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Tracking Cell Wall-Anchored Proteins in Gram-Positive Bacteria

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The Bacterial Cell Wall

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2727))

Abstract

Cell wall-anchored surface proteins are integral components of Gram-positive bacterial cell envelope and vital for bacterial survival in different environmental niches. To fulfill their functions, surface protein precursors translocate from cytoplasm to bacterial cell surface in three sequential steps: secretion across the cytoplasmic membrane, covalently anchoring to the cell wall precursor lipid II by sortase A, and incorporation of the lipid II-linked precursors into mature cell wall peptidoglycan. Here, we describe a series of immunofluorescence microscopy methods to track the subcellular localization of cell wall-anchored proteins along the sorting pathway. While the protocols are tailored to Staphylococcus aureus, they can be readily adapted to localize cell wall-anchored proteins as well as membrane proteins in other Gram-positive bacteria.

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References

  1. Foster TJ, Geoghegan JA, Ganesh VK, Hook M (2014) Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus. Nat Rev Microbiol 12(1):49–62. https://doi.org/10.1038/nrmicro3161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Schneewind O, Missiakas D (2019) Sortases, surface proteins, and their roles in Staphylococcus aureus disease and vaccine development. Microbiol Spectr 7(1). https://doi.org/10.1128/microbiolspec.PSIB-0004-2018

  3. Schneewind O, Model P, Fischetti VA (1992) Sorting of protein A to the staphylococcal cell wall. Cell 70(2):267–281

    Article  CAS  PubMed  Google Scholar 

  4. Schneewind O, Mihaylova-Petkov D, Model P (1993) Cell wall sorting signals in surface proteins of gram-positive bacteria. EMBO J 12(12):4803–4811

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Perry AM, Ton-That H, Mazmanian SK, Schneewind O (2002) Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring. J Biol Chem 277(18):16241–16248. https://doi.org/10.1074/jbc.M109194200

    Article  CAS  PubMed  Google Scholar 

  6. Ton-That H, Faull KF, Schneewind O (1997) Anchor structure of staphylococcal surface proteins. A branched peptide that links the carboxyl terminus of proteins to the cell wall. J Biol Chem 272(35):22285–22292. https://doi.org/10.1074/jbc.272.35.22285

    Article  CAS  PubMed  Google Scholar 

  7. Schneewind O, Fowler A, Faull KF (1995) Structure of the cell wall anchor of surface proteins in Staphylococcus aureus. Science 268(5207):103–106

    Article  CAS  PubMed  Google Scholar 

  8. Navarre WW, Ton-That H, Faull KF, Schneewind O (1998) Anchor structure of staphylococcal surface proteins. II. Cooh-terminal structure of muramidase and amidase-solubilized surface protein. J Biol Chem 273(44):29135–29142. https://doi.org/10.1074/jbc.273.44.29135

    Article  CAS  PubMed  Google Scholar 

  9. Ton-That H, Labischinski H, Berger-Bachi B, Schneewind O (1998) Anchor structure of staphylococcal surface proteins. III. Role of the FemA, FemB, and FemX factors in anchoring surface proteins to the bacterial cell wall. J Biol Chem 273(44):29143–29149. https://doi.org/10.1074/jbc.273.44.29143

    Article  CAS  PubMed  Google Scholar 

  10. Mazmanian SK, Liu G, Ton-That H, Schneewind O (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285(5428):760–763. https://doi.org/10.1126/science.285.5428.760

    Article  CAS  PubMed  Google Scholar 

  11. Ton-That H, Liu G, Mazmanian SK, Faull KF, Schneewind O (1999) Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc Natl Acad Sci U S A 96(22):12424–12429. https://doi.org/10.1073/pnas.96.22.12424

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ton-That H, Schneewind O (1999) Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction. J Biol Chem 274(34):24316–24320. https://doi.org/10.1074/jbc.274.34.24316

    Article  CAS  PubMed  Google Scholar 

  13. Mazmanian SK, Liu G, Jensen ER, Lenoy E, Schneewind O (2000) Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc Natl Acad Sci U S A 97(10):5510–5515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Frankel MB, Hendrickx AP, Missiakas DM, Schneewind O (2011) LytN, a murein hydrolase in the cross-wall compartment of Staphylococcus aureus, is involved in proper bacterial growth and envelope assembly. J Biol Chem 286(37):32593–32605. https://doi.org/10.1074/jbc.M111.258863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bierne H, Dramsi S (2012) Spatial positioning of cell wall-anchored virulence factors in Gram-positive bacteria. Curr Opin Microbiol 15(6):715–723. https://doi.org/10.1016/j.mib.2012.10.010

    Article  CAS  PubMed  Google Scholar 

  16. Dramsi S, Bierne H (2017) Spatial organization of cell wall-anchored proteins at the surface of Gram-positive bacteria. Curr Top Microbiol Immunol 404:177–201. https://doi.org/10.1007/82_2016_4

    Article  CAS  PubMed  Google Scholar 

  17. Pathak A, Bergstrand J, Sender V, Spelmink L, Aschtgen MS, Muschiol S, Widengren J, Henriques-Normark B (2018) Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification. Nat Commun 9(1):3398. https://doi.org/10.1038/s41467-018-05494-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Choo PY, Wang CY, VanNieuwenhze MS, Kline KA (2022) Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis. Mol Microbiol 119:1. https://doi.org/10.1111/mmi.15008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Forsgren A, Sjöquist J (1966) “Protein A” from S. aureus. I. Pseudo-immune reaction with human gamma-globulin. J Immunol 97(6):822–827

    Article  CAS  PubMed  Google Scholar 

  20. Scaffidi SJ, Shebes MA, Yu W (2021) Tracking the subcellular localization of surface proteins in Staphylococcus aureus by immunofluorescence microscopy. Bio-protocol 11(10):e4038. https://doi.org/10.21769/BioProtoc.4038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kim HK, Cheng AG, Kim HY, Missiakas DM, Schneewind O (2010) Nontoxigenic protein A vaccine for methicillin-resistant Staphylococcus aureus infections in mice. J Exp Med 207(9):1863–1870. https://doi.org/10.1084/jem.20092514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yu W, Missiakas D, Schneewind O (2018) Septal secretion of protein A in Staphylococcus aureus requires SecA and lipoteichoic acid synthesis. eLife 7. https://doi.org/10.7554/eLife.34092

  23. Zhang R, Shebes MA, Kho K, Scaffidi SJ, Meredith TC, Yu W (2021) Spatial regulation of protein A in Staphylococcus aureus. Mol Microbiol 116(2):589–605. https://doi.org/10.1111/mmi.14734

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sabala I, Jagielska E, Bardelang PT, Czapinska H, Dahms SO, Sharpe JA, James R, Than ME, Thomas NR, Bochtler M (2014) Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans. FEBS J 281(18):4112–4122. https://doi.org/10.1111/febs.12929

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Harry EJ, Pogliano K, Losick R (1995) Use of immunofluorescence to visualize cell-specific gene expression during sporulation in Bacillus subtilis. J Bacteriol 177(12):3386–3393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pinho MG, Errington J (2003) Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery. Mol Microbiol 50(3):871–881

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by NIH grant 1R35GM146993-01 and institutional start-up funds to W.Y.

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Authors and Affiliations

  1. Department of Molecular Biosciences, University of South Florida, Tampa, FL, USA

    Salvatore J. Scaffidi & Wenqi Yu

Authors
  1. Salvatore J. Scaffidi
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  2. Wenqi Yu
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Corresponding author

Correspondence to Wenqi Yu .

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Editors and Affiliations

  1. Division of Oral & Systemic Health Sciences, School of Dentistry, University of California, Los Angeles, Los Angeles, CA, USA

    Hung Ton-That

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Scaffidi, S.J., Yu, W. (2024). Tracking Cell Wall-Anchored Proteins in Gram-Positive Bacteria. In: Ton-That, H. (eds) The Bacterial Cell Wall. Methods in Molecular Biology, vol 2727. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3491-2_15

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  • DOI: https://doi.org/10.1007/978-1-0716-3491-2_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3490-5

  • Online ISBN: 978-1-0716-3491-2

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