University of Groningen
Amyloids - A functional coat for microorganisms
Gebbink, M.F.B.G.; Claessen, D.; Bouma, B.; Dijkhuizen, Lubbert; Wosten, H.A B; Wösten,
Han A.B.
Published in:
Nature Reviews Microbiology
DOI:
10.1038/nrmicro1127
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Gebbink, M. F. B. G., Claessen, D., Bouma, B., Dijkhuizen, L., Wosten, H. A. B., & Wösten, H. A. B. (2005).
Amyloids - A functional coat for microorganisms. Nature Reviews Microbiology, 3(4), 333 - 341. DOI:
10.1038/nrmicro1127
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REVIEWS
AMYLOIDS — A FUNCTIONAL COAT
FOR MICROORGANISMS
Martijn F. B. G. Gebbink*, Dennis Claessen‡, Barend Bouma*, Lubbert Dijkhuizen‡
and Han A. B. Wösten§
Abstract | Amyloids are filamentous protein structures ~10 nm wide and 0.1–10 µm long that
share a structural motif, the cross-β structure. These fibrils are usually associated with
degenerative diseases in mammals. However, recent research has shown that these proteins
are also expressed on bacterial and fungal cell surfaces. Microbial amyloids are important in
mediating mechanical invasion of abiotic and biotic substrates. In animal hosts, evidence
indicates that these protein structures also contribute to colonization by activating host
proteases that are involved in haemostasis, inflammation and remodelling of the extracellular
matrix. Activation of proteases by amyloids is also implicated in modulating blood coagulation,
resulting in potentially life-threatening complications.
*Department of
Haematology, Thrombosis
and Haemostasis Laboratory,
Institute of Biomembranes,
University Medical Center
Utrecht, Heidelberglaan 100,
3584 CX Utrecht,
The Netherlands.
‡
Department of
Microbiology, Groningen
Biomolecular Sciences and
Biotechnology Institute,
University of Groningen,
Kerklaan 30, 9751 NN Haren,
The Netherlands.
§
Microbiology, Institute of
Biomembranes, University
of Utrecht, Padualaan 8,
3584 CH Utrecht,
The Netherlands.
Correspondence to H.A.B.W.
e-mail:
[email protected]
doi:10.1038/nrmicro1127
Amyloid fibrils (BOX 1) have historically been associated
with pathology in a class of degenerative diseases
including Alzheimer’s disease and Creutzfeldt–Jakob
disease. However, recent data have shown that amyloid
fibril formation not only results in toxic aggregates but
also provides biologically functional molecules1. Such
functional amyloids have been identified on the surfaces
of fungi and bacteria. The proteins forming these fibrils
are not related; HYDROPHOBINS2 and CHAPLINS3–6 form amyloid fibrils on the surfaces of fungi and Gram-positive
streptomycetes, respectively, whereas CURLI and thin
aggregative fimbriae (TAFI, formerly known as SEF17)
form on the cell surfaces of Gram-negative Escherichia
and Salmonella species7 (FIG. 1). In this review, we discuss
how amyloids are assembled at microbial surfaces and
discuss their roles in bacterial and fungal invasion of
biotic and abiotic substrates. In particular, we consider
their potential role in human and animal infections. By
activating host proteases involved in the haemostatic
system, microbial amyloids have been implicated in
colonization of the host and might contribute to complications during sepsis. Evidence also indicates that they
might form a protective coat enabling certain pathogens
to evade the immune system. It is also possible that the
host immune system could recognize microbial amyloids
as a signal that indicates potential danger.
NATURE REVIEWS | MICROBIOLOGY
Amyloids and fungi
Hydrophobins and their encoding genes have been
identified in both the ascomycetes and the basidiomycetes — phyla that represent most species in the
fungal kingdom8. These proteins have not been found
in yeasts but they are expressed in dimorphic fungi.
The presence of hydrophobins is not associated with a
specific microbial lifestyle — they are produced by
SAPROPHYTIC fungi, pathogenic fungi and fungi that
establish a mutually beneficial symbiosis.
Functions of amyloid fibrils of hydrophobins. At
hydrophilic–hydrophobic interfaces class I hydrophobins
self-assemble into an amphipathic membrane that
consists of a mosaic of amyloid fibrils known as
rodlets (BOX 2). This membrane has multiple functions.
It allows fungi to escape an aqueous environment9,
confers hydrophobicity to air-exposed fungal surfaces10–18, and mediates the attachment of fungi to
hydrophobic solid substrates, such as the surface of a
host17,19,20. Hydrophobins thereby facilitate dispersion
of spores and contribute to invasion of abiotic and
biotic substrates.
Fungal spores are often produced by aerial structures,
which develop from hyphae that have escaped their
aqueous environment. Aerial growth is mediated by
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Box 1 | Formation of amyloid fibrils
Fibre axis
The aggregation of proteins into an
ordered fibrillar structure is causally
related to a class of degenerative
diseases. Recent evidence has
β-Sheet
indicated that the formation of
stacking
amyloid fibrils does not necessarily
result from a defect in the folding or
clearance pathways, but can be due
to normal biological
processes1,119,120.Although
4.7Å
amyloidogenic proteins are distinct
Inter
with respect to amino-acid sequence
β-strand
hydrogen
and native fold, the fibrils are
bond
structurally similar.Amyloid fibrils
have a diameter of 5–13 nm, are
straight, rigid and unbranched, and
share a structural motif, the cross-β
structure. This structure consists of β-sheets that are stacked in the direction
perpendicular to the fibril axis, with hydrogen bonds parallel or perpendicular to it (see
the figure). The similarity in structure implies a common mechanism of fibril formation
and that the fibrils themselves have common properties42,121. Indeed, all amyloids increase
the fluorescence of the dye thioflavin T, exhibit green–gold birefringence on binding the
dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red.
HYDROPHOBIN
Fungal secreted protein that
self-assembles at hydrophilic–
hydrophobic interfaces into an
amphipathic membrane. In the
case of class I hydrophobins this
membrane is very stable and
consists of amyloid fibrils.
CHAPLINS
Proteins secreted by
streptomycetes that selfassemble into an amphipathic
membrane at a
hydrophilic–hydrophobic
interface. Like the membrane of
hydrophobins, the chaplin
membrane consists of amyloid
fibrils and is very stable.
hydrophobins that are secreted by the hyphae into the
moist substrate. Through a process of self-assembly,
these structures reduce the water surface tension of the
substrate–air interface, which eliminates this physical
barrier and thereby allows the hyphae to grow into the
air9 (FIG. 2). The aerial hyphae continue to secrete
hydrophobins, which assemble at the hyphal surface.
The hydrophilic side of the amphipathic film is positioned next to the cell wall, whereas the hydrophobic
side is exposed10,11. As a result, aerial hyphae are water
repellent. Similarly, water-soluble hydrophobins assemble at the surfaces of spores that have developed from
differentiated aerial hyphae. The coating of spores by
hydrophobins facilitates their dispersal by wind. In fact,
the deployment of amyloid hydrophobins contributes
to the presence of an estimated several hundred, and
sometimes thousand, fungal spores per cubic metre of
air. These spores can attach to the surface of a plant or
an animal, or can enter the body by inhalation, a lesion
in the skin or an implant to which spores have adhered.
Certain plant pathogenic fungi start infection by
forming a structure — known as the APPRESSORIUM — at
the external surface (that is, the cuticle) of the host. To
penetrate the host by mechanical force, this structure
must attach firmly. The class I hydrophobin MPG1 of
the rice pathogen Magnaporthe grisea is involved in this
attachment process17,19. The mechanism by which
MPG1 mediates appressoria attachment to the host
surface is likely to be similar to that used by the SC3
hydrophobin of Schizophyllum commune in promoting
attachment of hyphae to hydrophobic Teflon. By selfassembling at the cell wall–Teflon interface20,21, SC3
bridges the incompatible surfaces of the hydrophilic
fungal cell wall and the hydrophobic substrate, allowing strong attachment20. So far, only MPG1 has been
shown to be directly involved in fungal pathogenicity.
However, it is possible that the function of this
hydrophobin is a general mechanism involved in
host–pathogen interactions. In support of this, the
hydrophobin gene ssgA of the insect pathogen
Metarhizium anisopliae is expressed during
in vitro appressoria formation22.
The role(s) of hydrophobins, once spores have
entered an animal or human, has been most extensively studied in Aspergillus fumigatus. This fungus
causes several respiratory diseases in immunocompromised patients. The hydrophobins RodA
(HYP1)18,23 and RodB24 are present on the conidial
surface of this pathogen. RodA is involved in rodlet
formation18,23 but the function of RodB is not
known24. The conidia of an A. fumigatus strain in
which the rodA gene was disrupted adhered equally
well to pneumocytes, fibrinogen and laminin when
compared with wild-type conidia, whereas adhesion
to collagen and albumin was reduced18. These data
indicate that RodA is not the main component that
determines adherence of conidia to host tissue, a conclusion that might have been expected as experiments
indicate that rodlets primarily mediate attachment to
hydrophobic surfaces. However, RodA does seem to
have an important role in allowing conidia to avoid
clearance by neutrophils and macrophages in the early
stages of infection. In an in vitro assay, more than 40%
of conidia lacking rodlets (∆rodA and ∆rodA∆rodB)
were killed by alveolar macrophages, compared with
~20% of the wild-type and ∆rodB spores24. These data
CURLI
Amyloid fibrils found at surfaces
of E. coli, mainly consisting of
the CsgA protein.
a
b
c
TAFI
Curli fibrils found on Salmonella
cell surfaces.
SAPROPHYTIC
Organisms that derive their
nutrients from dead organic
material.
APPRESSORIUM
A pre-penetration external
structure formed by the terminal
swelling of a fungal germ tube.
334
Figure 1 | Amyloid fibrils cover surfaces of microorganisms. Electron microscopic images of amyloid fibrils of hydrophobin at
the surface of an Aspergillus nidulans spore (a), chaplins at Streptomyces tendae spores (b) and curli covering Escherichia coli cells
(c). Note that chaplin and hydrophobin amyloids form membranes, whereas curli fibrils form a loose network. Scale bars represent
120 nm (a,b) and 60 nm (c). Parts a and b are reproduced, with permission, from REF. 155 © (1999) Elsevier Science. Part c is
reproduced, with permission, from REF. 7 © (2002) American Association for Advancement of Science.
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Box 2 | Assembly of hydrophobins into amyloid fibrils
The amino-acid sequences of hydrophobins are diverse, but all contain eight conserved
cysteine residues122 that form four disulphide bridges123–125. SC3 from Schizophyllum
commune is the best characterized hydrophobin2. Mature SC3 — after the secretion
signal sequence has been cleaved — consists of 112 amino acids and has a molecular
mass of 14 kDa126. At hydrophilic–hydrophobic interfaces it self-assembles into an
amphipathic protein film consisting of a mosaic of 10-nm-wide parallel amyloid fibrils,
called rodlets11,20,21,127. These rodlets are composed of two tracks of 2–3 protofilaments
and bind thioflavin-T and Congo red2. Rodlets are not formed in the aqueous
environment due to the presence of the disulphide bridges in SC3 (REF. 128).
Self-assembly of SC3 is accompanied by several conformational changes. The watersoluble form126,129 proceeds via an intermediate form with a greater amount of α-helical
structure (known as the α-helical state) to a stable end form with a greater amount of
β-sheet structure (known as the β-sheet state)126,130. SC3 in the β-sheet state initially has
no clear ultrastructure (β-sheet I state), but after prolonged incubation the protein
organizes itself into rodlets (β-sheet II state)2,130. These rodlets form a stable semipermeable membrane through which molecules >200 Da cannot pass (X. Wang and
G. T. Robillard, personal communication). This membrane lowers the water surface
tension from 72 mJ m–2 to 24 mJ m–2 and is therefore highly surface active9.
SC3 is not a paradigm for all hydrophobins. It is a typical example of a class I
hydrophobin131. Class II hydrophobins also assemble at hydrophilic–hydrophobic
interfaces, but they do not form amyloid fibrils2,132. Their membranes are much less
stable, possibly owing to their inability to form rodlets. It has been suggested that the
difference in amyloid formation between class I and class II hydrophobins is due to the
amino-acid sequence between the third and fourth cysteine residue126,133. Class II
hydrophobins have an intervening sequence of 11 residues, whereas class I
hydrophobins have 17–39 residues.
Air
Self assembly
Aqueaous
environment
α-Helical state
β-Sheet I state
β-Sheet II state hydrophobin
Water-soluble hydrophobin
MYCELIUM
A network of fungal or
streptomycete hyphae.
agree with the finding that in vivo growth of the
∆rodA strain of A. fumigatus is reduced in rat lungs
during the first 5 days of infection25. Assuming that
RodA forms a semi-permeable layer around spores
(like SC3; BOX 2), this protein film would not be
expected to hamper the diffusion of oxidative species
from the macrophage into the fungal cell. However, it
is possible that these reactive molecules are neutralized by reacting with the hydrophobins in the rodlet
layer24. Although RodA protects the spore during the
initial stages of infection, no differences were observed
in the mean length of survival of mice infected with
∆rodA spores compared with wild-type spores 18,25.
This observation may be explained by ongoing
swelling of spores caused by water uptake — the
resulting increase in spore volume disrupts the continuity of the hydrophobin membrane, which no
longer protects against oxidative species or shields
antigens located in the cell wall.
NATURE REVIEWS | MICROBIOLOGY
Amyloids and bacteria
Chaplin amyloids are present on the surfaces of the
Gram-positive actinomycetes Streptomyces spp.3
Genetic evidence indicates that chaplin genes are present
in other actinomycetes (including Thermobifida fusca5)
but are absent from Mycobacterium spp. and the closely
related Corynebacterium spp. Curli and Tafi amyloids
have been found on the surfaces of the Gram-negative
γ-proteobacteria Escherichia coli and Salmonella enterica7,
as well as Citrobacter spp.26 Genetic evidence has indicated that the genomes of other Enterobacteriaceae (for
example, Enterobacter spp. and, possibly, Shigella spp.)
also encode curli genes26–28. Whether amyloid fibrils
are more widespread within the bacterial domain is
not yet known.
Streptomycetes, such as Streptomyces coelicolor, are
soil-dwelling bacteria, whereas other related streptomycete species are pathogens of animals or plants. The
life cycle of streptomycetes is remarkably similar to that
of filamentous fungi. After a colonizing MYCELIUM has
been formed in an aqueous substrate, hyphae grow into
the air to form chains of exospores. These spores are dispersed and can give rise to a new colonizing mycelium.
E. coli and Salmonella spp. inhabit the intestinal tract of
humans and other warm-blooded animals and can
cause enteric disease, septicaemia or non-enteric localized diseases such as urinary tract infections, meningitis
or pneumonia.
Chaplin amyloids. Like hydrophobins, chaplins selfassemble into an amphipathic membrane that consists
of amyloid fibrils of these proteins (BOX 3). The functions
of the chaplin membrane are similar to those of
hydrophobin membranes — they mediate attachment
to hydrophobic surfaces29 and allow hyphae to escape
the aqueous environment3. The membrane is also
responsible for making the surface of hyphae and spores
hydrophobic3,4, which prevents aerial hyphae aggregating and probably facilitates the dispersal of spores by the
wind (FIG. 2). Streptomycete spores can be introduced
into humans or animals by a process similar to that of
fungal spores. These spores are abundant in damp environments, including buildings, and can result in occupants suffering inflammatory responses30,31 and even
immunotoxic effects32. In tropical and subtropical
regions, Streptomyces somaliensis causes actinomycetoma,
which is associated with progressive swelling and severe
skin deformations33. It is not yet clear whether chaplins
contribute to the development of this disease by, for
example, mediating attachment to, or colonization of,
host tissue.
Curli and Tafi amyloids. Curli and Tafi amyloid fibrils of
E. coli and Salmonella spp. are mainly composed of the
highly homologous subunit proteins CsgA and AgfA,
respectively (BOX 4). These structures are involved in the
attachment of the bacteria to inert solid surfaces and
also function in biofilm formation34,35. In the case of
Salmonella enterica serovar Enteritidis, Tafi fibrils have
been shown to promote bacterial cell–cell interactions
— a process that contributes to the thick biofilms
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REVIEWS
Monomeric hydrophobin
or chaplin
Hydrophobin or chaplin
in assembly
Air
Medium
Growing hyphae
Lowering of water surface tension
Branching hyphae
Coating of aerial hyphae
Surface amyloid
cross-β structure
Figure 2 | Formation of amyloid structures allows streptomycetes and filamentous fungi
to invade the air. The mechanism by which aerial structures are formed by streptomycetes and
filamentous fungi is remarkably similar. In both cases the water surface tension at the
substrate–air interface is reduced by proteins that self-assemble into an amphipathic rigid
membrane. This membrane is composed of amyloid fibrils of the hydrophobins and chaplins in
filamentous fungi and streptomycetes, respectively. Aerial hyphae continue to secrete
hydrophobins or chaplins and these molecules assemble at the hyphal surface making them
hydrophobic. Modified, with permission, from REF. 3 © (2003) Cold Spring Harbor Laboratory
Press, and REF. 9 © (1999) Elsevier Science.
formed by strains expressing these extracellular structures34. Curli and Tafi fibrils also function in bacterial
virulence; Tafi fibrils, like other fimbriae, probably fulfill
this role by mediating colonization of the small
intestine36–38. Moreover, curli fibrils have been shown to
have a role in the intracellular invasion of host cells39.
Interaction of microbial amyloid with host proteins
As mentioned above, amyloid fibrils share a structural
motif — the cross-β structure40,41 — which indicates
that these fibrils have common properties42. Indeed,
evidence is now accumulating that several mammalian
proteins bind amyloids irrespective of their amino-acid
sequence. Proteins that bind amyloids include
fibronectin, tissue-type plasminogen activator (tPA)
and factor XII. These proteins all contain a specific
module, the fibronectin type I domain, and several have
336
| APRIL 2005 | VOLUME 3
been shown to interact with amyloid (Bouma, B. et al.,
unpublished; FIG. 3). Interestingly, curli and Tafi fibrils
also bind fibronectin, tPA and factor XII43–45.
Fibronectin is a multi-domain, adhesive glycoprotein
that circulates in blood and is a component of the extracellular matrix (ECM). This glycoprotein supports cell
adhesion and has an important role in cell migration
during wound healing, cancer and bacterial infection.
Invasion of host cells by curli-coated E. coli could be
blocked by competitors (for example, RGD peptides) of
fibronectin46. It is proposed that invasion is a two-step
process — in the first step, the curli fibril interacts with
fibronectin and in the second step the fibronectincoated bacteria are internalized through interactions with
integrins46. This proposed mechanism is similar to the
process by which Group A streptococci are internalized
by eukaryotic cells47.
Both tPA and factor XII are serine proteases that are
part of the haemostatic system. This system consists of
fibrinolytic and coagulation cascades that control clot
dissolution and blood clotting, respectively (BOX 5). tPA
is the initiator of the fibrinolytic system, whereas factor
XII initiates the contact system of coagulation. Both
enzymes require an activator that functions as a scaffold, bringing enzyme and substrate into close contact.
Fibrin, present either in blood clots or as a component
of the ECM, is the classical cofactor for tPA. On binding tPA and plasminogen to fibrin, the rate of plasminogen activation is markedly increased48. It was
recently shown that fibrin and fibrin-derived peptides
adopt a cross-β structure and form amyloid fibrils49.
The formation of these fibrils correlates with tPA binding and stimulation of tPA-mediated plasminogen
activation. Interestingly, prototype amyloid peptides,
including amyloid-β (Aβ) and islet amyloid polypeptide (IAPP; which is associated with pancreatic β-cell
toxicity in type II diabetes), also bind tPA and can substitute for fibrin in plasminogen activation. These
results suggest that any amyloid can activate tPA, so
microbial curli, chaplin and hydrophobin amyloids
might also have plasminogen activation activity.
Furthermore, these microbial amyloids could also activate factor XII, which is structurally related to tPA.
Activation of the contact and fibrinolytic systems by
microbial surface-located amyloid could be a method
by which certain microorganisms exploit the host
haemostatic system.
Pathogens exploit the haemostatic system
The host fibrinolytic and contact systems are activated
during infection with bacteria, viruses, protozoa and
fungi (REFS 50–58 and refs therein). The resulting hostderived extracellular proteolytic activity contributes to
inflammation and the pathogenesis of sepsis43,51–53,59–72.
Activation of plasminogen by bacterial pathogens
results in proteolysis of the ECM73–77, allowing bacteria to penetrate the basement membrane67,78–81 and
leading to increased invasion of human endothelial82,
pharyngeal83 or HeLa cells84. Pathogens can synthesize
their own plasminogen activators or rely on host tPA
to generate plasmin on the cell surface (REFS 50,56 and
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M PROTEINS
A family of proteins expressed
by Streptococcus pyogenes that
mediate interaction with
human plasma proteins and
promote bacterial adherence to
epithelial cells.
Box 3 | Assembly of chaplins into amyloid fibrils
Streptomyces coelicolor contains eight chaplins5. The
Chaplin monomers
mature forms of five of these chaplins (ChpD–H) are
~55 amino acids long, whereas ChpA–C consist of
~225 amino acids. ChpA–C contain two so-called
chaplin domains (that is, sequences similar to those
of mature ChpD–H), a hydrophilic region and a cellwall-anchoring domain, which is recognized by
sortase enzymes that covalently attaches it to the
– Rodlins
+ Rodlins
growing peptidoglycan layer134,135. Linkage to the cell
wall would explain why ChpA–C cannot be extracted
from cell walls of aerial hyphae3. By contrast,
ChpD–H dissociate on treatment with
trifluoroacetic acid (TFA), and the resulting
monomers are water soluble. These unstructured
monomers assemble at the water–air interface into
amyloid fibrils with a diameter of 4–6 nm. The fibrils
form a rigid, light-reflecting film that reduces the
Fibrils
Rodlets
water surface tension from 72 to 26 mJ m–2 (REF. 3).
Water-soluble chaplins do not spontaneously
assemble at a hydrophobic solid substrate29. Instead, they adopt an α-helical conformation. Heating in diluted
detergent induces the protein to proceed to the β-sheet-rich amyloid form. The α -helical conformation could
therefore be an intermediate in the assembly process as observed for hydrophobins (BOX 2) and other amyloid
proteins2,130,136. Chaplins assemble in solution when a seed of the assembled form of the protein is added. So, in
contrast to hydrophobins, once a nucleus of amyloid has been formed, self-assembly of chaplins becomes
independent of a hydrophilic–hydrophobic interface. It is not yet known whether streptomycetes form nucleation
proteins as has observed for curli fibres (BOX 4).
Chaplin fibrils are organized in rodlets at the surface of S. coelicolor aerial hyphae and spores (FIG. 1b). These
rodlets consist of two tracks, each track containing two chaplin fibrils4. It has been proposed that the rodlins RdlA
and RdlB align the chaplin fibrils into rodlets (see figure). This hypothesis is based on the observation that
mutants in which either or both rdl genes have been deleted lack the rodlet structures but instead have fibrils at
their surfaces similar in appearance to those formed by chaplins in vitro. Figure modified, with permission, from
REF. 4 © (2004) Blackwell Publishing.
refs therein). Various microbial tPA receptors have
been described83,85,86–88, including curli amyloids of
E. coli 45,85,89. Interestingly, a curli-deficient mutant
strain of E. coli, which is no longer virulent, also fails
Box 4 | Formation of curli and Tafi amyloid
CsgA is the main structural component of curli of Escherichia coli 7,137. The mature
form of this protein is 131 amino acids in length (15 kDa) and is characterized by
several glycine repeats and the absence of cysteines137. A purified His-tagged version
of CsgA is initially soluble, but after incubation at 4°C for 4–12 hours the protein
forms amyloid fibrils that are similar to those observed at the surfaces of E. coli 7.
These fibrils are 4–7 nm and 6–12-nm wide, as shown by negative staining44 and
shadowing7 (FIG. 1c), respectively. For formation of curli fibrils in vivo, nucleation and
precipitation machinery is required that probably prevents self-assembly in the cell
and accelerates assembly at the cell surface. This machinery is encoded by csgB and
csgEFG138. Experimental evidence indicates that CsgB and CsgF nucleate CsgA fibres
at surfaces of cells7,139. As CsgB is homologous to CsgA it is possible that these
proteins co-assemble138. The lipoprotein CsgG, which localizes to the inner leaflet of
the outer membrane, was shown to be necessary for CsgA stabilization and
secretion7,140.
Tafi fibrils of Salmonella spp. are mainly composed of AgfA141, the amino-acid sequence
of which shares 74% identity and 86% similarity with that of E. coli CsgA142. The
mechanism of nucleation and aggregation of AgfA is identical to that of CsgA141–143. But
the physical appearance of Tafi can be different to curli, caused by the fact that Tafi
interacts avidly with cellulose produced by Salmonella spp.144 In the absence of this
polysaccharide, Tafi and curli fibres are indistinguishable from each other.
NATURE REVIEWS | MICROBIOLOGY
to bind tPA and activate plasminogen45,85,89, which
indicates that amyloid may be a virulence factor.
Several bacteria, including E. coli43,63, Salmonella
enterica serovar Typhimurium 63, Staphylococcus
aureus90 and Streptococcus pyogenes59,91,92, assemble
proteins from the contact system of clot dissolution
at their cell surface. Adsorption of these proteins to
the cell surface results in activation of factor XII and
subsequent release of bradykinin and other proinflammatory peptides that induce vasodilation,
vasopermeability and cytokine release43,63,90,92. The
mechanisms of assembly and activation of proteins of
the contact system are remarkably similar to those of
recruitment and activation of components of the
fibrinolytic system. In fact, the same cell-surface
receptors have been implicated — curli of E. coli43,63,
Tafi of Salmonella spp.63 and the M PROTEINS of S. pyogenes59,91. Mutant strains of E. coli or S. pyogenes lacking curli or M protein, respectively, fail to bind and
activate contact proteins43,63,91,92. So far, only curli and
Tafi are known to form amyloid. It remains to be
established whether M proteins and other receptors
for tPA and factor XII also form amyloid or whether
they activate these proteases by another mechanism.
The potential consequences of the interaction between
amyloid and factors of the haemostatic system are
illustrated schematically in FIG. 3.
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Box 5 | The haemostatic system
The haemostatic system
Extrinsic pathway
Intrinsic pathway
Fibrinolytic pathway
Tissue factor
controls blood clotting
K
XII
tPA
Ca2+
(coagulation) and clot
VII
VII
HK
dissolution (fibrinolysis). The
PK
XIIa
coagulation system comprises
Plg
Pls
the extrinsic and intrinsic
VIIla
XI
XIa
pathways.
PL
2+
The extrinsic pathway is
Ca
triggered by tissue factor that
IX
IXa
is exposed on cells on injury.
The intrinsic pathway is
Xa
X
initiated when blood contacts
2+
Ca , Va, PL
a negatively charged surface,
and is therefore also referred
Prothrombin
Thrombin
to as the contact system of
coagulation. Activation of
Fibrin degradation
both coagulation pathways
Fibrinogen
Fibrin
products
activates a cascade of proteases
that ultimately leads to the conversion of prothrombin into thrombin. Thrombin cleaves fibrinogen, which in turn
triggers fibrin polymerization and clot formation. The extrinsic pathway is considered the main route of coagulation to
prevent blood loss, whereas the intrinsic (contact) pathway has been postulated to function during inflammation.
Contact system
The contact system is activated by the conversion of factor XII into the active serine protease factor XIIa, induced by
binding of factor XII to an activator. In vitro activators often have negatively charged groups and include glass, kaolin,
dextran sulphate and phospholipids. The in vivo activator is unknown, but recent evidence indicates that amyloid may
be able to act in this regard (Bouma, B. et al., unpublished). Formation of factor XIIa ultimately yields the nona-peptide
bradykinin, which is released from high-molecular-weight kininogen (HK)145 by kallikrein (K). Bradykinin induces
vasodilation, hypotension, increased vasopermeability and broncho-constriction146, as well as the release of intracellular
vesicles containing tissue-type plasminogen activator (tPA)147,148. The latter is considered the main activator of
fibrinolysis. Factor XIIa and kallikrein also activate the complement system and stimulate neutrophils64,149.
Taken together, the contact system contributes to inflammatory reactions68,150.
Fibrinolytic system
Plasminogen (Plg) and its active form plasmin (Pls) are central components in fibrinolysis. Plasminogen is activated by
the serine proteases tPA or urokinase, both of which are produced by endothelial cells, among other cell types. Plasmin
has a broad substrate specificity and degrades not only fibrin but also other extracellular matrix components such as
laminin and fibronectin. In addition, plasmin activates pro-collagenases, allowing degradation of collagen matrices151,152,
and cleaves certain pro-hormones (for example, latent transforming growth factor-α)153. Given its broad specificity,
plasmin is believed not only to have a role in fibrinolysis but also in several physiological and pathophysiological
processes154. Indeed, it is associated with cell-mediated degradation of the extracellular matrix during inflammation,
wound healing, tissue remodelling, metastasis and invasion of tumour cells, neuronal cell migration and long-term
potentiation154. PK, prekallikrein; PL, phospholipids; Va, factor Va.
Microbial amyloid and the innate immune system
In addition to proteins that contain the fibronectin
type I module, other proteins also interact with amyloid, including components of the innate immune
system 93 such as the serum amyloid P component
(SAP)94–96, the scavenger receptors A, B1 (REFS 97,98),
CD36 (REFS 99–102) and RAGE (receptor for advanced
glycation end products)103–106. The physiological function of SAP is not completely understood, but it is
believed to control clearance of chromatin and apoptotic cells and prevents autoimmunity to chromatin
and apoptotic cells 107,108. SAP stabilizes amyloid
fibrils95, thereby contributing to the pathogenesis of
amyloidosis 109. SAP also interacts with bacteria
including S. pyogenes110 and E. coli111, and it has been
suggested that the binding of SAP to these bacteria is
mediated by surface-located amyloid. Interestingly,
338
| APRIL 2005 | VOLUME 3
by binding SAP, pathogenic bacteria evade neutrophil
phagocytosis and have enhanced virulence111.
So far, experiments indicate that the presence of
surface-located amyloid only benefits the microorganism. However, scavenger receptors of the innate
immune system might recognize amyloid as a
pathogen-associated molecular pattern. In support of
this hypothesis is the finding that scavenger receptor
type A is involved in phagocytosis of bacteria 112,113
and is known to bind amyloid97,114,115. Interestingly,
S. typhimurium with cell-surface-located Tafi is
cleared more rapidly from the host than strains that
do not express these amyloid fibrils116. However, Tafi
is also a virulence factor and provides the pathogen
with a selective advantage by increasing the efficiency
of colonization of the host intestine117. It could be that
in the case of Shigella, clearance of the microorganism
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REVIEWS
Bacterial surface amyloid
Fibronectin
tPA
Cell invasion
Factor XII
Extracellular matrix
breakdown and invasion
Fibronectin type I domain
EGF domain
Fibronectin type II domain
Kringle domain
Fibronectin type III domain
Protease
Cytokine release
and inflammation
Complications of infection
(sepsis)
Figure 3 | Consequences of the interaction between bacterial surface amyloid and the contact and fibrinolytic systems.
It is proposed that by binding type I domains of fibronectin, tissue-type plasminogen activator (tPA) and factor XII, different processes
and pathways may be activated by bacterial surface amyloid. Binding to fibronectin facilitates cell invasion, whereas binding to tPA
activates the fibrinolytic system, resulting in extracellular matrix degradation and pathogen invasion. Recent data suggests that the
contact system may be activated by binding of microbial amyloid to factor XII. This results in bradykinin formation, cytokine release
and an inflammatory response. The activated contact and fibrinolytic systems may result in complications during sepsis. Small
arrows indicate fibronectin type I domains that were tested and shown to mediate amyloid binding in vitro (Bouma, B. et al.
unpublished). EGF, epidermal growth factor-like.
by the immune system outweighs the advantage of
increased pathogenicity. This could explain, in part,
why many strains of this pathogen have lost the ability
to express curli fibrils118.
Conclusions
Within the past decade, several proteins that do not have
amino-acid sequence similarity have been shown to
form amyloids on the surfaces of mycelial fungi, Grampositive streptomycetes and the Gram-negative E. coli
and Salmonella species. So far, hydrophobins are the only
proteins known to form amyloids on fungal surfaces. In
fungi that do not express hydrophobins (for example,
the human pathogen Candida albicans), other proteins
may fulfill this role. Within the domain Bacteria, two
classes of proteins — chaplins and curli/tafi — have
been shown to form cell-surface-located amyloids.
These findings support the idea that the presence of amyloid structures on the cell surfaces of microorganisms is
widespread.
Amyloids that are present on microbial cell surfaces
function in the invasion of abiotic and biotic substrates.
By reducing the water surface tension, these structures
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Acknowledgements
Our research on amyloids is supported by The Netherlands
Thrombosis Foundation, the International Society of Alzheimer’s
Research (ISAO), the Biopartner Programme of the Dutch Science
Foundation (to M.F.B.G.G.), and the Dutch Programme EET
(Economy, Ecology and Technology), a joint initiative of the Ministries of
Economic Affairs, Education, Culture and Sciences and of Housing,
Spatial Planning and the Environment (EETK01031) (to L.D.).
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www.ncbi.nlm.nih.gov/Entrez
Magnaporthe grisea | Staphylococcus aureus | Streptomyces
coelicolor | Streptococcus pyogenes
SwissProt: http://www.expasy.org/sprot
AgfA | CsgA | MPG1 | RodA | SC3 | tPA
Access to this interactive links box is free online
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