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Staphylococcus aureus alpha toxin activates Notch in vascular cells

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Abstract

Staphylococcus aureus infection is one of the leading causes of morbidity in hospitalized patients in the United States, an effect compounded by increasing antibiotic resistance. The secreted agent hemolysin alpha toxin (Hla) requires the receptor A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10) to mediate its toxic effects. We hypothesized that these effects are in part regulated by Notch signaling, for which ADAM10 activation is essential. Notch proteins function in developmental and pathological angiogenesis via the modulation of key pathways in endothelial and perivascular cells. Thus, we hypothesized that Hla would activate Notch in vascular cells. Human umbilical vein endothelial cells were treated with recombinant Hla (rHla), Hla-H35L (genetically inactivated Hla), or Hank’s solution (HBSS), and probed by different methods. Luciferase assays showed that Hla (0.01 µg/mL) increased Notch activation by 1.75 ± 0.5-fold as compared to HBSS controls (p < 0.05), whereas Hla-H35L had no effect. Immunocytochemistry and Western blotting confirmed these findings and revealed that ADAM10 and γ-secretase are required for Notch activation after inhibitor and siRNA assays. Retinal EC in mice engineered to express yellow fluorescent protein (YFP) upon Notch activation demonstrated significantly greater YFP intensity after Hla injection than controls. Aortic rings from Notch reporter mice embedded in matrix and incubated with rHla or Hla-H35L demonstrate increased Notch activation occurs at tip cells during sprouting. These mice also had higher skin YFP intensity and area of expression after subcutaneous inoculation of S. aureus expressing Hla than a strain lacking Hla in both EC and pericytes assessed by microscopy. Human liver displayed strikingly higher Notch expression in EC and pericytes during S. aureus infection by immunohistochemistry than tissues from uninfected patients. In sum, our results demonstrate that the S. aureus toxin Hla can potently activate Notch in vascular cells, an effect which may contribute to the pathobiology of infection with this microorganism.

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Abbreviations

Hla:

Alpha toxin

EC:

Endothelial cell

HUVEC:

Human umbilical endothelial cell

EDTA:

Ethylenediaminetetraacetic acid

ADAM10:

A disintegrin and metalloproteinase domain-containing protein-10

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Acknowledgements

We would like to thank Jan Kitajewski, Carrie Shawber, Henar Cuervo, and Darrell Yamashiro; The University of Chicago Imaging Core; The University of Chicago Human Tissue Resource Center; The University of Chicago Flow Cytometry Facility; Cancer Center Support Grant (P30CA014599). Funding for this project was provided by the Pediatric Cancer Foundation and The University of Chicago. This work was supported by NIH award AI097434 to J.B.W. G.R.S. has received support through NIH award 5 T32 GM7197 and the Genetics and Regulation Training Program at the University of Chicago.

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Authors and Affiliations

  1. Section of Pediatric Surgery, Department of Surgery, The University of Chicago, Chicago, IL, USA

    Sonia L. Hernandez, Mildred Nelson, Naina Bagrodia, Ann M. Defnet, Bianca Lec, Jared Emolo, Rebecca Kirschner, Lydia Wu, Henry Biermann, Stephanie Shen & Jessica J. Kandel

  2. Department of Microbiology, The University of Chicago, Chicago, IL, USA

    Georgia R. Sampedro & Juliane Bubeck Wardenburg

  3. Department of Pediatrics, Washington University at St. Louis, St. Louis, MO, USA

    Juliane Bubeck Wardenburg

Authors
  1. Sonia L. Hernandez
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  2. Mildred Nelson
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  11. Stephanie Shen
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  12. Juliane Bubeck Wardenburg
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Contributions

Conception and design: SLH, JJK, JB-W. Development of methodology: SLH, MN, GS, BL, RK. Acquisition of data: SLH, MN, GS, LW, BL, NB, AMD, JE, HB, RK, SS. Analysis and interpretation of data: SLH, MN, LW, BL, GS, NB, AMD, JE, JJK, RK, SS. Writing, review, and/or revision of the manuscript: SLH, JJK, JB-W. Administrative, technical, or material support: SLH, MN, LW, BL, NB, AMD, JE, RK, SS.

Corresponding author

Correspondence to Sonia L. Hernandez.

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Ethical approval

All studies involving animals were in accordance with the ethical standards and approved by the IACUC at the University of Chicago. All procedures performed in studies involving human participants were in accordance with the ethical standards of the University of Chicago IRB and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Supplementary Fig. 1

Quantitative PCR showing HUVEC transiently transfected with ADAM10 siRNA significantly reduced their ADAM10 expression 48 hours after transfection relative to Untreated, Non-targeting, and GAPDH siRNA pool transfected controls. p value represents t test. Graphs represent mean and standard deviation. (EPS 501 KB)

Supplementary Fig. 2

Hla’s HUVEC stimulation leads to nuclear localization of cleaved Notch1. 100&#x00D7; magnification of Hla stimulated cells, detailing nuclear localization of cN1 in HUVEC. cN1 levels are higher than DMSO, but lower than EDTA. The ADAM10 inhibitor GI254023X abrogates Hla/s activation of Notch. (EPS 3890 KB)

Supplementary Fig. 3

S. aureus infection activates Notch in liver perivascular cells. A) Liver sections from a patient with culture-positive S. aureus infection (right panels) had increased Notch expression (red) in EC (green, white arrows), compared to a non-infected control (left panels). B) Notch reporter mice with subcutaneous infection of S. aureus expressing Hla (right panel, WT Hla) resulted in Notch activation of &#x03B1;-SMA cells 16 days after infection, but a strain lacking Hla (left panel, &#x0394;Hla) does not. (EPS 4185 KB)

Supplementary material 4 (DOCX 13 KB)

Supplementary material 5 (DOCX 12 KB)

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Hernandez, S.L., Nelson, M., Sampedro, G.R. et al. Staphylococcus aureus alpha toxin activates Notch in vascular cells. Angiogenesis 22, 197–209 (2019). https://doi.org/10.1007/s10456-018-9650-5

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  • DOI: https://doi.org/10.1007/s10456-018-9650-5

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