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Flow Cytometry-Based Assays to Quantify Complement Deposition and Neutrophil Uptake of Group A Streptococcus

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Group A Streptococcus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2136))

Abstract

Quantification of complement deposition and subsequent neutrophil-mediated uptake provides an important way to assess the role of different bacterial factors in evasion of the host innate immune response. Here, we describe flow cytometry-based methods to allow quantification of deposition of the complement opsonin C3 on the bacterial surface and subsequent uptake by primary human neutrophils. The assays outlined below provide key methods to determine whether specific bacterial factors are involved in the evasion of complement-mediated immunity, using widely accessible reagents and equipment.

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References

  1. Janssen BJ, Huizinga EG, Raaijmakers HC et al (2005) Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 437(7058):505–511. https://doi.org/10.1038/nature04005

    Article  CAS  PubMed  Google Scholar 

  2. Laabei M, Ermert D (2019) Catch me if you can: Streptococcus pyogenes complement evasion strategies. J Innate Immun 11(1):3–12. https://doi.org/10.1159/000492944

    Article  CAS  PubMed  Google Scholar 

  3. Ermert D, Weckel A, Agarwal V et al (2013) Binding of complement inhibitor C4b-binding protein to a highly virulent Streptococcus pyogenes M1 strain is mediated by protein H and enhances adhesion to and invasion of endothelial cells. J Biol Chem 288(45):32172–32183. https://doi.org/10.1074/jbc.M113.502955

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Honda-Ogawa M, Ogawa T, Terao Y et al (2013) Cysteine proteinase from Streptococcus pyogenes enables evasion of innate immunity via degradation of complement factors. J Biol Chem 288(22):15854–15864. https://doi.org/10.1074/jbc.M113.469106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pandiripally V, Gregory E, Cue D (2002) Acquisition of regulators of complement activation by Streptococcus pyogenes serotype M1. Infect Immun 70(11):6206–6214. https://doi.org/10.1128/iai.70.11.6206-6214.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Carlsson F, Sandin C, Lindahl G (2005) Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway. Mol Microbiol 56(1):28–39. https://doi.org/10.1111/j.1365-2958.2005.04527.x

    Article  CAS  PubMed  Google Scholar 

  7. Ji Y, McLandsborough L, Kondagunta A et al (1996) C5a peptidase alters clearance and trafficking of group A streptococci by infected mice. Infect Immun 64(2):503–510

    Article  CAS  Google Scholar 

  8. Lynskey NN, Reglinski M, Calay D et al (2017) Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase. PLoS Pathog 13(8):e1006493. https://doi.org/10.1371/journal.ppat.1006493

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hyams C, Trzcinski K, Camberlein E et al (2013) Streptococcus pneumoniae capsular serotype invasiveness correlates with the degree of factor H binding and opsonization with C3b/iC3b. Infect Immun 81(1):354–363. https://doi.org/10.1128/IAI.00862-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lynskey NN, Banerji S, Johnson LA et al (2015) Rapid lymphatic dissemination of encapsulated group A Streptococci via lymphatic vessel endothelial Receptor-1 interaction. PLoS Pathog 11(9):e1005137. https://doi.org/10.1371/journal.ppat.1005137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Turner CE, Dryden M, Holden MT et al (2013) Molecular analysis of an outbreak of lethal postpartum sepsis caused by Streptococcus pyogenes. J Clin Microbiol 51(7):2089–2095. https://doi.org/10.1128/JCM.00679-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Laarman AJ, Ruyken M, Malone CL et al (2011) Staphylococcus aureus metalloprotease aureolysin cleaves complement C3 to mediate immune evasion. J Immunol 186(11):6445–6453. https://doi.org/10.4049/jimmunol.1002948

    Article  CAS  PubMed  Google Scholar 

  13. Reglinski M, Gierula M, Lynskey NN et al (2015) Identification of the Streptococcus pyogenes surface antigens recognised by pooled human immunoglobulin. Sci Rep 5:15825. https://doi.org/10.1038/srep15825

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Correspondence to Nicola N. Lynskey .

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Lynskey, N.N. (2020). Flow Cytometry-Based Assays to Quantify Complement Deposition and Neutrophil Uptake of Group A Streptococcus. In: Proft, T., Loh, J. (eds) Group A Streptococcus. Methods in Molecular Biology, vol 2136. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0467-0_17

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  • DOI: https://doi.org/10.1007/978-1-0716-0467-0_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0466-3

  • Online ISBN: 978-1-0716-0467-0

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