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Molecular Immunology 44 (2007) 23–32
Review
Bacterial complement evasion
Suzan H.M. Rooijakkers, Jos A.G. van Strijp ∗
Experimental Microbiology, UMC Utrecht G04-614, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Received 1 June 2006; received in revised form 22 June 2006; accepted 27 June 2006
Available online 27 July 2006
Abstract
The human complement system is elemental to recognize bacteria, opsonize them for handling by phagocytes, or kill them by direct lysis.
However, successful bacterial pathogens have in turn evolved ingenious strategies to overcome this part of the immune system. In this review we
discuss the different stages of complement activation sequentially and illustrate the immune evasion strategies that various bacteria have developed
to evade each subsequent step. The focus is on bacterial proteins, either surface-bound or excreted, that block complement activation. The underlying
molecular mechanism of action and the possible role in pathophysiology of bacterial infections are discussed.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Complement; Bacteria; Evasion; Proteins; Inhibitors
1. Introduction
The complement system is an essential and effective part of
the innate immune system. It can rapidly recognize and opsonize
bacteria for phagocytosis by professional phagocytes or kill
them directly by membrane perturbations. Therefore it is to
no surprise that bacteria have evolved a whole array of highly
specific complement-modulating strategies. In this way bacteria can either stop or delay the detrimental effects of an innate
immune attack, thereby creating a window of opportunity to
divide and create a microenvironment that allows an even better survival. The human complement system consists of more
than thirty proteins in plasma and on cells. The complement
system is organized in three different initiation pathways that
all converge at one step: the cleavage of the central complement
protein C3 (Walport, 2001a,b). The three different complement
pathways are represented by the classical (CP), the lectin (LP)
and the alternative (AP) pathway. These pathways consist of different recognition molecules to sense a foreign substance. After
recognition, these pathways use similar activation mechanisms
to generate C3 convertases, the enzymes that cleave C3. The
attachment of C3b to acceptor cells is necessary to initiate phagocytosis, formation of the membrane attack complex (MAC) and
enhancement of humoral responses to antigens (Gasque, 2004).
∗
Corresponding author. Tel.: +31 30 2506528; fax: +31 30 2505863.
E-mail address: [email protected] (J.A.G. van Strijp).
0161-5890/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2006.06.011
In this overview we have divided the activation steps of the
complement system in the following sequential phases: ‘initial
steps’ (2), ‘convertases’ (3), ‘C3 and its degradation products’
(4), ‘the terminal pathway’ (5), ‘host regulators’ (6) and ‘receptors’ (7). In these chapters, the bacterial inhibitors that have been
identified for these subsequent steps will be discussed.
2. Modulation of the initial steps
The primary step in complement activation is the binding of
recognition molecules to the microbe. For the classical pathway
this is achieved through the binding of C1q to IgG or IgM that
is specifically bound to the surface of the microbe (Duncan and
Winter, 1988). C1q is complexed with two serine proteases, C1r
and C1s (Sim and Laich (2000)). When the globular heads of C1q
bind to an activator, the associated C1r undergoes auto activation
and activates C1s. Activated C1s then cleaves C4 to generate
C4b and the anaphylatoxic peptide C4a. Exposed within the
C4b is a highly labile internal thiol ester, which is able to react
with hydroxyl groups (creating an ester bond) and amino groups
(creating an amide bond) (Law and Dodds, 1997). C2 binds to
surface-bound C4b and activated C1s of the nearby C1 complex cleaves C2 to release the small C2b fragment and form the
classical pathway C3 convertase, C4b2a.
The lectin pathway (LP) is highly analogous to the CP and
its activation also results in formation of C4b2a (Matsushita
and Fujita, 2001). However, the recognition molecules for the
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S.H.M. Rooijakkers, J.A.G. van Strijp / Molecular Immunology 44 (2007) 23–32
lectin pathway recognize microbial sugars instead of immune
complexes. The recognition molecules of the LP are mannanbinding lectin (MBL) and ficolin (L-, H- or M-ficolin) (Fujita,
2004). These lectins are structurally similar to C1q. MBL binds
in a Ca2+ -dependent manner to the target via its C-type lectin
domains, which recognize neutral sugars (preferentially mannose, N-acetylglucosamine and fucose) on the surfaces of a range
of microorganisms, such as Neisseria and Leishmania species
(Holmskov et al., 2003). l-Ficolin binds GlcNAc and peptidoglycan. In circulation, both MBL and ficolins are associated
with several proteases called MBL-associated serine proteases
(MASPs) (Matsushita et al., 2000). MASP-1, MASP-2 and
MASP-3 are structurally similar to C1r and C1s. However, only
MASP-2 is known to cleave complement components C4 and
C2 and thereby generate C4b2a.
The alternative pathway (AP) mainly functions as an amplification loop of the CP and LP after surface-bound C3b is created.
However, it can also be spontaneously activated by hydrolysis
of the internal thioester bond in C3 (0.005%/min) (Sahu and
Lambris, 2001), forming the so-called C3H2 O. Just like particlebound C3b, this C3H2 O can participate in the formation of the
AP C3 convertase. Both surface-bound C3b and C3H2 O can
bind factor B. The resulting complexes are recognized by factor
D (Xu et al., 2001). Unlike other complement serine proteases
that are present as zymogens in the serum, factor D circulates
in its active form. Factor D cleaves factor B to release Ba and
yield the activated C3H2 O Bb complex, an unstable fluid-phase
C3 convertase, or a surface-bound C3bBb complex.
Bacteria have evolved several modulators of these initial steps
in complement activation. So far no inhibitors of the lectin pathway components have been described but, since this pathway
has been discovered only recently, we envision that bacterial
modulators will emerge in the coming years.
Although many biological roles have been attributed to
Staphylococcal protein A (SpA), its capacity to bind the Fc
part of IgG is still the most established one (Silverman et al.,
2005). SpA is a type I membrane protein that is bound to the
cell wall of Staphylococcus aureus via its C-terminal cell-wallbinding region X. In the N-terminal half of the protein are its
IgG-binding domains E, D, A, B, and C. Through binding of
IgG, protein A blocks Fc-receptor mediated phagocytosis but
is also a highly efficient complement activation modulator by
interfering with binding of C1q (Verhoef et al., 2004; Forsgren
et al., 1966; Goward et al., 1993). SpA is surface-bound, but can
also be released in the surrounding environment during growth
of the peptidoglycan layer to which it is attached. A second
IgG-binding protein has been reported in S. aureus called Sbi
(Zhang et al., 1998). The protein consists of 436 amino acids
and exhibits an immunoglobulin-binding specificity similar to
protein A. Its role in complement modulation remains to be
established.
In other bacteria, proteins with similar functions have been
described. Protein G, a bacterial cell wall protein with comparable affinity for IgG, was isolated from group G streptococci. With
a molecular weight of 30 kDa, Protein G was found to bind all
human IgG subclasses but also rabbit, mouse, and goat IgG. On
the IgG molecule, the Fc part appears mainly responsible for the
interaction with protein G, although a low degree interaction was
also recorded for Fab fragments. IgM, IgA, and IgD, however,
showed no binding to protein G (Bjorck and Kronvall, 1984).
By the same group, Protein L was isolated from the surface of
the anaerobic bacterium Peptostreptococcus magnus (Bjorck,
1988). Although this protein binds both IgM and IgG, it seems
to have no affinity for the Fc-part of immunoglobulins but rather
for Fab parts. A role in complement inhibition could not be
demonstrated and its role is more likely as a B-cell superantigen
(Genovese et al., 2003).
The majority of group A streptococci (GAS) express, next
to the well known M proteins, structurally similar M-related
proteins, Mrp and Enn, which have been described as IgGand IgA-binding proteins. Subsequent analysis of phagocytosis by flow cytometry indicates that, if present, both mrp
and emm gene products contribute to phagocytosis resistance
by decreasing bacterial binding to granulocytes (Podbielski et
al., 1996). GAS also produce two immunoglobulin-degrading
enzymes: the streptococcal cysteine proteinases, IdeS and SpeB,
that both cleave IgG specifically in the hinge region, and thus
removing the entire Fc region from IgG molecules that are
attached to the bacterium (Von Pawel-Rammingen and Bjorck,
2003).
Similarly, staphylococci hinder IgG-mediated effector functions by the excretion of Staphylokinase (SAK). S. aureus
expresses several plasminogen (PLG)-binding receptors at their
surface and this surface-bound PLG can be activated into plasmin (PL) by SAK. Surface-bound PL has the ability to cleave
both IgG and C3b. Recently we showed that PL, formed by
the conversion of PLG by SAK at physiological concentrations, leads to opsonin removal. PL cleaves human IgG from the
bacterial cell wall leading to impaired phagocytosis by human
neutrophils (Rooijakkers et al., 2005a). PL cleaves IgG at position Lys 222, and thus removes the entire Fc fragment, including
the glycosylation site (Asn 297) necessary for recognition by
C1q thereby inhibiting the activation of the classical pathway of
complement.
A similar strategy is employed by a protease from Porphyromonas gingivalis, a pathogen in human periodontitis. The prtH
gene encodes a 97-kDa active protease, which degrades C3 and
IgG. An allelic exchange mutant of P. gingivalis, in which the
prtH gene was inactivated, is less virulent in a mouse model
of bacterial invasiveness. Also, in comparison with its parent
strain, the mutant strain is less able to degrade C3 and accumulates significantly greater numbers of molecules of C3b and iC3b
on the bacterial surface during complement activation, resulting
in increased phagocytosis by human neutrophils as compared to
the wild type suggesting a function of the prtH gene product may
be important in evasion of host defense mechanisms (Schenkein
et al., 1995).
Also, bacteria can target C1 itself. The fish pathogen
Aeromonas salmonicida, encodes a 40 kDa C1q-binding outer
membrane protein. This 40 kDa porin binds C1q in an antibody
independent process, and its in vivo role in serum resistance
was established. The 40 kDa porin gene and/or protein was
present in all the A. salmonicida typical or atypical strains
tested (Merino et al., 2005). Two excreted enzymes from
S.H.M. Rooijakkers, J.A.G. van Strijp / Molecular Immunology 44 (2007) 23–32
Pseudomonas elastase (PaE) and alkaline protease (PaAP),
when incubated with highly purified C1q (0–5 h, 37 ◦ C) reveal
preferential sensitivity of the 28-kDa A-chain and 24-kDa
C-chain, of the C1q molecule, with PaAP being more potent
than PaE (Hong and Ghebrehiwet, 1992).
3. Modulation of convertases
The C3 convertases are bimolecular complexes that activate
C3 (Xu et al., 2001). Two different C3 convertases exist: C4b2a
and C3bBb representing the C3 convertases of the CP/LP and
AP, respectively. C4b2a and C3bBb are structurally and functionally similar (Sim and Laich, 2000). C4b and C3b are derived
from common ancestors and both molecules covalently attach
to microbial surfaces upon activation of its internal thioester.
Also, fB and C2 have a common precursor gene, share the same
domain organization and as part of the C3 convertases act as
similar proteases. Although C3 convertase activity resides in
one molecule (C2a or Bb), the capacity to cleave C3 is acquired
only through complex formation. Amplification of convertases
is regulated in several ways: first of all, the complexes itself are
instable, C2a and Bb fall off after a few minutes (Ponnuraj et al.,
2004). Secondly, serum and cell-bound regulators are known to
cause dissociation of C3 convertases on self surfaces (Kirkitadze
and Barlow, 2001). On microorganisms, decay of C3bBb can be
delayed by binding of the glycoprotein properdin. Dissociated
C2a and Bb are inactive and cannot re-associate with C4b and
C3b to form new convertases. The covalent binding of another
C3b molecule to C4b or C3b within a C3 convertase leads to the
formation of a C5 convertase, C4b2a3b or C3bBb3b (Lambris,
1988).
Staphylococcal complement inhibitor (SCIN) is a 10 kDa,
excreted protein that blocks all complement pathways: the lectin,
classical and alternative pathway. SCIN efficiently prevents
phagocytosis and killing of staphylococci and C5a production.
We found SCIN to specifically act on surface-bound C3 convertases, which has two major consequences. First SCIN stabilizes
both C3bBb as well as C4b2a at the surface of the bacterium.
Since the convertases are normally instable, the stabilization of
SCIN prevents generation of additional convertases. Secondly
and surprisingly, together with the stabilization, the binding of
SCIN to C3bBb and C4b2a impairs the enzymatic activity of
the convertases. This contrasts the action of properdin on convertases. SCIN binds activator-bound C3bBb, but not C3bB or
C3b. This and the fact that SCIN inactivates the convertase,
suggests that SCIN binds the active pocket of Bb. However,
earlier studies have clearly indicated that the conformation of
Bb induced by its cofactor C3b, is crucial for displaying activity
of the protease subunit Bb. So, a conformational change within
C3bBb by SCIN cannot be excluded (Rooijakkers et al., 2005b).
SCIN is highly human-specific. It does not inhibit complement
activation in all tested animals thus far including mice and
rats.
Many microbial modulators act indirectly on the convertases
since they attract human decay-accelerating proteins and thereby
destabilize the convertases. These will be discussed below under:
“Interactions with host regulators”.
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4. Modulation of C3 and its split products
C3 is the most abundant complement protein in serum
(1.2 mg/ml) and is comprised of an ␣ and ␤ chain (110 and
75 kDa, respectively) that are connected covalently by a single
disulfide bond and associated by non-covalent forces (Janssen
et al., 2005). One of the most intriguing features of C3 is its ability to attach covalently to acceptor molecules on cells surfaces.
This property is derived from the presence of an intramolecular thioester bond within the C3d region. The thioester bond is
protected within a hydrophobic pocket and is exposed only in
the C3b fragment upon cleavage of C3 by C3 convertases (Law
and Dodds, 1997). Once C3 is cleaved to C3b, the transiently
exposed thioester bond in C3b participates in a transacylation
reaction with nucleophilic groups present on cell surfaces. In
many biological systems the majority of C3b is linked via an
ester bond indicating a strong preference for the hydroxylated
targets. Proteolytic activation of native C3 by the C3 convertases leads to cleavage and generation of C3b (176 kDa) and
C3a (9 kDa). Activation of C3 results in the release of the small
chemo-attractant molecule C3a on one hand and in the deposition of C3b molecules on the microbial surface on the other
hand. C3b deposition is crucial for eradication of microbes since
C3b and the C3b degradation product iC3b mark the microbe
for efficient uptake by phagocytes. iC3b can be further degraded
by factors H and I to C3dg and finally to C3d, still attached to
the microbe (Lambris, 1988).
Staphylokinase (SAK) targets PLG to the staphylococcal surface and activates it into PL. PL cleaves human IgG as well
as human C3b and iC3b from the bacterial cell wall leading to
impaired phagocytosis by human neutrophils. It cleaves C3b in
both the ␣- and the ␤-chain. The decrease of C3b molecules
will indirectly diminish C3 convertases as well as C5 convertases (Rooijakkers et al., 2005a, c). Also the PrtH-encoded
97 kDa proteases from Porphyromonas gingivalis, degrades C3
(Schenkein et al., 1995). Further, the two Pseudomonas proteases that were described above as C1 degraders also target
C3. C3, after incubation with PaE and PaAP, was converted
from 190-kDa to a 120-kDa fragment. The 120-kDa piece
yielded three distinct bands on SDS-PAGE: an intact 75-kDa
beta-chain and two alpha-chain pieces of approximately 41and 26-kDa. NH2-terminal end sequence analysis localized
the 26-kDa fragment within the cysteine-rich 41-kDa, COOHterminal piece. The NH2-terminal end of the alpha-chain is completely degraded into small fragments (Hong and Ghebrehiwet,
1992).
The extracellular fibrinogen binding molecule (Efb) is a
15.6 kDa excreted molecule that was described earlier to bind
fibrinogen. The group of Brown found that Efb binds the C3d
region of C3 (Lee et al., 2004a). Efb blocks classical pathway
dependent opsonization and subsequent phagocytosis. Although
the present data on Efb do not demonstrate Efb binding to
bacterium-bound C3d, a role for Efb in modulation of C3dmediated recognition by CR2 on B-cells cannot be excluded. The
C3b binding site of Efb is distinct from its fibrinogen-binding
site, in fact, Efb can bind both molecules simultaneously (Lee
et al., 2004b).
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S.H.M. Rooijakkers, J.A.G. van Strijp / Molecular Immunology 44 (2007) 23–32
5. Modulation of the terminal pathway
The terminal complement pathway is the final cytolytic step
in the complement cascade. The “killer” molecule is the membrane attack complex (MAC), a lytic assembly of C5b, C6,
C7, C8 and multiple molecules of C9 (Ramm et al., 1982).
The terminal pathway starts when C5 is split into the chemoattractant C5a and C5b by the C5 convertase. C5b forms a
soluble bimolecular complex with C6 and the subsequent binding of C5b6 to C7 induces it to express a metastable site through
which C5b7 is inserted into target lipid bilayer membranes.
Subsequent incorporation of C8 and multiple C9 molecules
allows the complex to penetrate lipid bilayers creating complete transmembrane channels resulting in osmotic lysis of the
cell. The MAC directly kills Gram-negative organisms that
have an outer lipid membrane. However, Gram-positive bacteria resist this attack by the simple fact that their cell membrane is shielded by a thick cell wall (Joiner et al., 1983).
With the cleavage of C5, the important chemo-attractant C5a is
released. Together with C3a and bacterial formylated peptides,
C5a attracts phagocytes to the site of infection (Ramm et al.,
1982).
In S. aureus, 11 different staphylococcal superantigen-like
(SSLs) proteins were identified. Although these molecules are
closely related to the superantigens, they have different biological functions. SSL7 (23 kDa) is a complement inhibitor that
specifically binds to C5 (KD = 18 nM) and thereby prevents
complement-mediated lysis of erythrocytes or Escherichia coli
cells (Langley et al., 2005). SSL7 is not specific for the human
host, since it also binds primate, sheep, pig and rabbit C5.
Although the SSL7 binding site on C5 remains to be elucidated,
the authors propose the C5 cleavage site at the C5 ␣-chain as
a logical site. In this case, SSL7 would prevent cleavage of C5
into both C5b and C5a. From a bacterial point of view, inhibition
of C5a production is probably a more important complement
evasion strategy since S. aureus is resistant to C5b-9 cytolysis. Next to its role in complement evasion, SSL7 also binds
monomeric forms of human IgA1/IgA2 and the secretory form
of IgA. SSL7 prevented binding of serum IgA to Fc␣R1 (CD89)
on myeloid cells. All SSL genes were found to be located on
a 19-kb genetic cluster of the S. aureus pathogenicity island
SaPIn2.
Streptococcal inhibitor of complement (SIC) is a 31 kDa
excreted protein that fulfils many different roles in immune evasion by Group A Streptococci (GAS). SIC, exclusively found
in the highly virulent M1 type, was initially identified as a
terminal complement pathway inhibitor since it binds the soluble C5b-7 complex and thereby prevents its insertion into cell
membranes (Akesson et al., 1996; Fernie-King et al., 2001).
SIC functions similarly to the human MAC regulators, clusterin
and S-protein. Since streptococci are resistant to complementmediated cytolysis, complement inhibition was suggested not
to be the sole function of SIC. Next to its role in complement inhibition, SIC also counteracts the antibacterial actions
of secretory leukocyte proteinase inhibitor (sLPI) and lysozyme
(Fernie-King et al., 2002), inactivates human neutrophil alphadefensin (HNP-1) and LL-37 (Frick et al., 2003) and alters
cellular processes by binding the intracellular proteins Ezrin
and Moesin in epithelial cells and neutrophils (Hoe et al.,
2002).
A very unique property of SIC is that it is extraordinarily polymorphic (Stockbauer et al., 1998). A population-based
surveillance of GAS infections revealed that epidemic waves
were composed of strains expressing a heterogeneous array of
SIC variants (Hoe et al., 1999). From 892 different isolates, a
total of 162 different sic alleles were identified.
Two variants of SIC have been described in GAS. In serotype
M57, the gene for closely related to SIC (CRS) was found to be
located outside the mga regulon. CRS shares many characteristics of SIC, including its ability to bind C6 and C7. The protein
that is distantly related to SIC (DRS) has a limited sequence
similarity with the C-proximal half and its biological function
is unknown. DRS is present among M12 and M55 serotypes
(Hartas and Sriprakash, 1999).
The plasmid-encoded outer membrane protein TraT from E.
coli K12 strongly prevents the terminal pathway. TraT inhibited serum haemolytic activity at the step downstream of C5
activation and upstream of C7. TraT probably inhibits the assembly of C5b6 or causes structural changes that inactivate C5b6
(Pramoonjago et al., 1992).
Borrelia burgdorferi confers resistance to the terminal pathway by a 80 kDa surface protein that shares both antigenic and
functional similarities with human CD59, a natural membranebound inhibitor of MAC. Both CD59 and Borrelial CD59-like
inhibit cell lysis by preventing the polymerisation of C9 and
the formation of MAC. Despite its similarities, the CD59-like
molecule exhibits a number of structural and functional differences from human CD59; CD59-like is much larger than
CD59, indicating that it is not an acquired regulator. Furthermore, CD59-like interacts with native C8 and C9 while human
CD59 binds the same molecules only in context of the assembling MAC. Inhibition of MAC formation by a membrane bound
CD59-like represents an important survival mechanism in B.
burgdorferi (Pausa et al., 2003).
Both Group A (GAS) and group B streptococci (GBS) encode
a cell wall anchored C5a peptidase (scpA and scpB) (Wexler
and Cleary, 1985; Chmouryguina et al., 1996; Navarre and
Schneewind, 1999). This peptidase is an established virulence
factor as determined in animal models for scpA (Ji et al., 1996).
Mutant strains were better cleared than wild-type strains. Interestingly, ScpB is highly specific for human C5a (Bohnsack et al.,
1993) and an identical gene in Group G streptococci has been
shown to be restricted to strains that are capable of infecting
humans (Cleary et al., 1991). These C5a peptidases are unable
to cleave the whole C5 protein but do cleave the C5a fragment
(Cleary et al., 1992).
The 56-kilodalton protease (56 kDa protease) from Serratia
marcescens significantly and dose-dependently decreases the
chemotactic activity of activated human serum. Furthermore,
treatment of human recombinant C5a with 56 K protease at a
dose of 1 ␮g/ml resulted in a complete loss of chemotactic activity. In mice, the magnitude of infiltration of neutrophils into the
peritoneal cavity was much lower than that caused by a low
protease producing strain (Oda et al., 1990).
S.H.M. Rooijakkers, J.A.G. van Strijp / Molecular Immunology 44 (2007) 23–32
6. Interactions with host regulators
Since complement activation is potentially harmful to host
tissues, it is tightly regulated. At virtually every step of the complement cascade, fluid-phase or cell bound regulators control
complement activation. The first proteolytic step in the classical and lectin pathway is controlled by the serine protease
inhibitor C1 Inhibitor (C1-INH) (Sim and Laich, 2000). Further
downstream, C3 convertases are regulated by the so-called regulators of complement activation (RCA) including the plasma
proteins C4-binding protein (C4BP), factor H (FH), FH-like
protein (FHL-1) and the membrane proteins CR1, CR2, CD46
(MCP) and CD55 (DAF) (Kirkitadze and Barlow, 2001). RCA
proteins accelerate the decay of C3 convertases and furthermore
act as cofactors in the proteolytic degradation of C4b and/or C3b
by the soluble protease factor I (fI). Only one positive regulator
of complement is known; properdin is a multimeric protein that
enhances the stability of the alternative pathway C3 convertase
on microbial surfaces (Hourcade, 2006). The terminal complement pathway is regulated by CD59, vitronectin and clusterin
that all prevent insertion of C5b-7 into lipid membranes (Davies
and Lachmann, 1993).
Secreted protease of C1 esterase inhibitor from Enterohemorrhagic E. coli O157:H7 (StcE) is a 98 kDa zinc metalloprotease that is encoded by the large virulence plasmid pO157.
Lathem et al. initially showed that StcE specifically cleaves
C1-INH from its full length of 105 kDa into 60–65 kDa fragments (Lathem et al., 2002). Later on the authors described the
functional consequences of StcE interacting with C1-INH and
indicated that instead of inactivating C1-INH, StcE enhanced
the ability of the serpin to down-regulate the classical complement pathway (Lathem et al., 2004). StcE was found to trap
C1-INH to the cellular surface by binding both to the surface and the serpin at the same time. This increases the local
concentration of C1-INH and strengthens its inhibitory capacity. The proteolysis of C1-INH by StcE was not necessary to
increase its inhibitory actions. StcE was found to interact with
C1-INH at its glycosylated amino terminal domain, leaving
the serpin domain free to inactivate its target serine proteases.
Next to inhibition of classical complement-dependent lysis of
sheep erythrocytes, StcE interacting with C1-INH also offers
increased serum resistance to E. coli K-12. In summary, by
recruiting C1-INH to cell surfaces, StcE may protect E. coli
O157:H7 from complement mediated lysis and inflammatory
events.
Serratia marcescens also produces a protease that cleaves C1INH (Molla et al., 1989). The 56-kDa protease cleaves C1-INH,
but also alpha 2-antiplasmin, and antithrombin III into molecular
weights of approximately 8–10 kDa. The 56-kDa protease also
inactivated serum complement within 2–6 h.
A number of bacterial complement regulators evade complement attack by sequestration of human regulators to the bacterial
surface. Well-described examples are bacterial surface proteins
that bind human C4BP and FH/FHL-1. These regulators are
captured in such a way that they are still able to interact with
C3 convertases and function as cofactors in factor I cleavage of
C3b/C4b.
27
By binding C4BP (570 kDa), bacteria disturb activation of
the classical/lectin pathway.
Group A Streptococci bind to C4BP via the M protein family
members Arp and Sir (Jarva et al., 2003; Thern et al., 1995).
Neisseria gonorrhoeae captures C4BP via both its outer membrane porin (Por) and via type IV pili. Both isoforms of Por,
Por1A and Por1B, were shown to bind C4BP (Ram et al., 2001).
Moraxella catarrhalis interacts with C4BP via Ubiquitous surface protein A1 and A2 (UspA1 and UspA2) (Nordstrom et al.,
2004). Both UspA1 (88 kDa) and UspA2 (62 kDa) bind C4BP in
a dose-dependent manner and binding studies to single subunits
of C4BP revealed KD values of 13 and 1.1 ␮M for UspA150–770
and UspA230–539 , respectively.
The binding of Outer membrane protein A (OmpA, 35 kDa)
of E. coli K1 to human C4BP protects this bacterium from C3b
deposition and activation of downstream complement proteins
(Wooster et al., 2006). Finally, in clinical isolates of Bordetella
pertussis (Berggard et al., 1997) Filamentous Hemagglutinin
(FHA) was identified as a C4BP-binding molecule. In contrast
to the other bacterial C4BP-molecules, FHA could not protect
Bordetella from complement-mediated lysis via C4BP.
The binding sites in C4BP were identified for these
molecules. C4BP is composed of seven identical alpha-chains
(70 kDa) and one beta-chain (45 kDa) (Jenkins et al., 2006). The
alpha and beta chains consist of repeating domains of 60aa complement control protein (CCP) domains. Arp and Sir, but also
Neisserial type IV pili, bind the first two CCP modules of the
alpha-chain (Jenkins et al., 2006; Ram et al., 2001). Por binding sites in C4BP are exclusively in the CCP1 region of the
alpha chain. Both UspA1 and UspA2 bound the CCP2, CCP5
and CCP7 domains of the alpha chain. OmpA binds the CCP3
domain of the alpha-chain (Prasadarao et al., 2002).
Many bacteria also capture FH and/or FHL-1 to their surface
resulting in AP C3 convertase decay and inactivation of C3b by
factor I. FH (150 kDa) is composed of 20 short consensus repeats
(SCR). FHL-1 (42 kDa) consists of the first 7 SCR of FH in
combination with four additional amino acids at the C terminus.
Both FH (400 ␮g/ml) and FHL-1 (10–50 ␮g/ml) regulate the AP
C3 convertase, C3bBb, by accelerating its decay and displaying
cofactor activity.
Next to their interaction with C4BP, gonococcal Por
molecules and streptococcal M1 protein, also bind to FH (Ram
et al., 1999; Kihlberg et al., 1999). For Por1A, different loops
were shown to be involved in binding of C4BP or FH. Class 3
Por molecules in N. meningitidis have also been suggested as a
receptor for FH.
Factor H binding inhibitor of complement (Hic) of Streptococcus pneumoniae and Beta protein of Group B Streptococci
are cell-anchored proteins that bind to FH (Janulczyk et al.,
2000). Hic and beta protein are structurally related and were
found to bind similar regions in FH (SCR8–11 and SCR12–14)
(Jarva et al., 2004). The plasmid-encoded outer membrane protein YadA mediates serum resistance in Yersinia enterolitica
(China et al., 1993). The reduction of C3b deposition in YadA+
strains was ascribed to the specific binding of YadA to FH.
Omp100 belongs to a family of six major outer membrane proteins of Actinobacillus actinomycetemcomitans, a
28
S.H.M. Rooijakkers, J.A.G. van Strijp / Molecular Immunology 44 (2007) 23–32
pathogenic bacterium involved in periodontitis. Omp100 is a
versatile virulence factor involved in bacterial adhesion, invasion and serum resistance by trapping factor H (Asakawa et al.,
2003).
Fba is a surface protein of GAS that binds both FH and FHL-1
(Pandiripally et al., 2003). The binding site was localized in the
short consensus repeat 7 (SCR7), a domain common to both regulators. Since FHL-1 also functions in cell adhesion (Zipfel and
Skerka, 1999), Pandiripally et al found that Fba mediated binding of FHL-1 also promotes entry of streptococci into human
epithelial cells.
Borrelia burgdorferi produces several FH-binding proteins in
order to resist complement (Kraiczy et al., 2003). Complement
regulator-acquiring surface protein 1 (CRASP-1) is the dominant FH and FHL-1 binding protein of B. burgdorferi. Also
here, the main binding site was localized in the SCR7 of FH
and FHL-1. The Erp (OspE-F related lipoprotein) family members are encoded on members of the 32 kb circular plasmid-like
prophage family. Many Erp proteins serve as receptors for FH of
numerous vertebrate hosts (Miller and Stevenson, 2006; Hovis
et al., 2006).
Streptococcal pyrogenic exotoxin B (SPE B) is an excreted
cysteine protease, well known as an important virulence factor in GAS. Next to the described digestion of numerous host
proteins like immunoglobulin, kininogen, fibronectin, Tsao et
al. recently showed that SPE B also degrades serum properdin,
inhibiting complement activation via the alternative pathway
(Tsao et al., 2006). GAS opsonized with SPE B-treated serum
was more resistant to neutrophil killing.
7. Modulation of complement receptors
Complement receptors (CR) on leukocytes form an important
and integral part of the complement system. CR1 (CD35) binds
the opsonin fragments C3b and C4b and promotes phagocytosis
and clearance of antigen-antibody complexes (Gasque, 2004).
A receptor for C1q also promotes immune complex binding to
phagocytes. CR2 (CD21) is part of the B cell receptor complex;
binding of antigen-complement complexes to CR2 increases the
sensitivity of the B cell to antigen by up to a thousand fold. CR3
(CD11b/CD18, MAC-1) and CR4 (CD11c/CD18, p150.95) are
integrin molecules that allow monocytes, macrophages, neutrophils, and dendritic cells to adhere to blood vessel walls
and move into the tissues at the site of inflammation. Next to
that, especially CR3 is probably the primary responsible receptor for phagocytosis of opsonized bacteria by neutrophils since
CR3 recognizes iC3b. The C5a receptor (CD88) is responsible for chemotaxis and primes neutrophils and macrophages to
phagocytose complement-coated antigen even in the absence of
IgG. At high concentrations (over 10−6 M), C5a, upon interaction with the C5aR, can stimulate neutrophils to a metabolic
burst.
M5 protein of GAS interferes with CR3 (CD11b/CD18)dependent association between GAS and neutrophils, and
thereby blocks subsequent ingestion of the bacteria. Isolated human neutrophils killed an M-negative GAS mutant
(DeltaM5), but not the wild-type parent strain (M5). Differ-
ent Abs against CR3 blocked adhesion and killing of DeltaM5
bacteria, whereas the blocking of CR1 and CR4 had no effect
(Weineisen et al., 2004).
Group A Streptococci also secrete a protein with homology
to the alpha-subunit of CR3. The GAS Mac-1-like protein (Mac)
was secreted by most pathogenic strains, produced in log-phase
and controlled by a two-component gene regulatory system,
which also regulates transcription of other GAS virulence factors. Patients with GAS infection had titers of antibody specific
to Mac that correlated with the course of disease, demonstrating
that Mac was produced in vivo. Although Mac has homology
to CR3, its action is not related to complement modulation,
Mac binds CD16 (Fc␥RIIIB). This binding inhibits phagocytosis and production of reactive oxygen species, resulting in
decreased pathogen killing (Lei et al., 2001). Interestingly, Mac
is identical to IdeS, the IgG degrading enzyme of GAS (Von
Pawel-Rammingen and Bjorck, 2003).
The very early signs of bacterial invasion, C5a and formylated peptides (e.g. fMet-Leu-Phe), are recognized by the innate
immune system through two related receptors on neutrophils,
the C5aR and the formyl peptide receptor (FPR). The excreted
Chemotaxis inhibitory protein of S. aureus (CHIPS) is a 14.1kDa protein that reduces the neutrophil recruitment toward C5a
in a mouse peritonitis model, even though its activity is much
more potent on human than on mouse cells. CHIPS binds specifically to the C5aR and FPR and blocks C5a- and fMLP-induced
calcium mobilization. The apparent K(d) values of CHIPS for
the C5aR is 1.1 nM, similar to that of C5a itself (de Haas et
al., 2004). Using monoclonal anti-CHIPS Abs, peptides and
site directed mutagenesis it was demonstrated that the first and
third amino acid, both a phenylalanine, are essential for CHIPS
blocking the fMLP-induced activation of phagocytes, while the
C5aR binding site of CHIPS is located at the C-terminal end
(Haas et al., 2004). CHIPS does not affect activation of the
C5aR by a peptide mimic of the C5a C-terminus. Moreover,
CHIPS was found to bind only the C5aR N terminus. Deletion and mutation experiments within this C5aR N-terminal
expression system revealed that the binding site of CHIPS is
contained in a short stretch of nine amino acids (amino acids
10–18), of which the aspartic acid residues at positions 10, 15,
and 18 plus the glycine at position 12 are crucial. Binding studies
with C5aR/C3aR- and C5aR/IL8RA-chimeras confirmed that
CHIPS binds only to the C5aR N terminus without involvement
of its extracellular loops (Postma et al., 2004, 2005). CHIPS
fragment consisting of residues 31–121 (CHIPS31–121) has
the same activity in blocking the C5aR compared to full-length
CHIPS, but completely lacks FPR antagonism. CHIPS31–121
has a compact fold comprising an alpha-helix (residues 38–51)
packed onto a four-stranded anti-parallel beta-sheet. Comparison of CHIPS31–121 with known structures reveals striking homology with the C-terminal domain of staphylococcal
superantigen-like proteins (SSLs) 5 and 7, and the staphyloccocal and streptococcal superantigens TSST-1 and SPE-C.
Also, the recently reported structures of several domains of the
staphylococcal extracellular adherence protein (EAP) show a
high degree of structural similarity with CHIPS (Haas et al.,
2005).
S.H.M. Rooijakkers, J.A.G. van Strijp / Molecular Immunology 44 (2007) 23–32
Gingipains from Porphyromonas gingivalis have been implicated as C3 degraders. Lys-gingipain, but not Arg-gingipain,
is also active against the C5aR. It cleaves C5aR on human
neutrophils. The N-terminal region of C5aR (residues 9–29,
PDYGHYDDKDTLDLNTPVDKT) was readily cleaved by a
mixture of proteases from P. gingivalis. Next to Lys-gingipain
there appear to be additional proteinase(s) in the vesicles that
attack the cell surface molecule C5aR (Jagels et al., 1996).
8. Concluding remarks
In this overview we have encountered a vast amount of different proteins, partially overlapping in function and sequence or
structure, that all have the ability to divert the complement system. Now we will discuss some general aspects of these proteins;
arrangement in the genome, regulation of expression, presumed
redundancy, species specificity and the potential use as therapeutics in inflammatory disorders.
Both in S. aureus and Group A Streptococci, we encounter
genomic regions with multiple virulence factors specialized in
modulation of complement. In S. aureus, the genes for three
complement modulators SCIN, CHIPS and SAK cluster on
the conserved 3 end of beta-hemolysin (hlb)-converting bacteriophages, representing an immune evasion cluster (IEC) with
enterotoxin A. A total of 90% of S. aureus strains carry this IEC
in seven IEC variants. This IEC is easily transferred among S.
aureus strains by a diverse group of beta-hemolysin-converting
bacteriophages. (van Wamel et al., 2006). The M family of streptococcal surface proteins is composed of the related Emm (class
I and II), Mrp (FcrA), and Enn proteins. The so-called Mga
regulons contain M-family proteins mrp, emm, and enn followed by sic and scpA gene. The exact order and content of
the whole operon can vary but this is the basic architecture
(Navarre and Schneewind, 1999). Other complement modulators reviewed here, TraT and StcE, from E. coli, YadA from
Yersinia and the Erp family members of Borrelia are encoded
on plasmids or on plasmid-like prophages (Zhang and Marconi,
2005). All this indicates that the clustered genomic organization of these factors cannot be a coincidence; probably they
are (remnants of) mobile elements making bacteria virulent by
adding a complete set of immune evasion genes. The other option
is that their genomic co-localization is a result of a common
regulatory system that guides the expression timing of these
genes.
Indeed the expression of these genes is under strict control as
can be concluded from the following examples. The streptococcal terminal pathway modulator SIC, like the other genes on the
mga regulon, is excreted in high amounts during early and late
logarithmic growth phase during in vitro culture. In vivo evidence exists that SIC has a role in the early stages of infection.
There is no expression during stationary growth phase (Frick et
al., 2003). Similarly, we showed that the staphylococcal complement modulators SCIN and CHIPS are explicitly expressed
during early growth stage of in vitro bacterial growth implicated
with in vivo colonization phase, while the other complement
modulator SAK is expressed much later (Rooijakkers et al.,
2006).
29
The question is often posed whether it is costly redundancy
when bacteria make so many immune evasion molecules that
attack multiple steps of cascades even within one species. In
vitro it is often sufficient to block one step of a certain biological cascade to block the whole outcome of the cascade. In simple
animal models it is often the same: blocking one step using a
natural inhibitor or an antibody can be sufficient to block the
biological effects associated with that cascade. In Staphylococci
and Streptococci combined there are more than a dozen complement inhibitors described to date. Many people try to prove that
“their” molecule is the most important virulence factor since that
is the best blocker at the most crucial step of the complement
cascade. However, there is a lesson to be learned here. Viral
immune evasion has already shown us years ago that the best
way to block a cascade is to inhibit every subsequent step in that
cascade by different molecules. Antigen processing is attacked
in that very way by herpesviruses that evade adaptive immunity
(Ploegh, 1998; Novak and Peng, 2005; Wiertz et al., 1997). We
will identify far more evasion molecules in the years to come
and it will become clear that the best evasion strategy takes many
attacks at many points, even in a cascade.
The species specificity of many of these inhibitors is striking. For various bacteria it is clear that neither the pathogen
nor the encoded evasion molecules is host specific and these
molecules are easily studied in mouse models. In contrast, there
are also many evasion molecules that are highly specific for one
host and this is also true for complement evasion. Although the
complement system is evolutionary extremely conserved still
human specificity was found in complement evasion molecules
from human pathogens: CHIPS, C5a-peptidase, SAK, SCIN
and more. Since it is extremely difficult to study these evasion
molecules in animal models it is hard to predict the role of this
species specificity. Again in viral immune evasion strategies it
is clear that these molecules contribute to a great extent to the
host specificity of the pathogen itself (Dobbelstein, 2003; Ahn
et al., 1996).
From a therapeutically point of view, all proteins in this
overview have a high potential in anti-inflammatory therapy.
Per definition, molecules that help to evade acute innate immune
mechanism are anti-inflammatory compounds. Their high specificity correlates with their non-toxic nature. Especially for the
soluble molecules, it is tempting to speculate that they could
be used as injectables in severe acute inflammatory disorders.
Pre-existing antibodies against all these proteins will complicate
this approach but the targets that these molecules identify, to the
amino acid level and below, will prove to be crucial targets in
anti-inflammatory therapy.
Taken together, comprehending complement evasion is a crucial aspect of understanding bacterial pathogenesis. The more
proteins along with their exact mechanism of action are discovered, the more we will understand bacteria-host interactions in
general.
Acknowledgements
Discussed work that originated from the authors’ laboratory
was supported by grants from the Technology Foundation STW
30
S.H.M. Rooijakkers, J.A.G. van Strijp / Molecular Immunology 44 (2007) 23–32
(#UKG-6609), the Netherlands Genomics Initiative (NGI #05071-028), the European Union (#FP6-512093) and the Netherlands Organization for Scientific Research (NWO #9120.6020).
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