Cleavage of CXCL8 (IL8) by SpyCEP,

a Streptococcus pyogenes protease,

interrupts cell signalling in the host.

Dr Jenny Goldblatt

Supervisors: Professor Shiranee SriskandanDr James Pease

S.pyogenesFrom carriage to invasive infection

Immune Sequelae

Asymptomatic Carriage

Non-invasiveInfection

Invasive Infection

SystemicComplications

Estimated 700 million cases / year worldwide.

eg pharyngitis

Estimated 650,000 / year worldwide.

Increased incidence documented Europe, North America, New

Zealand. Eg Necrotising Fasciitis

Eg Scarlet Fever. increasing incidence documented in Asia Americas, Europe.

Eg Acute rheumatic fever, Rheumatic Heart

Disease - estimated prevalence

15-20 million worldwide, glomerulonephritis. Most

in socioeconomically deprived populations.

Immune Sequelae

Asymptomatic Carriage

Non-invasiveInfection

Invasive Infection

SystemicComplications

Estimated 700 million cases / year worldwide.

eg pharyngitis

Estimated 650,000 / year worldwide.

Increased incidence documented Europe, North America, New

Zealand. Eg Necrotising Fasciitis

Eg Scarlet Fever. increasing incidence documented in Asia Americas, Europe.

Eg Acute rheumatic fever, Rheumatic Heart

Disease - estimated prevalence

15-20 million worldwide, glomerulonephritis. Most

in socioeconomically deprived populations.

Q.What is

happening here?

S.pyogenes: necrotising fasciitis

100 μM

SpyCE

P

SpyCEP

SpyCE

P

SpyC

E P

Encoded in the genome of all S.pyogenes strains

Expression enhanced during invasion

Associated with necrotising disease in humans and miceEdwards, R.J., et al. J Infect Dis, 2005. 192(5): p. 783-90Turner, 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

Streptococcus Pyogenes Cell Envelope Protease (SpyCEP)

Necrotising fasciitis Paucity of neutrophils at infection site

100 μM

13 a.a.

SpyCEP

SpyCEP acts on ELR+ CXC chemokines(neutrophil recruitment and activation)

e.g. CXCL8 / IL8.

N-terminus: RECEPTORBINDING /ACTIVATION

C-terminal alpha helixGlycosaminoglycan (GAG) BINDING

Edwards, R.J., et al. J Infect Dis, 2005. 192(5): p. 783-90

What is the impact of C-terminal cleavage on chemokine function?

Q. Does cleavage of CXC chemokines interfere with signaling through their

receptors

CXCR1CXCR2

?

C-terminal alpha helixGAG BINDING

N-terminus:RECEPTORBINDING /

ACTIVATION

SpyCEP

CXCL8 incubated at 37o overnight with

and without SpyCEP

Western blot

Methods: Cleavage of CXCL8

Evaluation of cleavage

Pentapeptide antibody – raised against C-terminal 5aa of cleaved CXCL8Binds cleaved CXCL8

8KDa

Clea

ved

Inta

ct

Polyclonal antibody – commercially available Binds intact CXCL8

8KDa

Clea

ved

Inta

ct

Is SpyCEP - cleaved CXCL8 able to act as a chemoattractant?

Reduced potency and efficacy of cleaved CXCL8 as a chemoattractant

Log [CXCL8] / MCOUNT

PLC

GDPGTP

Cleaved CXCL8 does not stimulate calcium flux in HL60 cells differentiated as neutrophils

Calcium flux assay- Stimulation with intact vs cleaved

CXCL8

Does SpyCEP - cleaved CXCL8 activate its receptors?

Time

Rela

tive

fluore

scen

ce

Cleaved CXCL8 does not effectively displace intact CXCL8 from CXCR1 or CXCR2 transfectants

Is SpyCEP- cleaved CXCL8 able to bind its receptors?

Perc

en

tage b

indin

g o

f 1

25I-

CXC

L8(%

of

maxim

al bin

din

g)

CXCR1/2 transfecte

d cell

Experimental vs radiolabelled

CXCL8

Cell

Receptor

[CXCL8] / M

Does SpyCEP-cleavage of CXCL8 interfere with GAG binding?

Cleaved CXCL8 behaves similarly to monomeric CXCL8 in heparin binding assays

Intact and cleaved CXCL8 heparin binding

Hoogewerf et al Biochemistry 1997, 36, 13570-78

GAG attached to cell surface

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

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

Acknowlegements

Gram Positive Pathogenesis Group: Hammersmith

Campus

Pease Group, Leukocyte Biology Section: South Kensington Campus

a

Dimeric vs Monomeric CXCL8

WT (dimeric) CXCL8Monomeric CXCL8

Hoogewerf et al Biochemistry 1997, 36, 13570-78