended Data Fig. 1f

  2. Reporting Summary

  3. Supplementary Information

    This file contains Supplementary Information Sections 1-3, including a Supplementary Discussion, Supplementary Tables 1-3, Supplementary Figures 2- 3, NMR Spectra data and Supplementary References

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