Supplement bij veertiende jaargang, april 2006 - NVMM

experiments with SSL5 and P-selectin-Fc chimera or
antibodies directed against PSGL-1 were performed with
human neutrophils. Functional role for SSL5 was investigated
through rolling experiments with neutrophils
under flow on a P-selectin surface. Influence of SSL5 on
chemokine signaling was investigated in neutrophils and
U937 cells through a calcium mobilization assay.
Results: SSL5 inhibited binding of anti-PSGL-1 and Pselectin-Fc
to neutrophils by 90% and 60%, respectively.
SSL5 also strongly blocked adhesion and rolling of
neutrophils to immobilized P-selectin under static and flow
conditions comparably to anti-PSGL-1. Finally, SSL5 specifically
inhibited the ? and Neutrophila-interleukin-8 (IL-8),
growth-regulated oncogene alpha (GRO Activating Protein
2 (NAP2)-induced calcium mobilization in neutrophils and
U937 cells expressing the CXCR2.
Conclusions:
1) SSL5 binds PSGL-1 and interferes with PSGL-1-mediated
binding to P-selectin under static and flow conditions.
2) SSL5 inhibits CXCR2-induced cell activation.
3) SSL5 is an important immunomodulatory protein of
Staphylococcus aureus.
05.04
SCIn and CHIPS homologues are located on a new Immune
evasion Cluster in S. aureus
I. Jongerius, S.H.M. Rooijakkers, J.A.G. van Strijp
UMC Utrecht, Eijkman-Winkler Institute, Utrecht
Introduction: Staphylococcal Complement Inhibitor (SCIN)
and Chemotaxis Inhibitory Protein of Staphylococci
(CHIPS) are crucial for the staphylococcal defense against
our innate immune system. SCIN and CHIPS are located
on the first Immune Evasion Cluster (IEC) in Staphylococcus
aureus. A BLAST search revealed four unknown staphylococcal
proteins with high sequence homology to SCIN
or CHIPS. SCIN-B and SCIN-C, sharing 45% and 48%
homology with SCIN, also function as complement
inhibitors. The CHIPS-like proteins, Formylated Peptide
Receptor Like-1 (FPRL-1) Inhibitory protein (FLIPr) (28%
homology) and FLIPr-like (77% homology with FLIPr)
prevent activation of FPRL-1 or FPR&FPRL-1 respectively.
In this study we describe the unique location of SCIN and
CHIPS homologues on a new Immune Evasion Cluster
(IEC2) in S. aureus.
Methods: Database analyses were performed to characterize
IEC2. The prevalence of SCIN-B, SCIN-C, FLIPr, FLIPr-like
and neighboring genes in 90 clinical S. aureus strains was
determined by PCR. For functional analyses, we cloned and
expressed the protein with 33% homology to Extracellular
Fibrinogen Binding protein (Efb) (Efb-like) in Escherichia
coli. Efb-like was tested in whole blood phagocytosis assays
Ned Tijdschr Med Microbiol 2006; 4:Supplement
S23
using FITC-labeled S. aureus. Calcium mobilization was
performed to study effects on C5a production.
Results: We describe a new Immune Evasion Cluster
(IEC2) in S. aureus that carries the genes for SCIN-B (46%
of strains) or SCIN-C (33%), FLIPr (79%) or FLIPr-like
(4%), Alpha-toxin (100%), Efb (86% C3b modulator), Efblike
(58%), an unknown membrane protein (90%) and 3
putative exotoxins (93%). Efb-like was identified as a potent
immune evasion molecule since it effectively prevents
phagocytosis in whole blood. Since Efb-like prevents
generation of C5a, it is a new complement modulator.
Conclusion:
1) The immune evasion molecules SCIN-B, SCIN-C, FLIPr
and FLIPr-like are located on a new Immune Evasion
Cluster (IEC2) in S. aureus.
2) On this IEC2 we also found the genes for Alpha-toxin,
Efb, 4 unknown proteins and Efb-like.
3) Efb-like represents a new complement modulator in
S. aureus.
05.05
Complement inhibition by S. aureus
S.H.M. Rooijakkers1 , M. Ruyken1 , A. Roos2 , M.R. Daha2 ,
J.S. Presanis3 , R.B. Sim3 , W.J.B. van Wamel1 , K.P.M. van
Kessel1 , J.A.G. van Strijp1 1 2 UMC Utrecht, Eijkman-Winkler Institute, Utrecht, Department
of Nephrology, University Medical Center, Leiden, 3MRC Immunochemistry Unit, Department of Biochemistry,
University of Oxford, UK.
The complement system plays a pivotal role in host
defense but also contributes to tissue injury in several
diseases. Here we describe the discovery of Staphylococcal
Complement Inhibitor (SCIN), an excreted 9.8 kD protein
that blocks human complement by specific interaction
with C3 convertases (C4b2a and C3bBb). The assembly
of C3 convertases (C4b2a and C3bBb) is a prerequisite
for complement activation. The convertases catalyze C3b
deposition on activator surfaces. SCIN binds and stabilizes
C3 convertases, interfering with C3b deposition via the
classical, lectin and alternative complement pathway. This
leads to a dramatic decrease in phagocytosis and killing of
Staphylococcus aureus by human neutrophils. As a highly
active and small soluble protein that acts exclusively on
surfaces, SCIN is a promising anti-inflammatory molecule.
Furthermore, we address the role of other staphylococcal
proteins that modulate the complement system.