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Dr Jenny Goldblatt Supervisors: Professor Shiranee Sriskandan Dr James Pease

Dr Jenny Goldblatt Supervisors: Professor Shiranee Sriskandan Dr James Pease. Cleavage of CXCL8 (IL8) by SpyCEP , a  Streptococcus pyogenes  protease, interrupts cell signalling in the host. S.pyogenes From carriage to invasive infection. Asymptomatic Carriage.

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Dr Jenny Goldblatt Supervisors: Professor Shiranee Sriskandan Dr James Pease

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  1. Dr Jenny Goldblatt Supervisors: Professor ShiraneeSriskandan Dr James Pease Cleavage of CXCL8 (IL8) by SpyCEP, a Streptococcus pyogenes protease, interrupts cell signalling in the host.

  2. S.pyogenesFrom carriage to invasive infection Asymptomatic Carriage Eg Acute rheumatic fever, Rheumatic Heart Disease - estimated prevalence 15-20 million worldwide, glomerulonephritis. Most in socioeconomically deprived populations. Estimated 700 million cases / year worldwide. egpharyngitis Non-invasive Infection Eg Scarlet Fever. increasing incidence documented in Asia Americas, Europe. Systemic Complications Immune Sequelae Estimated 650,000 / year worldwide. Increased incidence documented Europe, North America, New Zealand. EgNecrotising Fasciitis Invasive Infection

  3. S.pyogenes: necrotising fasciitis Asymptomatic Carriage Q. What is happening here? Eg Acute rheumatic fever, Rheumatic Heart Disease - estimated prevalence 15-20 million worldwide, glomerulonephritis. Most in socioeconomically deprived populations. Estimated 700 million cases / year worldwide. egpharyngitis Non-invasive Infection Eg Scarlet Fever. increasing incidence documented in Asia Americas, Europe. Systemic Complications Immune Sequelae Estimated 650,000 / year worldwide. Increased incidence documented Europe, North America, New Zealand. EgNecrotising Fasciitis Invasive Infection

  4. Necrotising fasciitis Paucity ofneutrophils at infection site Streptococcus PyogenesCell Envelope Protease (SpyCEP) • Encoded in the genome of all S.pyogenesstrains • Expression enhanced during invasion • Associated with necrotising disease in humans and mice • Edwards, R.J., et al. J Infect Dis, 2005. 192(5): p. 783-90 • Turner, C.E., et al., J Infect Dis, 2009. 200(4): p. 555-63. • Kurupati, P., et al., Mol Microbiol, 2010. 76(6): p. 1387-97 SpyCEP SpyCEP SpyCEP SpyCEP 100 μM

  5. SpyCEP acts on ELR+ CXC chemokines(neutrophil recruitment and activation) e.g. CXCL8 / IL8. N-terminus: RECEPTOR BINDING /ACTIVATION SpyCEP 13 a.a. C-terminal alpha helix Glycosaminoglycan (GAG) BINDING 100 μM Edwards, R.J., et al. J Infect Dis, 2005. 192(5): p. 783-90

  6. What is the impact of C-terminal cleavage on chemokine function? Q. Does cleavage of CXC chemokines interfere with signaling through their receptors CXCR1 CXCR2 ? N-terminus: RECEPTOR BINDING / ACTIVATION SpyCEP C-terminal alpha helix GAG BINDING

  7. Methods: Cleavage of CXCL8 CXCL8 incubated at 37oovernight with and without SpyCEP Evaluation of cleavage Western blot Pentapeptide antibody – raised against C-terminal 5aa of cleaved CXCL8 Binds cleaved CXCL8 Polyclonal antibody – commercially available Binds intact CXCL8 Cleaved Intact 8KDa Cleaved Intact 8KDa

  8. Is SpyCEP - cleaved CXCL8 able to act as a chemoattractant? Log [CXCL8] / M Reduced potency and efficacy of cleaved CXCL8 as a chemoattractant COUNT

  9. Does SpyCEP - cleaved CXCL8 activate its receptors? PLC Relative fluorescence Calcium flux assay - Stimulation with intact vs cleaved CXCL8 GDP GTP Cleaved CXCL8 does not stimulate calcium flux in HL60 cells differentiated as neutrophils Time

  10. Is SpyCEP- cleaved CXCL8 able to bind its receptors? Experimental vsradiolabelled CXCL8 CXCR1/2 transfected cell Receptor Percentage binding of 125I-CXCL8 (% of maximal binding) Cell Cleaved CXCL8 does not effectively displace intact CXCL8 from CXCR1 or CXCR2 transfectants [CXCL8] / M

  11. Does SpyCEP-cleavage of CXCL8 interfere with GAG binding? Intact and cleaved CXCL8 heparin binding GAG attached to cell surface Hoogewerf et al Biochemistry 1997, 36, 13570-78 Cleaved CXCL8 behaves similarly to monomeric CXCL8 in heparin binding assays

  12. Conclusions • Cleavage of CXCL8 by SpyCEP leads to loss of effective CXCR1 and CXCR2 signalling • This is due to loss of effective receptor binding • The C-terminus of CXCL8 appears to be required for receptor binding • GAG binding is disrupted by SpyCEP • GAG binding at the cell surface and / or CXCL8 dimerisation may be required for effective ligand-receptor interaction

  13. Future work • Investigate the role of the C-terminus in receptor binding further • Does loss of CXCR1 and CXCR2 signaling cause neutrophil dysfunction, leading to damage to host tissues? SpyCEP

  14. Acknowlegements Gram Positive Pathogenesis Group: Hammersmith Campus Pease Group, Leukocyte Biology Section: South Kensington Campus a

  15. DimericvsMonomeric CXCL8 WT (dimeric) CXCL8 Monomeric CXCL8 Hoogewerf et al Biochemistry 1997, 36, 13570-78

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