Letter

Activation of band 3 mediates group A Streptococcus streptolysin S-based beta-haemolysis

  • Nature Microbiology 1, Article number: 15004 (2016)
  • doi:10.1038/nmicrobiol.2015.4
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Abstract

Streptococcus pyogenes, or group A Streptococcus (GAS), is a human bacterial pathogen that can manifest as a range of diseases from pharyngitis and impetigo to severe outcomes such as necrotizing fasciitis and toxic shock syndrome. GAS disease remains a global health burden with cases estimated at over 700 million annually and over half a million deaths due to severe infections1. For over 100 years, a clinical hallmark of diagnosis has been the appearance of complete (beta) haemolysis when grown in the presence of blood. This activity is due to the production of a small peptide toxin by GAS known as streptolysin S. Although it has been widely held that streptolysin S exerts its lytic activity through membrane disruption, its exact mode of action has remained unknown. Here, we show, using high-resolution live cell imaging, that streptolysin S induces a dramatic osmotic change in red blood cells, leading to cell lysis. This osmotic change was characterized by the rapid influx of Cl ions into the red blood cells through SLS-mediated disruption of the major erythrocyte anion exchange protein, band 3. Chemical inhibition of band 3 function significantly reduced the haemolytic activity of streptolysin S, and dramatically reduced the pathology in an in vivo skin model of GAS infection. These results provide key insights into the mechanism of streptolysin S-mediated haemolysis and have implications for the development of treatments against GAS.

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Acknowledgements

The authors thank R. Stahelin and S. Soni for discussions on this project and I. Spielman and C. Thomas for technical assistance. The authors also thank T. Orlova and W. Archer in the Integrated Imaging Facility at the University of Notre Dame for support with imaging. Finally, the authors thank members of the S. Lee laboratory for comments on this manuscript. This work was supported by a National Institutes of Health (NIH) Innovator Grant (DP2OD008468-01) awarded to S.W.L.

Author information

Author notes

    • Shaun W. Lee

    Present address: Department of Biological Sciences, University of Notre Dame, 100 Galvin Life Sciences Center, Notre Dame, Indiana 46556, USA.

Affiliations

  1. Department of Biological Sciences, University of Notre Dame, 100 Galvin Life Sciences Center, Notre Dame, Indiana 46556, USA

    • Dustin L. Higashi
    • , Jeffrey Luchetti
    •  & Shaun W. Lee
  2. Biology Department, Brooklyn College CUNY, New York 11210, USA

    • Nicolas Biais
  3. W.M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, Indiana 46556, USA

    • Deborah L. Donahue
    • , Jeffrey A. Mayfield
    • , Victoria A. Ploplis
    • , Francis J. Castellino
    •  & Shaun W. Lee
  4. Indiana University School of Medicine, South Bend, Indiana 46617, USA

    • Charles R. Tessier
  5. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA

    • Kevin Rodriguez
    • , Brandon L. Ashfeld
    • , Victoria A. Ploplis
    •  & Francis J. Castellino

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Contributions

D.L.H., N.B., V.A.P., F.J.C. and S.W.L. designed the overall project and experimental aims. D.L.H., N.B., D.L.D., J.A.M., C.R.T., K.R., B.L.A. and J.L. performed experimental work and analysed the results. D.L.H. and S.W.L. wrote the paper. All authors contributed to the proofreading and editing of the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Shaun W. Lee.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1–4

Videos

  1. 1.

    Supplementary Video 1

    Live cell imaging of RBC infected with wt GAS. Differential interference contrast (DIC) images (left panel) and haemoglobin fluorescence (right panel) were acquired from the same field every 10 seconds for 1 hour and assembled into a video using ImageJ. Scale bar: 22 μm.

  2. 2.

    Supplementary Video 2

    Live cell imaging of RBC infected with sagAΔcat. DIC images (left panel) and haemoglobin fluorescence (right panel) were acquired from the same field every 10 seconds for 1 hour and assembled into a video using ImageJ. Scale bar: 22μm.

  3. 3.

    Supplementary Video 3

    Live cell imaging of RBC in PBS control. DIC images (left panel) and haemoglobin fluorescence (right panel) were acquired from the same field every 10 seconds for 1 hour and assembled into a video using ImageJ. Scale bar: 22 μm.

  4. 4.

    Supplementary Video 4

    Live cell imaging of RBC infected with complemented strain (sagAΔcat + sagA). DIC images (left panel) and haemoglobin fluorescence (right panel) were acquired from the same field every 10 seconds for 1 hour and assembled into a video using ImageJ. Scale bar: 22 μm.

  5. 5.

    Supplementary Video 5

    Live cell imaging of RBC in hPBS treated with a SLS preparation from wt GAS. DIC images were acquired from the same field every 10 seconds for 1 hour and assembled into a video using ImageJ. Scale bar: 22 μm.

  6. 6.

    Supplementary Video 6

    Live cell imaging of RBC in hPBS treated with a SLS preparation from the complemented strain (sagAΔcat + sagA). DIC images were acquired from the same field every 10 seconds for 1 hour and assembled into a video using ImageJ. Scale bar: 22 μm.

  7. 7.

    Supplementary Video 7

    Live cell imaging of RBC in hPBS treated with a SLS preparation from the sagAΔcat. DIC images were acquired from the same field every 10 seconds for 1 hour and assembled into a video using ImageJ. Scale bar: 22 μm.

  8. 8.

    Supplementary Video 8

    Live cell imaging of RBC in hPBS treated with PBS. DIC images were acquired from the same field every 10 seconds for 1 hour and assembled into a video using ImageJ. Scale bar: 22 μm.

  9. 9.

    Supplementary Video 9

    Live cell imaging of MEQ-loaded RBCs treated with a SLS preparation from wt GAS. DIC images (left panel) and MEQ fluorescence (right panel) were acquired from the same field every 10 seconds for 30 minutes and assembled into a video using ImageJ. Scale bar: 6 μm.

  10. 10.

    Supplementary Video 10

    Live cell imaging of MEQ-loaded RBCs treated with a SLS preparation from the complemented strain (sagAΔcat + sagA). DIC images (left panel) and MEQ fluorescence (right panel) were acquired from the same field every 10 seconds for 30 minutes and assembled into a video using ImageJ. Scale bar: 6 μm.

  11. 11.

    Supplementary Video 11

    Live cell imaging of MEQ-loaded RBCs treated with a SLS preparation from the sagAΔcat. DIC images (left panel) and MEQ fluorescence (right panel) were acquired from the same field every 10 seconds for 30 minutes and assembled into a video using ImageJ. Scale bar: 6 μm.

  12. 12.

    Supplementary Video 12

    Live cell imaging of MEQ-loaded RBCs treated with PBS. DIC images (left panel) and MEQ fluorescence (right panel) were acquired from the same field every 10 seconds for 30 minutes and assembled into a video using ImageJ. Scale bar: 6 μm.