, Volume 22, Issue 1, pp 197–209 | Cite as

Staphylococcus aureus alpha toxin activates Notch in vascular cells

  • Sonia L. HernandezEmail author
  • Mildred Nelson
  • Georgia R. Sampedro
  • Naina Bagrodia
  • Ann M. Defnet
  • Bianca Lec
  • Jared Emolo
  • Rebecca Kirschner
  • Lydia Wu
  • Henry Biermann
  • Stephanie Shen
  • Juliane Bubeck Wardenburg
  • Jessica J. Kandel
Original Paper


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.


Notch Staphylococcus aureus Alpha-toxin HUVEC 



Alpha toxin


Endothelial cell


Human umbilical endothelial cell


Ethylenediaminetetraacetic acid


A disintegrin and metalloproteinase domain-containing protein-10



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.

Author 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.

Compliance with ethical standards

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.

Supplementary material

10456_2018_9650_MOESM1_ESM.eps (501 kb)
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)
10456_2018_9650_MOESM2_ESM.eps (3.8 mb)
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)
10456_2018_9650_MOESM3_ESM.eps (4.1 mb)
Supplementary Fig. 3S. 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)
10456_2018_9650_MOESM4_ESM.docx (14 kb)
Supplementary material 4 (DOCX 13 KB)
10456_2018_9650_MOESM5_ESM.docx (13 kb)
Supplementary material 5 (DOCX 12 KB)


  1. 1.
    Le J et al (2017) Epidemiology and hospital readmission associated with complications of Staphylococcus aureus bacteremia in pediatrics over a 25-year period. Epidemiol Infect 145(12):2631–2639CrossRefGoogle Scholar
  2. 2.
    Shenoy ES et al (2016) The impact of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) flags on hospital operations. Infect Control Hosp Epidemiol 37(7):782–790CrossRefGoogle Scholar
  3. 3.
    Inoshima N, Wang Y, Bubeck J, Wardenburg (2012) Genetic requirement for ADAM10 in severe Staphylococcus aureus skin infection. J Invest Dermatol 132(5):1513–1516CrossRefGoogle Scholar
  4. 4.
    Inoshima I et al (2011) A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice. Nat Med 17(10):1310–1314CrossRefGoogle Scholar
  5. 5.
    Wilke GA, Bubeck J, Wardenburg (2010) Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus alpha-hemolysin-mediated cellular injury. Proc Natl Acad Sci USA 107(30):13473–13478CrossRefGoogle Scholar
  6. 6.
    Powers ME et al (2012) ADAM10 mediates vascular injury induced by Staphylococcus aureus alpha-hemolysin. J Infect Dis 206(3):352–356CrossRefGoogle Scholar
  7. 7.
    Lozano C et al (2011) Staphylococcus aureus nasal carriage, virulence traits, antibiotic resistance mechanisms, and genetic lineages in healthy humans in Spain, with detection of CC398 and CC97 strains. Int J Med Microbiol 301(6):500–505CrossRefGoogle Scholar
  8. 8.
    van Tetering G et al (2009) Metalloprotease ADAM10 is required for Notch1 site 2 cleavage. J Biol Chem 284(45):31018–31027CrossRefGoogle Scholar
  9. 9.
    Rand MD et al (2000) Calcium depletion dissociates and activates heterodimeric notch receptors. Mol Cell Biol 20(5):1825–1835CrossRefGoogle Scholar
  10. 10.
    Uyttendaele H et al (1996) Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development 122(7):2251–2259Google Scholar
  11. 11.
    Al Haj Zen A, Madeddu P (2009) Notch signalling in ischaemia-induced angiogenesis. Biochem Soc Trans 37(Pt 6):1221–1227Google Scholar
  12. 12.
    Peschon JJ et al (1998) An essential role for ectodomain shedding in mammalian development. Science 282(5392):1281–1284CrossRefGoogle Scholar
  13. 13.
    Krebs LT et al (2000) Notch signaling is essential for vascular morphogenesis in mice. Genes Dev 14(11):1343–1352Google Scholar
  14. 14.
    Swiatek PJ et al (1994) Notch1 is essential for postimplantation development in mice. Genes Dev 8(6):707–719CrossRefGoogle Scholar
  15. 15.
    Hartmann D et al (2002) The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts. Hum Mol Genet 11(21):2615–2624CrossRefGoogle Scholar
  16. 16.
    VanDussen KL et al (2012) Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells. Development 139(3):488–497CrossRefGoogle Scholar
  17. 17.
    Wu Y et al (2010) Therapeutic antibody targeting of individual Notch receptors. Nature 464(7291):1052–1057CrossRefGoogle Scholar
  18. 18.
    Carulli AJ et al (2015) Notch receptor regulation of intestinal stem cell homeostasis and crypt regeneration. Dev Biol 402(1):98–108CrossRefGoogle Scholar
  19. 19.
    Ridgway J et al (2006) Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444(7122):1083–1087CrossRefGoogle Scholar
  20. 20.
    Noguera-Troise I et al (2006) Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Nature 444(7122):1032–1037CrossRefGoogle Scholar
  21. 21.
    Hellstrom M et al (2007) Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445(7129):776–780CrossRefGoogle Scholar
  22. 22.
    Chigurupati S et al (2007) Involvement of notch signaling in wound healing. PLoS ONE 2(11):e1167CrossRefGoogle Scholar
  23. 23.
    Thurston G, Kitajewski J (2008) VEGF and Delta-Notch: interacting signalling pathways in tumour angiogenesis. Br J Cancer 99(8):1204–1209CrossRefGoogle Scholar
  24. 24.
    Kangsamaksin T et al (2015) NOTCH decoys that selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth. Cancer Discov 5(2):182–197CrossRefGoogle Scholar
  25. 25.
    Nowotschin S et al (2013) A bright single-cell resolution live imaging reporter of Notch signaling in the mouse. BMC Dev Biol 13:15CrossRefGoogle Scholar
  26. 26.
    Baker M et al (2011) Use of the mouse aortic ring assay to study angiogenesis. Nat Protoc 7(1):89–104CrossRefGoogle Scholar
  27. 27.
    Becker RE et al (2014) Tissue-specific patterning of host innate immune responses by Staphylococcus aureus alpha-toxin. J Innate Immun 6(5):619–631CrossRefGoogle Scholar
  28. 28.
    Banerjee D et al (2015) Notch suppresses angiogenesis and progression of hepatic metastases. Cancer Res 75(8):1592–1602CrossRefGoogle Scholar
  29. 29.
    Murtomaki A et al (2014) Notch signaling functions in lymphatic valve formation. Development 141(12):2446–2451CrossRefGoogle Scholar
  30. 30.
    Gupta-Rossi N et al (2001) Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notch1 receptor. J Biol Chem 276(37):34371–34378CrossRefGoogle Scholar
  31. 31.
    Stahl A et al (2010) The mouse retina as an angiogenesis model. Invest Ophthalmol Vis Sci 51(6):2813–2826CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Sonia L. Hernandez
    • 1
    Email author
  • Mildred Nelson
    • 1
  • Georgia R. Sampedro
    • 2
  • Naina Bagrodia
    • 1
  • Ann M. Defnet
    • 1
  • Bianca Lec
    • 1
  • Jared Emolo
    • 1
  • Rebecca Kirschner
    • 1
  • Lydia Wu
    • 1
  • Henry Biermann
    • 1
  • Stephanie Shen
    • 1
  • Juliane Bubeck Wardenburg
    • 2
    • 3
  • Jessica J. Kandel
    • 1
  1. 1.Section of Pediatric Surgery, Department of SurgeryThe University of ChicagoChicagoUSA
  2. 2.Department of MicrobiologyThe University of ChicagoChicagoUSA
  3. 3.Department of PediatricsWashington University at St. LouisSt. LouisUSA

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