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REVIEW ARTICLE
Bacteria under stress by complement and coagulation
Evelien T.M. Berends1,*, Annemarie Kuipers1,*, Marietta M. Ravesloot1, Rolf T. Urbanus2 &
Suzan H.M. Rooijakkers1
1
Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands; 2Department of Clinical Chemistry and Haematology,
University Medical Center Utrecht, Utrecht, The Netherlands
*Equal contribution
Correspondence: Suzan H.M. Rooijakkers,
University Medical Center Utrecht, Medical
Microbiology, G04.614, Heidelberglaan 100,
3584 CX Utrecht, The Netherlands.
Tel.: +31887556535; fax: +31887555863;
e-mail: [email protected]
Received 7 February 2014; revised 23 June
2014; accepted 14 July 2014. Final version
published online 3 September 2014.
DOI: 10.1111/1574-6976.12080
MICROBIOLOGY REVIEWS
Editor: Jos van Strijp
Keywords
innate immunity; complement; membrane
attack complex; coagulation; bacteria;
evasion.
Abstract
The complement and coagulation systems are two related protein cascades in
plasma that serve important roles in host defense and hemostasis, respectively.
Complement activation on bacteria supports cellular immune responses and
leads to direct killing of bacteria via assembly of the Membrane Attack
Complex (MAC). Recent studies have indicated that the coagulation system
also contributes to mammalian innate defense since coagulation factors can
entrap bacteria inside clots and generate small antibacterial peptides. In this
review, we will provide detailed insights into the molecular interplay between
these protein cascades and bacteria. We take a closer look at how these pathways are activated on bacterial surfaces and discuss the mechanisms by which
they directly cause stress to bacterial cells. The poorly understood mechanism
for bacterial killing by the MAC will be reevaluated in light of recent structural
insights. Finally, we highlight the strategies used by pathogenic bacteria to
modulate these protein networks. Overall, these insights will contribute to a
better understanding of the host defense roles of complement and coagulation
against bacteria.
Introduction
The complement and coagulation systems are two ancient
proteolytic cascades in plasma that play important roles
in host defense and hemostasis, respectively. Both systems
are more than 400 million years old and probably evolved
from a common ancestor, explaining the significant overlap in protein domains (Krem et al., 2000). Complement
and coagulation proteins circulate in the blood as inactive
precursors, but are activated upon contact with target
structures. The resulting proteolytic cascade generates
multiple protein cleavage products that trigger numerous
events important for inflammation and hemostasis.
Complement is an integral part of the innate immune
system, our primary host defense barrier against invading
bacteria (Walport, 2001; Ricklin et al., 2010). Whereas
the complement reaction exclusively takes place on the
bacterial surface, the resulting cleavage products are either
linked to the surface or released in fluid-phase. While the
released chemotactic peptides attract phagocytes from
the blood to the site of infection (Bardoel & Strijp, 2011),
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the massively deposited C3 fragments enhance bacterial
recognition by phagocytes and trigger phagocytosis.
Finally, the pore-forming complex (Membrane Attack
Complex or MAC) that inserts into the membrane
(M€
uller-Eberhard, 1986) results in direct bacterial killing.
The primary function of the coagulation system is to
respond to damage of blood vessels via formation of
fibrin, which seals wounds and stops bleeding (hemostasis). Although clotting is a well-known immune defense
mechanism in invertebrates, its role in mammalian host
defense is largely unknown. However, multiple lines of
evidence now suggest that coagulation is important for
the early innate immune response since it supports the
inflammatory response, generates antimicrobial peptides
and induces local entrapment of bacteria in fibrin clots
(Frick et al., 2007; Nickel & Renne, 2012).
In this review, we aim to provide detailed insights into
how complement and coagulation contribute to host
clearance of bacterial infections. In particular, we will
focus on the recognition mechanisms that drive activation
of these cascades on bacteria and discuss how these
FEMS Microbiol Rev 38 (2014) 1146–1171
1147
Molecular interplay of complement, coagulation and bacteria
systems directly induce stress to bacterial cells. The comparison of these related proteolytic cascades as innate
sensors provides us a unique opportunity to discuss the
relatively unknown role of coagulation as part of the
mammalian innate immune system. Finally, we also highlight how bacterial pathogens block or use these proteolytic cascades to overcome immune clearance. Since both
complement and coagulation are pathological during systemic infections, detailed insights into their activation
mechanisms and modulatory strategies developed by
bacteria could stimulate research into new treatment
strategies for infectious diseases.
on target cells. Recognition molecules generally bind
evolutionarily conserved structures that are only present
on microbes and absent on host cells. In the past years,
our knowledge on these recognition mechanisms of bacteria has increased significantly and below we will provide
an overview of bacterial structures that are recognized by
complement.
Recognition of bacteria by the complement
system
Bacteria are roughly divided into Gram-positive and
Gram-negative bacteria, determined by their cell wall
characteristics (Fig. 1). Peptidoglycan (PG) is the common trait in the cell envelope of almost all bacteria and
important for cell integrity. It is composed of polysaccharide strands of alternating N-acetylglucosamine (GlcNAc)
and N-acetylmuramic acids (MurNAc) that are linked by
short peptides (reviewed in Vollmer et al., 2008). Grampositive bacteria possess one phospholipid membrane
covered by a thick PG layer (20–80 nm) (reviewed in
Silhavy et al., 2010) that commonly contains complex
polysaccharides and teichoic acids linked to the peptidoglycan (wall teichoic acids, WTAs) or the membrane
(lipoteichoic acids, LTAs) (Reichmann & Gr€
undling,
2011). In contrast, Gram-negative bacteria contain two
membranes (the inner (IM) and outer membrane (OM))
with a thin PG layer (1–7 nm) in between. The lipid
Bacteria under stress by complement
Complement activation is a step-wise process in which
protein binding and cleavage events occur in a welldefined order (Gros et al., 2008; Ricklin et al., 2010):
first, the recognition molecules of the three complement
pathways (classical, lectin, alternative) bind to the bacterial cell surface (Fig. 1); second, they activate associated
serine proteases that form C3 convertase enzymes
(Fig. 2); third, C3 convertases cleave the central complement protein C3 to label the target surface with C3b and
amplify the labeling process (Fig. 2b); fourth, C5 convertases convert C5 into C5a and C5b; fifth, C5b generates
the MAC (C5b-9) (Fig. 2a, Fig. 3). The recognition phase
is important for the specific activation of complement
MBL
MBL
H-ficolin
H-ficolin
Collectin-11
Collectin-11
LPS
LPS/PS
L-ficolin
?
LPS/?
Properdin
FEMS Microbiol Rev 38 (2014) 1146–1171
GlcNAc
L-ficolin
?
LPS/PS
Fig. 1. How bacteria are recognized by
complement. Complement recognition
molecules of the CP (C1q), LP (MBL, ficolins,
collectin-11), and AP (properdin) bind to
various structures on Gram-positive and Gramnegative bacteria (recognition motifs in italics).
Next to direct binding, C1q also indirectly
recognizes bacteria via antibodies, CRP or SAP.
MBL, mannose-binding lectin; SAP, serum
amyloid P; CRP, C-reactive protein; IgG,
immunoglobulin G; LPS, lipopolysaccharide;
PS, polysaccharides; WTA, Wall Teichoic acid;
GlcNAc, N-acetyl glucosamine; LTA,
Lipoteichoic acid.
WTA
GlcNAc/PS
Gram– Gram+
LTA
LPS
C1q
SAP
PS
C1q
LPS
SAP
C1q
PS
PG
C1q
SAP
WTA
proteins proteins
C1q
SAP
P
CRP
IgG
IgG
C1q
C1q
C1q
C1q
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Published by John Wiley & Sons Ltd. All rights reserved
1148
E.T.M. Berends et al.
(a)
AP amplification
B
C2a
C4b
Recognition
Bb
C3a
C3b C3b C3b
C3b
C3 convertases
C3b deposition
C2a
C4b C3b
C5a
C5 convertases
C5b
MAC
(b)
C3a
C33
C
C3b
C3 cleavage C3b attachment
Fig. 2. The complement reaction on bacteria. (a) Recognition molecules initiate the complement cascade by their associated serine proteases
(C1s and MASP-2, purple) that cleave C4 and C2 to generate a C4b2a complex on the bacterial surface. This complex is a C3 convertase that
cleaves C3 into the released anaphylatoxin C3a and C3b, which binds covalently to the bacterial surface. C3b molecules initiate formation of
another C3 convertase (C3bBb) that amplifies the C3b labeling process (AP amplification). C3b molecules also generate C5 convertases by
binding onto or near the C3 convertases. This initiates a substrate switch, which causes convertases to cleave C5 into soluble C5a and C5b that
forms a complex with C6-9 to generate the MAC. This figure was partly modified from Gros et al., 2008. (b) Conversion of C3 by the C3
convertase. C3 holds a reactive thioester bond (red dot) that is hidden within the C3 molecule but exposed upon its cleavage into C3b. The acyl
group of the thioester moiety forms a covalent bond with a hydroxyl or amine group, which allows covalent attachment of C3b to the target
surface.
(a)
C5b-6
(b)
sC5b-7
mC5b-7
Model A
C5b-8
C5b-9
MAC
Model B
OM
PG
IM
Fig. 3. Assembly and bactericidal mechanisms of the MAC. (a) Newly formed C5b reacts with C6 to form the stable C5b6 complex. Binding of
C7 results in a hydrophobic complex that targets the membrane (mC5b-7). Membrane insertion is initiated upon binding of C8 (C5b-8) after
which 12–18 copies of C9 polymerize to form the pore-forming ring structure (MAC). (b) Several hypotheses for MAC-mediated killing of Gramnegative bacteria. Model A: the MAC simultaneously disrupts the OM and IM via insertion into e.g. Bayer’s junctions. Model B: the MAC or its
precursors (C5b-8 or C5b-9n) rupture the OM, which allows passage of C5b-9 components (creating another MAC), C9 alone (or fragments of
C9), or other factors into the periplasm to disrupt IM integrity. This process may require additional enzymes to breakdown the PG layer.
composition of the OM is heterogeneous and contains
glycolipids called lipopolysaccharide (LPS) (Silhavy et al.,
2010).
It has long been recognized that isolated PG, which is
unique to bacteria, is a potent activator of the complement system (Verbrugh et al., 1980; Ma et al., 2004). In
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Published by John Wiley & Sons Ltd. All rights reserved
the context of living bacteria, however, PG is less
exposed; it is hidden underneath the outer membrane
(OM) of Gram-negative bacteria or shielded by
PG-anchored polysaccharides and teichoic acids in Grampositives. Therefore, complement also recognizes other
conserved bacterial structures (Fig. 1). The recognition
FEMS Microbiol Rev 38 (2014) 1146–1171
1149
Molecular interplay of complement, coagulation and bacteria
molecules of the lectin pathway (LP) are the mannose
binding lectin (MBL), ficolins (Ficolin-1, Ficolin-2, and
Ficolin-3) and collectin-11 (Matsushita & Fujita, 2001;
Holmskov et al., 2003; Fujita et al., 2004; Ma et al.,
2013). These large multimeric complexes resemble a
‘bunch of tulips’ since they hold a collagen-like domain
build up from three identical polypeptide chains that
assemble in a coiled-coil structure at the base and
multiple specific carbohydrate-binding domains at the
top. MBL and collectin-11 contain a C-type carbohydrate-recognition domain (CRD) (Holmskov et al., 1994)
that allows binding to carbohydrate and glycoconjugate
structures on many different microbes (reviewed in Jack
et al., 2001b). The main bacterial targets of MBL are LPS
in Gram-negative and WTA in Gram-positive bacteria
(Polotsky et al., 1996; Jack et al., 2001a; Park et al.,
2010). Collectin-11 was recently identified and described
to bind Escherichia coli, Pseudomonas aeruginosa (Hansen
et al., 2010), and Streptococcus pneumoniae (Ali et al.,
2012). In ficolins, the CRD consists of a fibrinogen-like
domain that recognizes both sugar motifs (Matsushita
et al., 2013) and acetylated groups present in for instance
GlcNAc and GalNAc (Matsushita & Fujita, 2001). The
binding to GlcNAc, a major constituent of PG, is believed
to be important for the binding to Gram-positive bacteria
(Ma et al., 2004).
The C1q molecule, which is structurally similar to
MBL and ficolins in the LP, mediates activation of the
classical pathway (CP). C1q is classically known to bind
to Fc regions of IgG or IgM antibodies on the microbial
surface. For some bacteria like Neisseria meningitidis, antibodies are essential for an effective complement response
(Granoff, 2009). Furthermore, C1q binds bacteria via
pentraxins, evolutionarily conserved plasma proteins that
recognize foreign antigens and altered-self ligands. The
major pentraxins in human plasma are the short pentraxins C-reactive protein (CRP) and serum amyloid P (SAP)
(reviewed in Du Clos, 2013) and the long pentraxin 3
(PTX3) (Ma et al., 2011). CRP interacts with phosphocholine (PC) that is present in a number of bacterial structures including the teichoic acid of S. pneumoniae
(Volanakis, 2001). SAP binds a variety of bacterial cell
wall structures such as carbohydrates, LPS, and peptidoglycan (Hind et al., 1985; Noursadeghi et al., 2000; An
et al., 2013). Alternatively, C1q can bind directly to the
bacterial surface to activate the CP. C1q was found to
bind to a variety of microbial structures including the
lipid A region of LPS (Cooper & Morrison, 1978; Tan
et al., 2011) and different outer membrane proteins on
Gram-negative bacteria (Loos & Clas, 1987; Albertı et al.,
1993; Mintz et al., 1995; Merino et al., 1998), and LTA
on Gram-positive bacteria (Loos et al., 1986). Moreover,
C1q can bind to various carbohydrates (Pa€ıdassi et al.,
FEMS Microbiol Rev 38 (2014) 1146–1171
2008), which potentially mediates direct binding to
bacteria.
Recently, it was shown that the alternative pathway
(AP), generally viewed as an amplification mechanism of
the CP and LP, also directly recognizes bacteria via the
properdin molecule. It has been reported that properdin,
a stabilizing protein for the convertases of the AP,
functions as a pattern recognition receptor for bacteria
(Spitzer et al., 2007; Kemper et al., 2010). Properdin
binds to LPS of certain Gram-negative bacteria but also
to LPS-defective E. coli and Salmonella typhimurium
(Spitzer et al., 2007). Also, properdin was reported to
bind and activate complement on Chlamydia pneumoniae
(Cortes et al., 2011). However, since other studies have
questioned these data, the direct recognition of bacteria
via the AP is still under debate (Harboe et al., 2012). Of
note, activation of the AP can also occur at a low-level
via the spontaneous ‘tick-over’ process in which hydrolysis of C3 creates the C3b-like molecule C3H2O (Pangburn
et al., 1981; Ricklin et al., 2010). This activation process
is down regulated on host cells but quickly amplified on
bacterial cells.
Thanks to the large variety of recognition mechanisms,
almost all bacteria will be recognized by the complement
system. Many studies have shown that the relative importance of each pathway may be different for every bacterium. Importantly, the contribution of each pathway may
change during an active infection due to upregulation of
the acute phase response (CRP and MBL) and the
(increased) production of antibodies. Recently, an interesting study reported that binding of MBL to WTA of Staphylococcus aureus occurs exclusively in infants with low
levels of anti-WTA antibodies. In adults, higher levels of
anti-WTA antibodies compete with MBL for WTA binding
and trigger activation of the classical pathway (Park et al.,
2010). Whether such a pathway-shift results in a more
effective complement response is presently unknown.
Complement reaction on bacteria
Whereas the initial phase of complement recognition is
strictly dependent on the composition of the bacterial
surface, the next steps of the complement pathway occur
similarly on all target surfaces (Fig. 2a). The recognition
molecules do not possess enzyme activity but circulate in
complex with serine proteases responsible for activation
of the proteolytic cascade. The recognition molecules of
the LP (MBL, Ficolins, and Collectin-11) circulate in
complex with the MBL-associated serine proteases
(MASPs) while C1q is complexed with the C1r and C1s
proteases. The homologous MASP-2 and C1s proteases
can both cleave C4 and C2 to generate a C3 convertase
enzyme that consists of surface-bound C4b and protease
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1150
C2a (Fig. 2a). This convertase catalyzes the covalent
deposition of C3b onto the bacterial surface and release
of the small anaphylatoxin C3a. The covalent binding of
C3b, a critical step in the complement response, is
mediated by its reactive thioester. Thanks to the crystal
structures of C3 and C3b (Janssen et al., 2005, 2006), we
now understand how this thioester is hidden within C3
but becomes exposed in C3b as a result of extensive conformational changes (Fig. 2b). Although the acyl group of
the thioester is thought to form a covalent bond with any
hydroxyl or amine group (Law & Dodds, 1997; Janssen &
Gros, 2007), this reaction is probably less random than
generally believed (Law et al., 1981, 1984; Law & Dodds,
1997). Most C3b is linked by an ester bond, which
indicates that it reacts better with hydroxyl groups than
amine groups (Law et al., 1984). Binding occurs preferentially to hydroxylated sugar groups, but also to amino
acids in proteins of which tyrosine, threonine, and serine
show the highest reactivity (Tyr>Thr>Ser) (Sahu &
Pangburn, 1995).
The deposited C3b molecules initiate the alternative
pathway (AP) amplification loop because C3b itself forms
the basis of the AP C3 convertase enzyme (C3bBb).
Surface-bound C3b forms a complex with Factor B, which
is then converted by Factor D (Gros et al., 2008). The
resulting C3bBb enzyme converts new C3 molecules into
C3b (Fig. 2a and b) and thus rapidly amplifies the number of deposited C3b molecules. This is crucial for initiation of the terminal pathway, since high densities of C3b
are required to change the affinity of the convertase for
C5 (Rawal & Pangburn, 2001; Pangburn & Rawal, 2002),
which can then cleave C5 into the chemo-attractant C5a
and soluble C5b that initiates assembly of the MAC.
Host defense functions of complement on
bacteria
Complement activation products can exert stress to bacterial cells, either indirectly by supporting cellular immune
responses or directly by damaging bacterial membranes.
Before we zoom into the direct antibacterial mechanisms,
which are the focus of our review, we will shortly
summarize the indirect actions of complement. A very
important role of complement is to facilitate phagocytosis
of bacteria by labeling (or opsonizing) their surface with
C3b and its degradation product iC3b, which are
recognized by complement receptors 1 (CR1), 3 (CR3), 4
(CR4), and CRIg on phagocytic cells (Vik & Fearon,
1985; Krych-Goldberg & Atkinson, 2001; Helmy et al.,
2006; Bajic et al., 2013). Also, the release of anaphylatoxins C5a and C3a is important to trigger a pro-inflammatory status. Moreover, neutrophils and monocytes (C5a),
but also eosinophils and mast cells, are attracted to the
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E.T.M. Berends et al.
site of infection through interaction with the C5a (C5aR)
or C3a receptor (C3aR) (Haas & Strijp, 2007). Moreover,
complement activation can orchestrate adaptive immunity
by enhancing antigen presentation via interaction of the
C3b degradation product C3d with CR2 on B cells and
follicular dendritic cells (reviewed in Carroll & Isenman,
2012). In addition, complement activation products influence T cell immunity via stimulation of antigen presenting cells (APCs) and T cells (for a complete overview see
Kemper & Atkinson, 2007; Dunkelberger & Song, 2010).
Direct stress: the Membrane Attack Complex
Direct stress of complement to bacterial cells is mainly
exerted through formation of the MAC that kills Gramnegative bacteria. Although the fluid-phase anaphylatoxin
C3a and its derivative C3ades-Arg were also reported to
exhibit direct antibacterial activity (Nordahl et al., 2004),
we will focus on the molecular mechanisms of MAC
assembly and lysis of bacteria.
Assembly
The MAC is a multiprotein complex that comprised single copies of C5b, C6, C7, and C8 together with 12–18
copies of C9 (Podack et al., 1980; M€
uller-Eberhard,
1985). MAC formation is initiated by the cleavage of C5
into C5b by the C5 convertase (Fig. 2a). In contrast to C3
and C4, C5 does not hold a reactive thioester for covalent
linkage to the bacterial surface (Cooper & M€
uller-Eberhard, 1970). Newly formed C5b has an unstable binding
site with a half-life of c. 2.5 min (Cooper & M€
uller-Eberhard, 1970) and needs to react with C6 to form the stable
C5b6 complex (Fig. 3a) (DiScipio et al., 1983). Subsequent binding of C7 renders the complex lipophilic and
mediates binding to the target membrane (Preissner et al.,
1985). Initial membrane insertion is mediated by the
binding of C8 to the C5b-7 complex (Monahan & Sodetz,
1981; M€
uller-Eberhard, 1986; Stewart et al., 1987) with
the C8 domain inserting into the lipid bilayer (Steckel
et al., 1983). The C5b-8 complex then facilitates recruitment of C9 and catalyzes its polymerization into a
pore-forming ring structure that inserts into the target
membrane (Fig. 3a) (Podack & Tschopp, 1982b; Tschopp,
1984; M€
uller-Eberhard, 1985; Tschopp et al., 1985).
Structure and pore-formation
The MAC is a SDS-stable ring-structure (Podack &
Tschopp, 1982a) with an inner diameter of c. 100 A, an
outer diameter c. 200 A, and a length of c. 160 A
(Podack & Tschopp, 1982b; DiScipio & Hugli, 1985;
M€
uller-Eberhard, 1985). Components C6-C9 are all
FEMS Microbiol Rev 38 (2014) 1146–1171
Molecular interplay of complement, coagulation and bacteria
homologous and share a 40 kDa Membrane Attack Complex Perforin (MACPF) domain (Tschopp et al., 1986;
Rosado et al., 2008), which is flanked by different regulatory domains at the C- and N-termini (Esser, 1994). This
MACPF domain, which is essential for membrane insertion of C8a, C8b, and C9 (Steckel et al., 1983), is also
found in perforin (Tschopp et al., 1986), a cytolytic
protein released by cytotoxic T lymphocytes or natural
killer cells to form pores in target cells (e.g. virus-infected
host cells) (Bolitho et al., 2007). The structures of C8 and
C8a have recently provided new insights into formation
and composition of the MAC pore (Hadders et al., 2007;
Lovelace et al., 2011). Since the MACPF domain of C8a
is structurally similar to bacterial cholesterol-dependent
cytolysins (CDCs), MACPF proteins are believed to use a
similar mechanism for membrane insertion as CDC. Like
CDC monomers, MACPF proteins contain domains that
refold from soluble into transmembrane b-hairpins
(TMH) that oligomerize to form a circular pore (Tweten,
2005; Hadders et al., 2007; Lovelace et al., 2011). In the
MAC, C9 is the main component making up the pore
although the other MAC proteins insert partially in the
membrane with C8 as the predominant protein (Hu
et al., 1981; Podack et al., 1981; Steckel et al., 1983). Not
all MACPF proteins function as lytic toxins: molecules
like C6 and the b domain of C8 do not insert into membranes to form lytic pores (Rosado et al., 2008), they
rather have a supporting role in formation of polymeric
C9 within the MAC.
Bactericidal mechanisms of the MAC
Although the mode of assembly and structural organization of the MAC are well characterized, the exact mechanism by which the MAC kills bacterial cells remains
poorly understood. The MAC is reported to kill Gramnegative bacteria while Gram-positive bacteria are resistant (Joiner et al., 1984). However, the most commonly
used model systems for MAC lysis are erythrocyte membranes and artificial lipid bilayers. These single-membrane
‘cells’ are much more sensitive to lysis by pore-forming
proteins than Gram-negative bacteria that possess two
membranes separated by a periplasmic space containing
peptidoglycan. Furthermore, bacteria also have membrane
repair mechanisms (Garcıa et al., 2006). Below, we discuss the proposed bactericidal mechanisms of the MAC
and evaluate these hypothetical models in light of the
makeup of a Gram-negative cell.
Molecular composition of the bactericidal MAC
While the complete ring-structured C5b-9 complex is
generally considered the active ‘killer’ component, many
FEMS Microbiol Rev 38 (2014) 1146–1171
1151
reports have shown that the premature C5b-8 complex
also has membranolytic activity. Although there is no
direct evidence for C5b-8 to assemble into a pore, this
complex can cause leakage of synthetic lipid vesicles
(Zalman & Muller-Eberhard, 1990). Also in lipid bilayers
and erythrocytes, C5b-8 can increase ion conductance
(Shiver et al., 1991) or cause cell lysis, respectively
(Tamura et al., 1972). However, the C5b-8 complex does
not efficiently kill Gram-negative bacteria, again underlining the difference between lysis of bacteria vs. erythrocytes and lipid bilayers. Interestingly, although C9 is
absolutely required for bacterial killing, its polymerization
into a SDS-stable ring does not seem essential [observed
for both E. coli (Joiner et al., 1985) and S. typhimurium
(Joiner et al., 1989)].
Hypothetical models for MAC insertion into the
Gram-negative cell envelope
Since damage to the OM alone is unlikely to cause bacterial death (Wright & Levine, 1981a; Tomlinson et al.,
1990), the MAC should be able to disturb the IM as well.
Currently, a few hypotheses exist on how the MAC gains
access to the IM (Fig. 3b). With recent information on
the exact dimensions of the MAC (Aleshin et al., 2012;
Hadders et al., 2012), we can now further contemplate
the likelihood of the proposed events. Both the inner and
outer membranes of Gram-negatives are c. 75 A thick
and they are separated by a c. 100 A periplasmic space,
creating a total cell envelope of c. 250 A (DiRienzo et al.,
1978). The first hypothesis considers that one C5b-9
complex would span both membranes (transmural pore)
(Born & Bhakdi, 1986). However, with its length of
c. 160 A, it is physically impossible for the MAC to cross
the entire cell envelope of Gram-negative bacteria. In
addition, only the lower part of the MAC (containing the
hydrophobic TMHs) is believed to insert the membrane,
making it even less likely for the MAC to span the entire
cell envelope. Bayer et al. proposed that the Gram-negative cell wall contains regions, Bayer’s junctions, where
the IM and OM are in close contact (Bayer, 1968), creating hotspots for MAC perturbation (Fig. 3b, Model A)
(Wright & Levine, 1981a,b). Although today the existence
of these sites remains controversial (Kellenberger, 1990;
Ruiz et al., 2006), they have been proposed as sites where
the MAC inserts into the cell envelope. Wright and
Levine postulated simultaneous disturbance of the inner
and outer membrane by the MAC at these junctions
(Wright & Levine, 1981a,b). The second hypothesis
proposes that the MAC disrupts the OM but that the
lethal activity is executed at the IM (Wright & Levine,
1981b; Dankert & Esser, 1987; Esser, 1994) by allowing
access of MAC or other membrane-damaging proteins to
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1152
the periplasmic space and eventually the IM (Fig. 3b,
Model B). Such a mechanism would require these components to pass through the PG layer that covers the
inner membrane. It was previously believed that the natural pores in the PG matrix were c. 2 nm, allowing
proteins of maximum c. 25 kDa to diffuse (Demchick &
Koch, 1996). However, a recent study indicated the presence of PG pore sizes in the range of 10 nm in E. coli
(Turner et al., 2013) allowing diffusion of proteins up to
c. 64 kDa. Still, the MAC proteins are all larger (C5b
c. 179 kDa, C6 c. 102 kDa, C7 c. 97 kDa, C8 c. 150 kDa,
and C9 c. 73 kDa), making it plausible that PG degrading
enzymes are essential for these components to reach the
IM. In analogy, lysis of E. coli via bacteriophage holins
can also exclusively occur in the presence of phage endolysins that break the PG layer (Catal~ao et al., 2013). Many
studies were performed analyzing the role of lysozyme
(muramidase), an enzyme abundantly present in body
fluids including the serum, in the bactericidal reaction of
the MAC (reviewed in Taylor, 1983). Initial damage of
the OM might allow access of serum-derived lysozyme to
the periplasmic space where it can degrade the peptidoglycan layer (Wright & Levine, 1981a). Lysozyme was
found to accelerate the bacteriolytic effects of the MAC,
although blocking of lysozyme did not abrogate the bactericidal process completely indicating that the enzyme is
not absolutely required for MAC-mediated killing of
bacteria (Martinez & Carroll, 1980). Even though the C9
molecule without C5b-8 has no toxic effect on bacteria,
studies introducing C9 in the periplasm of Gram-negative
bacteria showed that the C9 molecule can be lethal by
itself when it has access to the inner membrane (Dankert
& Esser, 1987; Wang et al., 2000). This hypothesis was
strengthened by experiments in which plasmid-derived
full-length C9, targeted to the periplasm, led to killing of
Gram-negative bacteria whereas C9 expression in the
cytoplasm did not have this effect (Wang et al., 2000). It
is not known whether C9 causes lysis of the inner membrane in its native conformation. The authors considered
the possibility that C9 might be converted into a more
toxic form by enzymes present in the periplasmic space
(Dankert & Esser, 1986; Wang et al., 2000).
E.T.M. Berends et al.
the presence of an uncoupler of oxidative phosphorylation
completely prevented MAC-mediated killing (Taylor &
Kroll, 1983). In line with this, another hypothesis that has
not been suggested before is that upon MAC formation,
the bacteria might activate a self-death pathway like
previously described for mammalian peptidoglycan recognition proteins (PGRPs) (Kashyap et al., 2011).
The MAC on Gram-positive bacteria
For long it has been known that Gram-positive bacteria
are protected from killing by the MAC, supposedly due to
their thick PG layer that prevents MAC insertion into the
membrane (Joiner et al., 1984). Intriguingly, we recently
found that the MAC deposits on several Gram-positive
bacteria as SDS-stable polymeric C9 structures (Berends
et al., 2013). To our surprise, these complexes were
located at specific regions on the bacterial cell. For
instance, on Streptococcus pyogenes (group A streptococcus, GAS), C5b-9 deposited near the division septum,
whereas on Bacillus subtilis the complex was located at the
poles (Berends et al., 2013). This specific C5b-9 deposition
did not affect bacterial viability. Although this remains to
be elucidated, it might indicate that the MAC has a nonclassical role in immune defense against these microbes.
Bacteria under stress by coagulation
The coagulation cascade can be activated via different
pathways that all converge in a common pathway for clot
formation (Sun, 2006). Two pathways can trigger coagulation: the contact system (intrinsic pathway) and the tissue factor (extrinsic) pathway. The tissue factor pathway
responds to blood vessel damage via exposure of tissue
factor, a subendothelial protein that is normally shielded
from contact with blood components. Since this pathway
plays no clear role in host defenses, it will not be further
discussed here. Microbe-specific activation of coagulation
was suggested to occur via the FXII-dependent contactdriven pathway, which results in formation of the
pro-inflammatory peptide Bradykinin (BK) (Fig. 4a) and
clotting via activation of factor XI (Fig. 4b).
The lethal event
Contact activation on bacteria
Following pore-formation, it is believed that bacteria are
killed due to reduction of the membrane potential (Wright
& Levine, 1981a), analogous to pore-forming colicins, a
group of toxins secreted by some strains of E. coli (Wright
& Levine, 1981a; Cramer et al., 1995; Cascales et al.,
2007). Alternatively, bacteria might participate actively in
their loss of viability by metabolic events (Kroll et al.,
1983). Incubation of bacteria with active complement in
The contact system is activated upon binding of FXII to
negatively charged structures, as was demonstrated in
coagulation assays using dextran sulfate or silica (Citarella
et al., 1997; Yoshida et al., 2013). Since these structures
are abundantly present on bacteria, FXII can bind to
bacterial surfaces (Ben Nasr et al., 1996; Herwald et al.,
1998). Target-binding of FXII results in low-level autoactivation into FXIIa, which then converts prekallikrein
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Rev 38 (2014) 1146–1171
1153
Molecular interplay of complement, coagulation and bacteria
(a)
PK
HK
BK
K
HK
FXIIa
FXII
K
HKa
Amplification
`C
(b)
FX
FIX
FXI
PT
HK
FIXa
FXIa
Thrombin
HK
FXIIa
FXII
FXa
(c)
Fibrinogen
Thrombin
Fibrinopeptides
Fibrin monomer
Fibrin dimer
Fibrin polymerization
FXIII
Clot stabilization
Fig. 4. Activation of coagulation on bacteria. The contact activation pathway is initiated by the binding of FXII to negatively charged bacterial
structures. FXII is then auto-activated into FXIIa, which either triggers the kallikrein-kinin system (a) or the clotting cascade (b). (a) FXIIa converts
PK, which circulates in complex with HK, into active kallikrein. Kallikrein cleaves HK to release BK and activates FXII to amplify the contact
system. (b) FXIIa cleaves FXI, which also circulates in complex with HK. FXIa subsequently converts FIX into FIXa. FIXa will then trigger the
common pathway of coagulation by converting FX into FXa, which leads to conversion of prothrombin into thrombin that cleaves fibrinogen.
(c) Formation of fibrin clots. Activated thrombin cleaves fibrinogen into fibrin monomers that are capable of interacting with adjacent fibrin
molecules and thereby form long fibers and finally a fibrin clot. This clot can be stabilized by FXIII or thrombin-activated FXIIIa.
FEMS Microbiol Rev 38 (2014) 1146–1171
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
1154
(PK) into active plasma kallikrein (Fig. 4a) (Wuepper &
Cochrane, 1972; Samuel et al., 1992; Citarella et al.,
1997). Kallikrein can cleave the associated high molecular
weight kininogen (HK) to release the pro-inflammatory
peptide bradykinin (BK). Furthermore, kallikrein amplifies the contact system by cleavage of FXII (Schmaier &
McCrae, 2007). At least 75–90% of PK circulates in an
equimolar complex with HK (Mandle et al., 1976; Scott
& Colman, 1980), which is important for effective cleavage of PK by FXIIa (Colman & Schmaier, 1997). The
activated form of HK (HKa) has an increased capacity to
bind negatively charged surfaces and provides a surface
for further activation of the cascade (Scott & Silver, 1984;
Reddigari & Kaplan, 1988). Escherichia coli curli and
S. typhimurium fimbriae can activate the contact system
due to interactions with FXII and HK. All contact factors
were assembled on these fibrous structures and mutant
strains lacking either curli or fimbriae did not activate the
cascade (Ben Nasr et al., 1996; Herwald et al., 1998). Also
LPS was described to provide a surface for contact activation by binding to domain 5 in HK, which is responsible
for binding to negatively charged surfaces (Perkins et al.,
2008). For S. aureus it was proposed that negatively
charged teichoic acids induce contact activation (Kalter
et al., 1983; Mattsson et al., 2001). Apart from surfaceassociated molecules, fluid-phase bacterial products can
also initiate the contact system. For instance, LPS is
released from the bacterial surface during cell growth and
can then activate the contact system by binding HK in
solution (Perkins et al., 2008). Also, it has been described
that excreted bacterial polyphosphates can trigger coagulation through the contact pathway by binding to FXII
and to a lesser extent to HK (Smith et al., 2006), depending on the length of the polymer chains (Smith et al.,
2010). In a murine infection model, it was shown that
bacterial polyP induces vascular leakage through release
of BK in a FXII-dependent manner (M€
uller et al., 2009).
Next to its role in cleavage of PK and HK, contact activation also results in clotting (Fig. 4b). Surface-activated
FXIIa can also cleave coagulation factor XI, a homolog of
PK that circulates in complex with HK as well. As
explained in Fig. 4b, activated FXI (FXIa) triggers the
common pathway of coagulation in which subsequent
activation of FIX, FX, and prothrombin results in thrombin-catalyzed cleavage of fibrinogen (Fig. 4b and c).
Fibrinogen is a highly abundant (4 mg mL 1) and large
plasma protein (340 kD) that consists of two sets of three
polypeptide chains (Aa, Bb, and Cc) organized in three
distinct domains: two outer D-domains and a central
E-domain connected by coiled coil segments (Kollman
et al., 2009). Thrombin drives fibrin formation by cleavage of peptide bonds in both the N-terminal Aa chain
and the N-terminal Bb-chain that are located in the
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
E.T.M. Berends et al.
E-domain (Fig. 4c) (Blomb€ack et al., 1978). Cleavage of
the Aa-chain results in release of fibrinopeptide A and
exposure of a polymerization site known as EA. This
newly exposed EA-site subsequently interacts with the
constitutively exposed Da-site on the b-chain segment of
the D-domain of adjacent fibrin(ogen) molecules, leading
to formation of a staggered overlapping double-stranded
fibrin fibril. Cleavage of the Bb-chain by thrombin and
the associated release of fibrinopeptide B is much slower
and leads to exposure of the EB polymerization site that
interacts with constitutive Db-sites on the b-chain region
of the D-domain. This interaction induces a conformational change in the D-domain that allows lateral association of fibrin fibers (Yang et al., 2000). Fibrin fibers are
subsequently stabilized by factor XIII or thrombinactivated FXIIIa (Siebenlist et al., 2001), a transglutaminase that introduces covalent intermolecular bonds
between C-terminal lysine and glutamine residues of adjacent c-chains (Chen & Doolittle, 1971). Recently, it was
shown that contact activation by bacterial fimbriae and
curli indeed results in cleavage of fibrinogen and subsequent fibrin formation (Persson et al., 2003).
The contact system: an innate recognition
pathway?
Due to its redundant function for efficient hemostasis,
the contact system has been proposed to be part of the
innate immune system. Interestingly, the recognition
mechanisms of the contact system seem less specific than
those of the complement system and innate immunity in
general. In order to respond to a large group of microbes,
the innate immune system uses a variety of proteins that
recognize evolutionarily conserved structures on non-self
surfaces. For example, toll-like receptor 4 (TLR4) recognizes Gram-negative bacteria by binding to LPS, the
formylated peptide receptor (FPR) responds to all living
bacteria by sensing formylated peptides (exclusively produced by bacteria) (Bardoel & Strijp, 2011) and the complement system uses a variety of lectins to bind conserved
bacterial sugars or teichoic acids. In contrast, the contact
system is not activated by conserved bacterial structures
but recognizes bacteria via their negatively charged surface. Still there appears to be some specificity since contact activation does not normally occur in the circulation
and seems to be limited to foreign surfaces or human
cells in a procoagulant state.
Host defense functions of coagulation on
bacteria
The coagulation system is recognized to be important
to the immune response of invertebrates. Several
FEMS Microbiol Rev 38 (2014) 1146–1171
1155
Molecular interplay of complement, coagulation and bacteria
invertebrate species contain a coagulation cascade that
senses invading microorganisms and neutralizes them via
clotting (Osaki & Kawabata, 2004). Horseshoe crab clotting factors are stored in the granules of hemocytes
(phagocytic cells) that are released upon sensing bacterial
components like LPS. This triggers a proteolytic cascade
that eventually leads to cleavage and polymerization of
the clottable protein coagulogen that embeds bacteria in
insoluble aggregates (Osaki & Kawabata, 2004). In
addition to clottable proteins, the immune system of
invertebrates uses transglutaminases that are highly
homologous to FXIII to stabilize clots containing bacteria.
In Drosophila melanogaster, a lack of transglutaminases
led to an immune defect since microbes were no longer
entrapped in clots (Wang et al., 2010).
In contrast to invertebrates, the mammalian coagulation system was long considered to be exclusively
important for hemostasis. However, several studies now
indicate that coagulation factors also contribute to the
effective elimination of bacteria in mammals. Activation
of coagulation on bacteria triggers multiple biological
effects important for host defense. While some of these
effectors induce direct stress on the bacteria, others
support the cellular immune response. Before we zoom in
to the direct antimicrobial effects of coagulation, we will
briefly summarize the indirect immune functions of
coagulation. One important consequence of coagulation
at the site of infection is the recruitment of innate
immune cells via BK that binds kinin receptors (B1R and
B2R) on endothelial, smooth muscle, and innate immune
cells (Frick et al., 2007). BK induces vascular leakage and
interacts with macrophages to release chemo-attractants.
Furthermore, a number of immune cells express proteaseactivated receptors (PARs) that are cleaved by coagulation
proteases (thrombin and FXa) to initiate inflammation
(Shpacovitch et al., 2008). Finally, activation of fibrinogen
is accompanied by the release of the fibrinopeptides A
and B, which are also chemo-attractants (Skogen et al.,
1988).
Fibrin formation
The best-defined direct action of coagulation on bacteria
is the formation of fibrin at the site of infection. When
bacteria become immobilized inside the fibrin network,
they are prevented from spreading into the surrounding
tissues. It has been known for decades that this local
entrapment reduces bacterial dissemination into the
bloodstream (Rotstein, 1992). Mice deficient in fibrinogen
are therefore more susceptible to infections with GAS due
to decreased fibrin production (Sun et al., 2009). For
some bacteria like S. aureus and E. coli, it was found that
this immobilization inside a fibrin clot is FXIII dependent
FEMS Microbiol Rev 38 (2014) 1146–1171
(Wang et al., 2010) and that bacteria are directly attached
to the fibrin fibers via multiple binding sites cross-linked
by FXIII. In the absence of FXIII, they appear to be
loosely assembled within the clot without direct interaction with the fibers. Furthermore, it has been described
that FXIII triggers entrapment of GAS at the site of infection, which subsequently leads to killing of the bacteria
due to generation of plasma-derived antimicrobial peptides (Loof et al., 2011). In a murine skin infection
model, these bacteria were also shown to cluster within
fibrin networks in the presence of FXIII, whereas in the
absence of FXIII they are distributed throughout the
infection site. This leads to a higher influx of neutrophils
and thereby increased inflammation. Furthermore, a systemic response, and therefore bacterial dissemination, was
induced when FXIII was absent (Loof et al., 2011).
Furthermore, fibrin polymerization mediates clearance of
E. coli and S. aureus from the peritoneal cavity and limits
the growth of Listeria monocytogenes in the liver
(Ahrenholz & Simmons, 1980; Flick et al., 2004; Mullarky
et al., 2005).
Antimicrobial peptides
Another direct action of coagulation in host defense
against bacteria is the production of antimicrobial peptides (AMPs). First of all, BK itself was found to exhibit
antimicrobial activity in vitro (Kowalska et al., 2002)
although it has been debated if this is relevant in vivo
(Frick et al., 2006). Also, coagulation proteins are sensitive to cleavage by neutrophil-derived proteases resulting
in peptides with antimicrobial activity (Nordahl et al.,
2005). For instance, thrombin-derived C-terminal peptides (TCP) were produced when fibrin clots or human
plasma was incubated with neutrophil elastase, which
could induce lysis of microbial membranes (Papareddy
et al., 2010). TCPs were found to be antibacterial against
the Gram-positive S. aureus, as well as to Gram-negative
species P. aeruginosa and E. coli. Membrane permeabilization by the TCP GKY25 was confirmed by electron
microscopy (Papareddy et al., 2010). Although it was
shown that GKY25 acts on the bacterial membrane, the
precise mechanism remains to be elucidated. HKH20, a
peptide derived from domain 5 of HK, also displays antibacterial activity against both Gram-positive (S. aureus
and Enterococcus faecalis) and -negative species (E. coli
and P. aeruginosa) (Nordahl et al., 2005; Ringstad et al.,
2007). Antibacterial activity of this peptide and its
truncated variants was correlated to the ability to cause
leakage in model lipid bilayers, indicating that disruption
of membrane integrity is the mechanism that leads to
HKH20-mediated bacterial killing (Ringstad et al., 2007).
The effect of HKH20 is similar to that of the HK domain
ª 2014 Federation of European Microbiological Societies.
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1156
3 derived peptide, NAT26. This peptide kills bacteria that
activate the contact system, GAS AP1, S. aureus Cowan I,
and S. typhimurium SR11B (Frick et al., 2006) and their
cell walls were disintegrated when analyzed by electron
microscopy. Furthermore, also this peptide causes leakage
of anionic liposomes by disrupting the membrane (Frick
et al., 2006). Finally, the fibrinogen-derived peptide
GHR28 also exhibits antibacterial activity against the
Gram-positive species Streptococcus agalactiae (GBS) and
S. aureus, but not against E. faecalis and the Gram-negative bacterium E. coli (P
ahlman et al., 2013).
Crosstalk between complement and
coagulation
There is a significant amount of crosstalk between the
complement and coagulation systems and this has been
reviewed in a number of excellent papers (Markiewski
et al., 2007; Oikonomopoulou et al., 2012). Here, we
highlight a few of these interactions that, in our opinion,
are of special interest in light of host defenses against
bacterial infections. First, it was reported that several
complement proteases activate the final stage of coagulation to form fibrin clots. A major role has been suggested
for the MASPs, which circulate in complex with MBL or
ficolins and are thus activated on bacterial surfaces via
pattern recognition. Next to the role of MASP-2 in activation of complement, this protease was also found to
cleave prothrombin into thrombin thereby promoting
fibrin clot formation (Krarup et al., 2007). Although the
prothrombin turnover rate was only 5% compared to the
cleavage by FXa, activation occurred at physiological concentrations of prothrombin, which seemed specific since
MASP homologues did not mediate this cleavage. Furthermore, MBL-MASP complexes bound to S. aureus
could generate fibrin that was covalently linked to the
bacterial surface via FXIII. This suggests that MBL-MASP
may lead to localized fibrin formation. In addition to the
action of MASP-2 on coagulation, MASP-1 is also
involved in clotting. MASP-1 has thrombin-like activity
and activates FXIII and fibrinogen to release fibrinopeptides and to induce clot formation (Krarup et al., 2008).
Although it is tempting to speculate that the two MASPs
might act together to cause localized formation of fibrin,
a study comparing both actions indicated that clotting via
MBL/ficolin-MASP is predominantly catalyzed by
MASP-2 cleavage of prothrombin (Gulla et al., 2010).
MASP-1 and MASP-2 can also cleave HK, but release of
BK is only shown for MASP-1 (Dob
o et al., 2011).
Although the physiological importance of MASPs in coagulation clearly needs further investigations, the fact that
these proteases are targeted to bacterial surfaces via pattern recognition molecules might trigger a more specific
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
E.T.M. Berends et al.
and localized clotting reaction compared to ‘contact’
activation.
Vice versa, coagulation proteases can activate the complement system. A number of coagulation proteases, such
as thrombin, FXa, FIXa, and FXIa, directly cleave the central complement components C3 and C5 into their bioactive fragments (Huber-Lang et al., 2006; Amara et al.,
2010). In addition, it has been shown that the CP can be
directly initiated by activation of C1r by FXIIa (Ghebrehiwet et al., 1981; Kaplan & Ghebrehiwet, 2010) and that
kallikrein has Factor-D-like activity since it converts Factor B to form the AP convertase (Hiemstra et al., 1985).
Similar to MASP-mediated coagulation activation, the relevance of these findings in vivo is presently unknown.
However, it is very intriguing that some of these cleavage
reactions generate alternate complement activation products that have different biological activities. In particular,
thrombin was reported to cleave both the MAC components C5 and C9 into alternative products. First, thrombin cleaves C9 into a 37 kDa C9b molecule (Biesecker
et al., 1982) that theoretically can cross the peptidoglycan
layer. In contrast to native C9, this carboxyl-terminal
fragment of C9 is able to disturb the membrane potential
of membrane vesicles in absence of C5b-8 (Dankert & Esser, 1986). Second, thrombin efficiently cleaves C5 at a
different cleavage site than the C5 convertase, generating
C5T (Krisinger et al., 2012). This cleavage does not
directly lead to release of C5a or C5b, but after cleavage
of C5T by the complement C5 convertase, a different C5b
molecule was formed that seemed to form a more potent
MAC than the conventional C5b-9 complex.
Overall, the crosstalk between complement and coagulation cascades may enhance innate immunity by generating more specific, localized and effective antibacterial
reactions.
Complement and coagulation in
bacterial disease
Whereas complement and coagulation are evolved to act
locally at the site of infection and prevent bacterial dissemination, both systems are often pathological during
systemic infections. The Systemic Inflammatory Response
Syndrome (SIRS), a hallmark of sepsis, is characterized by
excessive activation of complement and coagulation (De
Jong et al., 2010). Septic patients have high plasma levels
of complement activation products (C3a, C4a and C5a)
of which especially C5a is believed to cause an overwhelming inflammatory response that plays a role in the
outcome of sepsis. Also coagulation markers like soluble
tissue factor and thrombin-antithrombin complexes
(TATc) are strongly increased while endogenous inhibitors of coagulation and the fibrinolytic system are
FEMS Microbiol Rev 38 (2014) 1146–1171
1157
Molecular interplay of complement, coagulation and bacteria
consumed (van der Poll & Opal, 2008). In severe sepsis,
coagulation causes disseminated intravascular coagulation
(DIC), a serious condition in which microvascular
thrombosis, bleeding and microvascular fibrin deposition
lead to multiple organ failure, the most common cause of
death. Whereas pathogens can directly activate the coagulation system through the contact system, thrombin
generation in DIC is mediated by the extrinsic pathway
of coagulation (Taylor et al., 1991; Pixley et al., 1993;
Levi et al., 1994). The systemic inflammatory response
associated with sepsis disturbs the hemostatic balance,
with increased levels of IL-6 leading to tissue factor
expression on vascular cells (van der Poll et al., 1994)
and release of TNFa leading to impaired fibrinolysis (van
der Poll et al., 1991). Several experimental studies are
now exploring whether therapeutic intervention with
complement and coagulation can reduce the mortality of
sepsis. Blocking C5a with antibody treatment reduced
mortality of experimental models of Gram-negative sepsis
(reviewed in Guo & Ward, 2005). Also in a baboon
model of E. coli sepsis, complement inhibition at the level
of C3 prevented organ damage and pathological coagulation (Silasi-Mansat et al., 2010). Still, sepsis is a complex
disease and not all forms of sepsis are the same. For
instance, whereas C5a has a negative role in Gram-negative sepsis, it seems essential for the immune response
during S. aureus sepsis (Von K€
ockritz-Blickwede et al.,
2010). In analogy, inhibition of coagulation via natural
anticoagulants has led to contrasting results in the treatment of sepsis (Esmon, 2004). Thus, even though
knowledge of complement and coagulation provides new
avenues for the treatment of sepsis, more insights seem
required for the pathophysiology of sepsis caused by
different bacteria.
How bacterial pathogens deal with
stress invoked by complement and
coagulation
Since complement and coagulation cause stress to bacterial cells, bacterial pathogens have developed strategies to
counteract these pathways. For the complement system,
inhibition can occur at almost every level of the cascade.
For example, S. aureus secretes different proteins that
block C1q binding (Kang et al., 2013), convertase activity
(Rooijakkers et al., 2005; Jongerius et al., 2007), and C5
cleavage (Langley et al., 2005; Bestebroer et al., 2010).
Since these mechanisms have been subject of many excellent reviews (Rautemaa & Meri, 1999; Lambris et al.,
2008; Zipfel et al., 2013) and ‘direct’ complement actions
are the focus of this review, we will here exclusively
discuss strategies evolved by Gram-negative bacteria that
are aimed at inhibiting the MAC (Table 1). Furthermore,
FEMS Microbiol Rev 38 (2014) 1146–1171
we will discuss how bacterial pathogens block coagulation
or degrade fibrin clots and produce virulence factors to
specifically induce coagulation (Table 2).
Bacterial evasion of the MAC
Surface modification
A long known mechanism by which Gram-negative bacteria resist MAC-mediated killing is by modification of the
LPS molecules. LPS forms a physical and chemical barrier
around the OM and is build up of three regions; the lipid
moiety in the outer leaflet of the OM called lipid A; the
core oligosaccharide containing 10–12 sugars; and on the
exterior a polysaccharide chain of repeating units, the
O-chain. Bacteria lacking the O-chain grow in roughlooking colonies, therefore termed ‘rough’ while wild-type
species are termed ‘smooth’ (reviewed in Caroff et al.,
2002). It has been recognized for decades that the
O-chain is involved in complement resistance since
smooth-type bacteria are usually serum-resistant whereas
the rough bacteria are serum-sensitive. The O-chain does
not prevent complement activation or C5 cleavage but
probably sheds the MAC from the OM and/or sterically
hinders its insertion into the OM (Joiner et al., 1982a,b).
For several bacterial species, it was shown that bacteria
with longer O-chains are better protected against the
MAC. Furthermore, a higher density of O-chains
increases MAC resistance (Grossman et al., 1987; Merino
et al., 1992; Hong & Payne, 1997; Murray et al., 2005).
LPS expressed by N. meningitidis, but also N. gonorrhoeae
and Haemophilus influenzae, lack the O-specific sidechain. Alternatively, most disease-associated strains of
N. meningitidis produce two distinct short carbohydrate
chains, in which a lacto-N-neotetraose (LNT) moiety is
present. The LNT epitope serves as the major site for
sialylation that reduces immunogenicity and is associated
with increased serum-resistance (Estabrook et al., 1997).
However, the importance of sialylation remains controversial, indicating that this strategy is of minor importance for MAC evasion by meningococci (Schneider
et al., 2007).
Another bacterial mechanism to evade MAC-dependent
killing is formation of a capsule. Although a bacterial
capsule can prevent complement activation in general, we
will solely discuss how a capsule blocks the MAC specifically. Neisseria meningitidis expresses an (a2?8)-linked
polysaccharide capsule that also contains sialic acids
(Kahler et al., 1998). In most serotypes this capsule did
not affect C3b deposition (Vogel et al., 1997), but specifically prevented MAC insertion in the OM (Schneider
et al., 2007). Recently it was reported that capsule
expression in meningococci is enhanced by increased
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1158
E.T.M. Berends et al.
Table 1. MAC evasion by Gram-negative bacteria
Microorganism
Microbial structure
Category
Function
Reference
Borrelia burgdorferi
CD59-like
CRASP-3, -4, -5
CRASP-1
Inhibition of MAC assembly
Binding of CFHR1
Blocks MAC assembly
Pausa et al. (2003)
Haupt et al. (2007)
€m et al. (2013)
Hallstro
Borrelia garinii
Escherichia coli
CRASPs
?
Mimick or attract host regulators
Mimick or attract host regulators
Expression of inhibitory surface
proteins
Mimick or attract host regulators
Mimick or attract host regulators
Binding of CFHR1
Capture soluble CD59 to inhibit
MAC assembly
Inhibition of MAC assembly
Van Burgel et al. (2010)
Rautemaa et al. (1998)
Binding of vitronectin
Binding of vitronectin
Capture soluble CD59 to inhibit
MAC assembly
Binding of vitronectin
Protects the OM from MAC
assembly to mediates
serum-resistance
Binding of vitronectin
Leduc et al. (2009)
€m et al. (2006)
Hallstro
Rautemaa et al. (2001)
TraT
Haemophilus ducreyi
Haemophilus influenza
Helicobacter pylori
DrsA
Hsf
?
Expression of inhibitory surface
proteins
Mimick or attract host regulators
Mimick or attract host regulators
Mimick or attract host regulators
Klebsiella pneumoniae
?
O-chain variation
Mimick or attract host regulators
Surface modification
Moraxella catarrhalis
UspA2
Mimick or attract host regulators
Neisseria gonorrhoeae
Neisseria meningitidis
OpaA
Msf
Opc
NhhA
LNT sialylation
Mimick or attract host regulators
Mimick or attract host regulators
Mimick or attract host regulators
Expression of inhibitory surface
proteins
Surface modification
Capsule expression
Surface modification
Porphyromonas
gingivalis
Rickettsia conorii
Salmonella minnesota
APS layer
Surface modification
OmpB
O-chain variation
Mimick or attract host regulators
Surface modification
Salmonella montevideo
O-chain variation
Surface modification
Salmonella
typhimurium
O-chain variation
Surface modification
Rck
Expression of inhibitory surface
proteins
Surface modification
Shigella flexneri
O-chain variation
Yersinia enterocolitica
YadA
Ail
Expression of inhibitory surface
proteins
Expression of inhibitory surface
proteins
temperature, which occurs for instance during inflammation when the bacteria need to interfere with immune
killing (Loh et al., 2013). Also the outer surface of Porphyromonas gingivalis bears a glycan layer containing
anionic polysaccharides (APS) that mediate direct resistance against the MAC. This APS did not inhibit MAC
deposition on the bacteria (Slaney et al., 2006) but probably prevents the MAC from inserting into the OM.
ª 2014 Federation of European Microbiological Societies.
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Binding of vitronectin
Binding of vitronectin
Binding of vitronectin
Blocks MAC deposition
Protects from serum-mediated
killing
Protects the OM from MAC
insertion
Protects the OM from MAC
insertion
Binding of vitronectin
Protects the OM from MAC
insertion and mediates
serum-resistance
Protects against MAC-mediated
killing
Protects against MAC-mediated
killing
Blocks C9 polymerization
Protects against MAC-mediated
killing
Inhibits C9 binding, thereby
MAC formation
Serum-resistance, potentially
blocks C9 polymerization
Pramoonjago et al. (1992)
Ringn
er et al. (1992)
Merino et al. (1992)
Attia et al. (2006) and
Singh et al. (2010a,b)
Duensing & Putten (1998)
Griffiths et al. (2011)
Sa E Cunha et al. (2010)
€linder et al. (2008)
Sjo
Estabrook et al. (1997)
Schneider et al. (2007)
Slaney et al. (2006)
Riley et al. (2013)
Joiner et al. (1982a,b)
Grossman et al. (1987)
Murray et al. (2005)
Heffernan et al. (1992)
Hong & Payne (1997)
Pilz et al. (1992)
Bliska & Falkow (1992)
Expression of inhibitory surface proteins
Gram-negative bacteria also express surface-localized
proteins to inhibit MAC formation. TraT is a plasmidencoded OM protein of E. coli K12 that mediates
MAC-resistance at the level of C5b6. TraT is believed to
block C5b6 assembly or structurally alter the complex
into an inactive form (Pramoonjago et al., 1992). The
FEMS Microbiol Rev 38 (2014) 1146–1171
1159
Molecular interplay of complement, coagulation and bacteria
Table 2. How bacterial pathogens modulate the coagulation system
Microorganism
Microbial structure
Target
Effect
Reference
Staphylococcus
aureus
Clumping factor
Coagulase
Fibrinogen
Prothrombin
and fibrinogen
Fibrinogen and C3b
Adherence to host tissue and bacterial aggregation
Activation of prothrombin to form fibrin clots
Fitzgerald et al. (2006)
Friedrich et al. (2003)
Inhibition of phagocytosis, neutrophil adherence to
fibrinogen and platelet aggregation
Activation and aggregation of platelets
Delay in clotting times
Activation of prothrombin and FXIII to form and
stabilize fibrin clots
Inhibition of complement deposition
Assembly and activation of contact system
Inhibition of fibrinolysis
Inhibition of HK binding to the bacterial surface
and inhibition of antimicrobial peptides of HK
Assembly of contact system and release of BK
Ko et al. (2011, 2013) and
Shannon & Flock (2004)
Fitzgerald et al. (2006)
Itoh et al. (2013)
Cheng et al. (2010) and
Thomer et al. (2013)
Smeesters et al. (2010)
Efb
FnbpA
SSL10
vWbp
Group A
streptococci
Group G
streptococci
Salmonella
typhimurium
Escherichia coli
Gram negative
bacteria
Various bacterial
species
M protein
Fibrinogen
Prothrombin
Prothrombin,
fibrinogen and FXIII
Fibrinogen and HK
SclA and SclB
SIC
TAFI
HK
FOG
HK
PG
HK
Fimbriae
LPS
FXII, HK and
fibrinogen
FXII, HK and
fibrinogen
HK
Activation of contact system
PolyP
FXII and HK
Activation of contact system and release of BK
FV
Acceleration of thrombin formation and clot
stabilization
Curli
Assembly of contact system
Inhibition of HK-derived antimicrobial peptides
Assembly and activation of contact system
Assembly and activation of contact system
OM protein NhhA of N. meningitidis mediates bacterial
colonization and was also recognized to be important for
complement resistance. NhhA prevents MAC deposition
to the bacterial surface although it was not determined at
what level in the complement cascade the protein has its
effect (Sj€
olinder et al., 2008). Borrelia burgdorferi, the
causative agent of Lyme disease, expresses the OM-localized complement regulator-acquiring surface proteins
(CRASP) family. The CRASP-1 (or CspA) protein was
recently identified to inhibit MAC attack by binding C7
and C9, thereby inhibiting assembly and membrane
insertion of the MAC (Hallstr€
om et al., 2013). Yersinia
enterocolitica virulence is associated with the expression of
an autotransporter protein in the OM, Yersinia adhesin A
(YadA). The protein mediates protection against
MAC-mediated killing by inhibiting C9 deposition on the
bacteria. However, it was not completely elucidated
whether inhibition of MAC assembly was not just a result
of inhibition at the level of C3 (Pilz et al., 1992). More
recently, binding of C3b or iC3b by YadA was described
whereby Factor H, normally promoting complement
decay on host cells, was attracted to the bacterial
surface to enhance bacterial survival (Schindler et al.,
2012). Another outer membrane protein expressed by
FEMS Microbiol Rev 38 (2014) 1146–1171
Bengtson et al. (2009)
Akesson et al. (2010)
Wollein Waldetoft
et al. (2012)
Wollein Waldetoft
et al. (2012)
Herwald et al. (1998)
Ben Nasr et al. (1996) and
Herwald et al. (1998)
Perkins et al. (2008)
€ller et al. (2009) and
Mu
Smith et al. (2006)
Morrissey et al. (2012)
Y. enterocolitica, Ail, also conferred complement resistance
when it was expressed in E. coli (Bliska & Falkow, 1992).
Potentially, Ail acts by inhibiting C5b-9 assembly. A
structurally and functionally related membrane protein
expressed by S. typhimurium, Rck, interferes with MAC
formation by inhibiting C9 polymerization (Heffernan
et al., 1992).
Mimicking or attracting host regulators
To protect the host from self-damage, complement is
tightly regulated by multiple fluid-phase and cell-bound
proteins (for a review see Zipfel & Skerka, 2009). Bacteria
often hijack these complement regulators to resist
MAC-mediated killing (reviewed in Blom et al., 2009).
CD59 is a membrane-bound glycophosphatidylinositol
(GPI) lipid-anchored glycoprotein that protects host cells
from the MAC by binding C5b-8 and preventing incorporation of polymeric C9. On the other hand, CD59
interferes with C8 and C9 insertion into the membrane
(Zalman et al., 1986; Meri et al., 1990; Rollins & Sims,
1990; Letho & Meri, 1993; Farkas et al., 2002). The OM
protein CD59-like of B. burgdorferi is functionally similar
to human CD59 since it interacts with C8 and C9 to
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Published by John Wiley & Sons Ltd. All rights reserved
1160
inhibit MAC assembly (Pausa et al., 2003). Escherichia coli
and Helicobacter pylori were found to capture the soluble
form of CD59, which is often shed from host cell
membranes (V€akev€a et al., 1994), to their OM and
thereby prevent MAC-mediated killing (Rautemaa et al.,
1998, 2001).
Another human regulator of MAC is the abundant
serum protein vitronectin (S-protein, 250–450 lg mL 1).
This protein, that also regulates coagulation (Preissner,
1991; Felding-Habermann & Cheresh, 1993), can bind
fluid-phase C5b67 complexes and prevent formation of
an active MAC (Milis et al., 1993; Podack et al., 1977).
Bacteria can capture vitronectin to prevent MAC-mediated killing (reviewed in Singh et al., 2010b). Meningococcal surface fibril (Msf) (Griffiths et al., 2011), OM
protein Haemophilus surface fibril (Hsf) of H. influenzae
(Hallstr€
om et al., 2006), surface protein UspA2 of Moraxella catarrhalis (Attia et al., 2006; Singh et al., 2010a),
DrsA of Haemophilus ducreyi (Leduc et al., 2009), and
autotransporter protein OmpB expressed by Rickettsia
conorii (Riley et al., 2013) all mediate recruitment of
vitronectin to the bacterial surface, thereby mediating
serum resistance. Since bacterial acquisition of vitronectin
also fulfills additional roles in pathogenesis like bacterial
adherence (Singh et al., 2010b), the role of vitronectin
binding proteins in MAC evasion was not always studied
[e.g. vitronectin binding to the surface of H. pylori
(Ringner et al., 1992), OpaA of N. gonorrhoeae (Duensing
& Putten, 1998), and Opc protein of N. meningitidis (Sa
E Cunha et al., 2010)]. However, it cannot be excluded
that these bacterial vitronectin-binding proteins also
mediate serum resistance.
Finally, bacteria can regulate the MAC by attracting the
complement Factor H-related protein 1 (CFHR1), a C5
convertase inhibitor present in the circulation at a concentration of 20–100 lg mL 1 (Timmann et al., 1991;
Heinen et al., 2009). CFHR1 is related to Factor H, the
C3 convertase regulator that binds C3b (Wu et al., 2009).
CFHR1 specifically regulates the AP C5 convertase by
binding both C3b and C5 (Skerka et al., 1991; Timmann
et al., 1991). Moreover, formation and membrane insertion of the MAC is controlled by CFHR1 (Heinen et al.,
2009). The B. burgdorferi CRASP-3, -4, and -5 proteins
were all found to attract CFHR1, thereby mediating
serum-resistance of B. burgdorferi (Haupt et al., 2007).
Some strains of the different genospecies Borrelia garinii
express CRASPs that can also bind CFHR1 to mediate
MAC escape (Van Burgel et al., 2010).
Polyphosphates
Microorganisms store long polymers of phosphate
(polyphosphates or polyP) that are essential to bacterial
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
E.T.M. Berends et al.
motility and survival (Rao & Kornberg, 1996; Kim et al.,
2002). Recently, it was discovered that polyP, when
excreted, specifically inhibit MAC assembly on target
surfaces by binding and destabilizing the C5b6 complex
(Wat et al., 2014). Accordingly, it was described that
mutants of N. meningitidis lacking exopolyphosphatase,
that cleaves polyP, were protected from MAC-mediated
killing (Zhang et al., 2010). This indicates that the production of long polymers of polyP forms an additional
mechanism for bacteria to deal with stress invoked by the
MAC, although more proof of this concept has yet to be
provided.
Bacterial modulation of coagulation
Mechanisms to block coagulation
One mechanism bacteria use to block coagulation is to
prevent contact activation. In GAS, the secreted protein
SIC (streptococcal inhibitor of complement) inhibits the
binding of HK to the bacterial surface thereby downregulating the contact system. Furthermore, SIC inhibits
the antimicrobial activity of HK-derived peptides
(Akesson et al., 2010; Frick et al., 2011). Staphylococcus
aureus secretes the staphylococcal superantigen-like
protein 10 (SSL10) that delays clotting by binding to prothrombin. This interaction does not inhibit thrombin
activity, but blocks the binding of prothrombin to
platelets, which is important for its activation (Itoh et al.,
2013). No other bacterial inhibitors of contact activation
have been described so far but yet to be defined S. aureus
surface proteins can also block contact activation
(Mattsson et al., 2001).
Degradation of fibrin clots
Many pathogenic bacteria have developed mechanisms to
exploit the human fibrinolytic system to escape the physical boundaries of a fibrin clot. Fibrinolysis is normally
mediated by the serine protease plasmin, which is formed
by the conversion of plasminogen by the plasminogen
activators tPA and uPA (Reichel et al., 2012). Many
bacteria can attract plasminogen to their surface where it
can be converted into active plasmin by host activators to
subsequently induce fibrinolysis. Borrelia burgdorferi
expresses a variety of plasminogen receptors on their
surface, including the outer surface proteins (OspA and
OspC) and Erp proteins (ErpA, ErpC, ErpP), to establish
plasmin formation during all stages of infection (Brissette
et al., 2009). Other examples of bacterial plasminogen
receptors include the H. influenzae surface protein E (PE)
(Barthel et al., 2012) and surface-bound enolases of several other bacteria (Pancholi & Fischetti, 1998; Bergmann
FEMS Microbiol Rev 38 (2014) 1146–1171
1161
Molecular interplay of complement, coagulation and bacteria
et al., 2001; Chung et al., 2011; Floden et al., 2011). Next
to expression of plasminogen-activating molecules at the
surface, some bacteria also produce secreted plasminogen
activators. Well-known are the streptococcal streptokinase
(STK), S. aureus staphylokinase (SAK), and Yersinia pestis
Pla. Both STK and SAK form a complex with plasminogen
resulting in its autolytic cleavage into a form of plasmin
that is less sensitive to inactivation by a2-antiplasmin.
They are highly specific for human plasminogen (Sun
et al., 2004). Pla does not form a complex but proteolytically converts plasminogen to plasmin. It has been shown
that Pla is also capable of cleaving and inactivating a2-antiplasmin, which will result in uncontrolled fibrinolysis
(Kukkonen et al., 2001). Since plasmin cleaves a broad
range of substrates, including extracellular matrix proteins,
surface-associated plasmin activity may have evolved to
not only degrade fibrin clots but also facilitate bacterial
spread through host tissues (Brissette et al., 2009).
Mechanisms to promote coagulation
In contrast to mechanisms described above, bacteria also
induce the coagulation system to either release vasoactive
peptides or promote clotting. Enhancement of coagulation can be accomplished via several different mechanisms. A well-characterized mechanism by which
S. aureus promotes clotting is via the production of coagulases. Staphylococcus aureus secretes two of these coagulases, named coagulase and von Willebrand factor
binding protein (vWbp), which are both able to bind and
activate prothrombin (McAdow et al., 2012). The binding
of coagulases induces a conformational change in prothrombin that leads to its non-proteolytic activation into
thrombin. In this way, coagulases induce the conversion
of fibrinogen to fibrin and the formation of a clot (Friedrich et al., 2003). It was recently described that both
coagulase and vWbp are necessary for the formation of
abscesses in which the bacteria can replicate and are protected from the host immune cells. Furthermore, they
have been demonstrated to be important virulence factors
in vivo (Cheng et al., 2010) that promote S. aureus agglutination in the bloodstream (McAdow et al., 2011).
Finally, a recent study shows that vWbp is also able to
bind FXIII and that the vWbp-prothrombin complex first
associates with fibrinogen and then with FXIII. This leads
to non-proteolytic activation of FXIII and thereby clot
stabilization (Thomer et al., 2013). Clotting may be an
advantage for bacteria since they can use clots to hide
from phagocytes (Guggenberger et al., 2012). For
S. aureus, it was suggested that the coagulase-induced
fibrin clots are structurally different from conventional
clots. Mouse models have shown that these clots are beneficial for escaping phagocytic clearance in the tissues and
FEMS Microbiol Rev 38 (2014) 1146–1171
promote bacterial growth and dissemination through the
blood (McAdow et al., 2012).
Next to the secreted coagulases, bacterial pathogens
also express fibrinogen-binding proteins that confer bacterial clotting. The S. aureus clumping factor A (ClfA) is
a surface-bound protein that binds fibrinogen and causes
bacteria to clump. A different S. aureus surface protein
that binds fibrinogen is the fibronectin binding protein A
(FnbpA). This protein forms fibrinogen bridges between
S. aureus and platelets leading to activation and aggregation of platelets, ultimately resulting in clot formation.
Since FnbpA and ClfA bind similar regions of fibrinogen,
it is expected that ClfA can also activate platelets in a
similar way (Fitzgerald et al., 2006).
In addition to induction of clotting, bacteria can also
promote activation of the contact pathway. One way to
promote contact activation is to enhance the binding of
HK to the bacterial surface where it is more susceptible
to activation by host proteases. M1 protein of GAS binds
HK via domain 5 (for binding to negatively charged surfaces) and domain 6 (for binding to PK). In addition, the
fibrinogen-binding protein (FOG) and protein G of group
G streptococci (GGS) are capable of binding HK, albeit
with distinct functions. FOG, but not protein G, is capable of releasing BK from HK whereas protein G inhibits
the specific release of the AMP NAT26 that results from
cleavage of HK (Wollein Waldetoft et al., 2012). Furthermore, bacteria can activate the contact system by secreting proteases that can for instance activate FXII and PK
or cleave HK to release BK (for a review see Tapper &
Herwald, 2000; Nickel & Renne, 2012). Well-known for
this activity are the cysteine proteinases staphopain A
(ScpA) and staphopain B (SspB) from S. aureus that act
directly on HK in human plasma to release BK (Mattsson
et al., 2001; Imamura et al., 2005; Wollein Waldetoft
et al., 2012). Staphopain A (ScpA) can thereby induce
vascular leakage in vivo (Imamura et al., 2005).
Finally, bacteria also promote coagulation by inactivation of endogenous regulators. Thrombin activatable
fibrinolysis inhibitor (TAFI) is a negative regulator of
fibrinolysis that is activated by both plasmin and thrombin. GAS uses collagen-like surface proteins A and B
(SclA and SclB) to recruit TAFI to its surface (P
ahlman
et al., 2007). GAS can also attract both plasmin and
thrombin to its surface and thus locally activate TAFI,
which results in inhibition of fibrinolysis (Bengtson et al.,
2009).
Misuse of coagulation proteins for immune
escape
Finally, bacteria use the coagulation system to modulate
immune cells. Recent investigations by our group revealed
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
1162
that the Extracellular fibrinogen binding protein (Efb) of
S. aureus covers bacteria with an anti-opsonic shield of
fibrinogen that effectively blocks phagocytosis (Ko et al.,
2013). Efb uses its C-terminal domain to bind C3b molecules on the surface of S. aureus. At the same time, it can
attract fibrinogen (via its N-terminal domain) to the bacterial surface and thereby generate a layer of fibrinogen
around the bacterial cell that effectively blocks phagocyte
recognition of opsonic antibodies and C3b (Ko et al.,
2013). Efb binding to fibrinogen also prevents aggregation
of platelets and attachment of neutrophils to fibrinogen
(Ko et al., 2011; Shannon & Flock, 2004). Surfaceexpressed M protein of GAS can also bind fibrinogen to
cover the bacterial surface. The M protein-fibrinogen
complexes decrease complement deposition and might
also avoid antibody binding by steric hindrance (Smeesters et al., 2010). The formation of these complexes was
shown to lead to neutrophil activation, which neither M
protein nor fibrinogen by itself is capable of. The coiled
coil structure within the M1 protein was responsible for
the binding and assembly of four fibrinogen molecules,
which creates a M1-fibrinogen network, distinct from a
conventional fibrin clot (Macheboeuf et al., 2011). Furthermore, it was shown that the virulence of GAS
depends both on the presence and functionality of M
protein as well as the presence of host fibrinogen, indicating that both host and bacterial factors contribute to
virulence (Uchiyama et al., 2013).
Conclusions
Our insights into the molecular interplay between complement, coagulation and bacteria have increased significantly over the past years. Not only do we know more
about their modes of recognition but also the way these
systems induce stress to bacterial cells has become more
lucent. Still, the poorly understood basis for MAC-dependent killing of Gram-negative bacteria remains an important challenge to elucidate in the future. Although the
importance of coagulation in clearance of infections is
now established, a key question remains to whether coagulation is mainly activated via bystander inflammatory
reactions at sites of vascular injury or whether this occurs
specifically on bacteria via the contact activation pathway.
The evolutionary conservation of this pathway certainly
suggests an important function and evidence for its role
in innate immunity is steadily accumulating. Still, the
pro-coagulatory mechanisms of some bacterial pathogens
suggest that coagulation is not always harmful to bacteria.
Pathogens seem to induce clot formation to escape the
immune system or induce contact activation to release
the vasoactive component BK, which could be an
advantage since increased vascular permeability promotes
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
E.T.M. Berends et al.
bacterial dissemination. Since bacterial modulators of
complement and coagulation contribute to bacterial virulence, they may provide new targets for infection control.
Overall, the multiple strategies evolved by pathogenic bacteria to modulate complement and coagulation are strong
indicators of a constant battle between bacteria and these
two protein networks during infections.
Acknowledgements
The authors were supported by grants of the Netherlands Organization for Scientific Research (NWO-Vidi, #91711379)
to SHMR&ETMB) and the Dutch Heart Foundation
(2010T068) to RTU. We thank Toon Verhoeven and Michiel van Gent for excellent assistance with graphical design
and Reindert Nijland and Daphne Stapels for critical
reading of the manuscript.
Authors’ contribution
E.T.M.B. and A.K. contributed equally to this work.
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