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THE PATHOGENESIS OF
STREPTOCOCCAL INFECTIONS: FROM
TOOTH DECAY TO MENINGITIS
Timothy J. Mitchell
The development of bacterial disease has been likened to a molecular arms race, in which the
host tries to eliminate the bacteria, while the bacteria try to survive in the host. Although most
bacteria do not cause disease, some cause serious human infection in a large proportion of
encounters. Between these two extremes are bacteria that can coexist with humans in a carriage
state but, under appropriate circumstances, cause disease. The streptococci exemplify this
group of organisms, and by studying them we can begin to address why bacteria cause such a
wide spectrum of disease.
ENDOCARDITIS

Inflammation of the lining of


the heart and its valves.

Division of Infection and


Immunity, Institute of
Biomedical and Life Sciences,
University of Glasgow,
Glasgow G12 8QQ, UK.
e-mail:
t.mitchell@bio.gla.ac.uk
doi:10.1038/nrmicro771

Many species of streptococci are members of the


commensal microflora, bacteria that are present on
the mucosal surfaces of humans or animals and generally cause no harm. However, as well as being carried
asymptomatically, these species can also cause a range
of serious infections. The diseases caused by the streptococci range from dental caries and pharyngitis (sore
throat) through to life-threatening conditions such as
necrotizing fasciitis and meningitis. How this group of
organisms causes such a range of conditions is an
intriguing question. The genomes of several streptococci have now been sequenced and this information
is being used to understand how these microorganisms interact with their hosts and how gene content
affects their ability to cause particular diseases. This
review will focus on the comparative biology of four
species of streptococci: Streptococcus mutans,
Streptococcus agalactiae, Streptococcus pyogenes and
Streptococcus pneumoniae. The diseases that these
organisms cause and some of the virulence factors that
they produce are listed in TABLE 1. These four species
have been selected because they cause a range of diseases and because genome sequence information is
available for them.
To cause disease, a bacterial pathogen needs to fulfil
several basic requirements. First, it must be able to
adhere to a tissue surface and compete with the normal

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flora present on that surface. For S. mutans, this


involves attaching to the tooth surface and to other
bacteria present in the biofilm on the surface of the
tooth. For S. agalactiae, S. pyogenes and S. pneumoniae,
the points of attachment are the mucosal surfaces and
the skin. In the case of S. agalactiae, the main sites of colonization that are relevant to pathogenesis are the vaginas and recta of pregnant women, whereas S. pyogenes
colonizes the skin and pharynx. S. pneumoniae is normally found attached to the mucous membranes of the
nasopharynx, but causes disease when aspirated into
the lower respiratory tract.
Once attached, these organisms usually need to invade
the host and replicate. S. mutans rarely causes invasive
disease (although it can cause ENDOCARDITIS), but does cause
damage at the surface of the tooth; the other streptococci
considered here can cause invasive disease. To survive in
the hostile environment of the host, bacterial pathogens
must acquire nutrients (which can be sequestered in host
stores) and avoid the host immune system, which is activated by the presence of invading microorganisms. To be
considered as a pathogen, the bacterium must then damage the host in some way. In the case of S. mutans, the
mechanism of damage is clearly defined: this streptococcal species produces acid that damages the enamel of the
teeth. For the other three organisms considered here, the
situation is more complex. S. pyogenes produces an
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Table 1 | Streptococcal diseases and virulence factors


Organism

Diseases

Virulence factors

Streptococcus mutans

Dental caries, endocarditis

Adhesins: antigen I/II (SpaP), glucosyltransferases


and glucan-binding proteins A and C, WapA, SloC,
PavA-like protein
Acid production

Streptococcus agalactiae

Neonatal sepsis and meningitis;


systemic infection in immunocompromised individuals

Capsule
Laminin-binding protein
- and -proteins
Fibronectin-binding proteins: FbsA, PavA-like protein
C5a-peptidase
Other LPXTG-anchored proteins
Haemolysin (CylE)

Streptococcus pyogenes

Pharyngitis; cellulitis; scarlet fever;


streptococcal toxic-shock syndrome;
necrotizing fasciitis; rheumatic fever
as sequela; glomerulonephritis as
sequela

Capsule
Fibronectin-binding proteins: SfbI, SfbII, FBP54
(PavA-like), F2, PFBP
C5a-peptidase
Mac
SIC
Haemolysins: SLO, SLS
Pyogenic exotoxins: SpeA, SpeC, SpeG, SpeH,
SpeJ, SpeK, SpeL, SSA, SMEZ, SMEZ2
GRAB
DNAses A, B, C and D
Streptokinase
Hyaluronidase
SpeB (cysteine protease)

Streptococcus pneumoniae Otitis media; bacteraemia;


pneumonia; meningitis

Capsule
Cell wall
Pneumolysin
Surface proteins: LPXTG-anchored, choline
anchored (PspA, PspC, autolysin), PavA, PsaA
Hydrogen peroxide
NADH oxidase
Superoxide dismutase

The list of virulence factors is not exhaustive and represents selected examples. FbsA, fibrinogen-binding protein from S. agalactiae;
FBP54, fibronectin-binding protein 54; GRAB, G-related 2-macroglobulin-binding protein; Mac, Mac1-like protein; PavA, pneumococcal
adhesion and virulence A; PFBP, pyogenes fibronectin-binding protein; Psa, pneumococcal surface antigen; Psp, pneumococcal surface
protein; Sfb, streptococccal fibronectin-binding protein; SIC, streptococcal inhibitor of complement; SLO, streptolysin O; SloC, S. mutans
LraI operon C; SLS, streptolysin S; SMEZ, streptococcal mitogenic exotoxin Z; SpaP, streptococcal protein antigen P; Spe, streptococcal
pyogenic exotoxin; SSA, streptococcal superantigen A; WapA, wall-associated protein A.

TWO-COMPONENT SIGNALTRANSDUCTION PATHWAY

A signal-transduction system
that consists of a sensor protein
that senses and responds to an
external signal, and which acts
on a response-regulator protein
that transmits the signal to other
components of the cell.
LECTIN

A cell-agglutinating protein of
non-immune origin, which
binds carbohydrates without
modifying them.
PASSIVE IMMUNIZATION

The induction of immunity by


the transfer of immunoglobulins
or T cells.

220

impressive array of enzymes and toxins that are involved in


the disease process, whereas disease caused by S. pneumoniae
can be thought of as an overactivation or dysregulation of
the host inflammatory response. As the three potentially
invasive streptococci can also be carried asymptomatically,
there must be triggers that allow disease to occur. These
can include bacterial factors (for example, acquisition of a
particularly virulent strain) and host factors, such as
reduced immune function or lack of pre-existing antibodies
against the bacterium.
In this review, the mechanisms of disease associated
with each of these four streptococcal species will be
considered in turn. The comparative genomics and
gene-expression analysis of these organisms will then
be discussed.
Streptococcus mutans

S. mutans is the main causative agent of human dental


caries, one of the most common infectious diseases that
affect humans. Approximately 300 different bacterial
species have been found to be associated with dental
plaque, but only the presence of S. mutans has been
linked with the causation of dental caries1.
S. mutans has several factors that allow it to accumulate within the dental biofilm and produce and
tolerate the acids that cause caries. These are summarized

| DECEMBER 2003 | VOLUME 1

in FIG. 1. The key event in the production of the lesions


associated with dental caries is the production of
acid2. S. mutans expresses virulence factors as part of
the microbial biofilm that constitutes dental plaque.
TWO-COMPONENT SIGNAL-TRANSDUCTION PATHWAYS have an
important role in environmental sensing, and mutagenesis experiments have shown that one such system is
important in biofilm growth3.
S. mutans uses two methods of attachment: sucrose
independent and sucrose dependent. In the absence of
sucrose, the organism can adhere to salivary agglutinin, other bacteria, the extracellular matrix and
epithelial cell-surface receptors using ionic and LECTINlike interactions. S. mutans expresses several main
adhesins, including streptococcal protein antigen P
(SpaP; also known as antigen I/II)4, which can bind to
a specific salivary component called salivary agglutinin glycoprotein (SAG). Isogenic mutants that lack
antigen I/II are less adherent to saliva or to SAGcoated surfaces, and PASSIVE IMMUNIZATION of monkey
or human subjects with antibodies against antigen
I/II results in reduced levels of colonization with
S. mutans5. Antigen I/II is anchored to the cell surface
via an LPXTG motif, and this anchoring process
requires a SORTASE enzyme (BOX 1). Isogenic mutants that
lack the sortase A enzyme cannot anchor the protein to

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Other bacteria in biofilm

Complement
factor H

PavA-like

Secreted: haemolysin
proteases

Glucan-binding
proteins (A and C)

-protein

ABC transporter

Glucan

Fibronectin binding

FbsA

PsaA
Cell-associated
glucosyltransferases

-protein

WapA

GBS

S. mutans

Sucrose
Sortase

Sortase

ABC transporter
Lmb (binds to laminin)
Capsule

PavA-like protein
(fibronectin binding)

Glycolysis

Haemolysin CylE
Acid
SAG

Sugars

C5a peptidase
Sortase

Antigen I/II

Other LPXTGanchored proteins

Damage
Tooth surface

d
Fibronectin-binding
SfbI
proteins
SfbII
FBP54

MHC

F2

Superantigen
PFBP
C5a
peptidase

TCR

DNAses A, B, C, D
Streptokinase
Hyaluronidase
Cysteine protease (SpeB)
EndoS
GRAB (binds
protease inhibitor)

GAS

Superantigenic
toxins

Capsule

Signal
Sortase
Cytokines

SIC
Mac
interferes with
phagocytosis

Streptococcal
toxic-shock syndrome

Haemolysins
SLO, SLS
cause tissue
damage
and ulcers

M-protein
Binds:
Factor M
Fibronectin
Fibrinogen
Albumin

Choline-binding
proteins
Binds to
factor H, C3

PspC

Binds to polymeric
immunoglobulin
receptor
Sortase

Autolysin
PspA

Inhibition of host defence,


inflammation, apoptosis

Pneumolysin

ABC transporter

S. pneumoniae

PsaA
Capsule

Neuraminidase

LPXTG-anchored
proteins

Release of
pneumolysin
by autolysin

PavA
(fibronectin
binding)
Hyaluronidase

Figure 1 | Summary of the main virulence factors of four streptococcal species. a | For Streptococcus mutans, the main causative agent of dental caries, the
main virulence factor is the production of acid as part of the bacterial biofilm that constitutes dental plaque. S. mutans also expresses a variety of cell-surface and
secreted virulence factors, including a haemolysin. b | The polysaccharide capsule has been identified as an important virulence factor for the group B streptococci
(GBS), which are the leading cause of neonatal sepsis, pneumonia and meningitis in the United States and western Europe. GBS also express a variety of surface
proteins that allow evasion of the host immune response, and a haemolysin (CylE), which mediates invasion and tissue damage. c | Group A streptococcus (GAS) is
often termed the most versatile of the streptococcal pathogens, and this is reflected in the huge array of virulence factors that are produced; these allow evasion of
the host immune response and cause tissue damage. Toxins and tissue-degrading enzymes might have an important role in the severe diseases that are associated
with GAS infection, such as necrotizing fasciitis. GAS also produces superantigenic toxins which stimulate T cells to proliferate and produce cytokines, and which
are associated with streptococcal toxic-shock syndrome and the cytotoxins streptolysin O (SLO) and streptolysin S (SLS). d | Streptococcus pneumoniae (the
pneumococcus) is a commensal microorganism of the upper respiratory tract; however, if it gains access to the brain or lower respiratory tract, it can induce massive
inflammation the features of meningitis and pneumonia. Important virulence factors for the pneumococcus include the capsule, choline-binding proteins and a
haemolysin (pneumolysin). ABC, ATP-binding cassette; EndoS, endoglycosidase S; FBP54, fibronectin-binding protein 54; FbsA, fibrinogen-binding protein from
S. agalactiae; Lmb, laminin-binding protein; Mac, Mac1-like protein; MHC, major histocompatibility complex; PavA, pneumococcal adhesion and virulence A; PFBP,
pyogenes fibronectin-binding protein; PsaA, pneumococcal surface antigen A; Psp, pneumococcal surface protein; SAG, salivary agglutinin glycoprotein; Sfb,
streptococcal fibronectin-binding protein; SIC, streptococcal inhibitor of complement; TCR, T-cell receptor; WapA, wall-associated protein A.

SORTASE

An enzyme that links proteins to


the cell wall.
HYDROXYAPATITE

The main mineral component of


teeth.
GLUCOSYLTRANSFERASE

Any hexosyltransferase enzyme


for which the glycosyl group
transferred is glucosyl.

the cell surface and show reduced adhesion to salivacoated HYDROXYAPATITE, as well as reduced colonization of
rat teeth in vivo6.
In the presence of sucrose, cell-wall-associated
GLUCOSYLTRANSFERASES mediate the tight attachment of
S. mutans to the tooth surface by synthesizing glucans7.
Glucans interact with surface-associated glucanbinding proteins to promote cellcell aggregation.
Mutation of the genes encoding S. mutans glucosyltransferases and glucan-binding proteins can alter

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plaque structure and cariogenesis810. It has recently


been shown that an orphan response regulator (tarC)
controls the expression of glucosyltransferase S and
glucan-binding protein C. In a TarC-negative mutant,
the biofilm architecture was altered relative to that
associated with wild-type S. mutans and the mutant was
hypocariogenic in GERM-FREE RATS11.
The other adhesins that are produced by S. mutans
include wall-associated protein A (WapA)12, S. mutans
LraI operon C (SloC)13, glucan-binding proteins A

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Box 1 | LPXTG surface proteins and sortases


Most Gram-positive genomes contain proteins that have an LPXTG amino-acid motif
near to the carboxy-terminal end of the protein. These proteins usually also contain a
typical secretion signal and are secreted to the outside of the cell. As the protein is
secreted, the LPXTG motif is recognized by a so-called sortase enzyme. This enzyme
links the protein to the peptidoglycan of the bacterial cell wall via the threonine residue
of the LPXTG motif. The protein is therefore covalently anchored to the cell surface.
The availability of complete genome sequences allows these proteins to be identified by
computer analysis.

GERM-FREE RATS

Rats bred and maintained in


such conditions that they
contain no microorganisms.
SIGNATURE-TAGGED
MUTAGENESIS

A negative selection method for


the identification of virulence
genes. The technique involves
mutation of microbial genes by
random insertion of a tagged
transposon. After growth of a
mixed population in a host,
mutated genes that are absent
and hence required for virulence
are identified with the help of
the tag.
OPSONOPHAGOCYTOSIS

Increased uptake of bacteria by


host cells due to binding of
antibody or complement.
-HAEMOLYSIS

Zone of clearing around


bacterial colonies grown on
blood agar, caused by lysis of red
blood cells.
HAEMOLYSIN

Material that lyses red blood


cells.

222

and C (REFS 14,15) and a protein that has similarity to


the fibronectin-binding protein (FBP) PavA (pneumococcal adhesion and virulence A) from S. pneumoniae16.
Homologues of PavA are present in all four of the streptococcal species discussed in this review (TABLE 2). SloC
belongs to the lipoprotein receptor antigen I family
(LraI)13 and is contained within an operon that
encodes an ATP-binding cassette (ABC) transport system. Two similar systems have been identified in
S. pneumoniae psaBCAD and adcCBA17 in which
they have been shown to be involved in the transport of
divalent cations. The psa operon has also been shown to
be important in the virulence of S. pneumoniae18,19, and
is probably involved in attachment18,20. Whether LraI
family proteins are actually adhesins or instead have a
role in the regulation of adhesion through the transport
of metal ions is still unclear. Interestingly, an isogenic
mutant of S. mutans lacking sloC was not deficient in the
ability to cause caries, but showed reduced virulence in a
rat model of endocarditis13.
The metabolism of S. mutans is key to the pathogenesis of dental caries. Analysis of its genome sequence
shows that S. mutans can metabolize a wider variety of
carbohydrates than any other Gram-positive microorganism that has been sequenced so far. Genes for the
transport and metabolism of glucose, fructose, sucrose,
lactose, galactose, mannose, cellobiose, -glucosides, trehalose, maltose, raffinose, ribulose, mellobiose, starch,
isomaltosaccharides and possibly sorbose are present in
its genome21. The fermentation of these carbohydrates
is the principal source of energy for S. mutans. The
glycolytic pathway leads to the production of pyruvate,
which is then reduced to lactic acid, formate, ethanol
and acetate. The resulting acidification of the local environment is responsible for the damage that occurs in
caries. As the organism itself needs to be able to grow in
this environment, it is acid tolerant. Acid tolerance is
based on the presence of a membrane-bound, acidstable, proton-translocating F0F1 ATPase that can
maintain the intracellular pH at 7.5 (REF. 2).
Streptococcus agalactiae

The group B streptococci (GBS) which are all strains


of S. agalactiae are the main cause of neonatal sepsis,
pneumonia and meningitis in western Europe and the
United States, and are emerging pathogens in immunocompromised adults22. GBS can be subclassified into
serotypes according to the immunogenic type of their
polysaccharide capsule. There are currently nine known
serotypes; serotype III is particularly important because

| DECEMBER 2003 | VOLUME 1

it causes the majority of infections in neonates23.


Colonization of the recta and vaginas of pregnant
women can cause infection of the amniotic cavity.
Newborns are colonized during delivery; invasion probably occurs through the neonatal lung, and the organism can spread systemically to cause sepsis. GBS can
invade both alveolar epithelial and endothelial cells 24,25.
GBS express a wide range of products that are implicated in virulence (FIG. 1) and SIGNATURE-TAGGED MUTAGENESIS
has identified a range of genes that are involved in the
ability of the organism to survive in the host26. The
polysaccharide capsule is important in protecting the
organism from complement by preventing deposition
of the opsonic fragment C3b (REF. 27). The complement
pathway is a key element of the host immune system
and consists of a series of serum proteins that can be
activated in a cascade mechanism (FIG. 2). The activation
of this pathway leads to the production of opsonins
(which attach to the bacterium and target it for destruction), as well as chemotactic molecules that attract
immune cells to the site of infection.
GBS can attach to human laminin using lamininbinding protein (Lmb), a lipoprotein that has similarity
to the LraI adhesin family28 (see above). Homologues of
Lmb are present in the genomes of each of the four
species of streptococci discussed here. Other virulence
factors associated with GBS are the - and -proteins.
The role of the -proteins in the pathogenesis of infection is unclear, but disruption of the C antigen gene
attenuates the virulence of GBS29 and streptococcal
-protein has been shown to bind to the crystallizable
fragment of IgA (IgA-Fc) and to the complement-control
protein factor H. There are several FBPs in the GBS
genome. One of these, FbsA (fibrinogen-binding protein from S. agalactiae)30 (gene number gbs1087 in the
genome of NEM316 (REF. 31)), protects GBS from
OPSONOPHAGOCYTOSIS, whereas the other (gbs1263) is
similar in sequence to PavA, a known virulence factor
of S. pneumoniae (TABLE 2). GBS can also express a range
of other surface proteins, which were identified in the
genome sequence. Glaser and co-workers31 analysed
the genome of the serotype III GBS strain NEM316
and found 30 open reading frames that encode surface
proteins bearing a cell-wall-sorting-signal motif. One of
these surface proteins is the C5a peptidase, which
cleaves the complement factor C5a, a chemoattractant
for neutrophils32.
GBS are -HAEMOLYTIC when grown on blood agar,
and this has been shown to be a result of the production of a HAEMOLYSIN (CylE)33. The use of isogenic
mutants has shown that CylE production is associated
with damage to lung epithelial and endothelial cells,
brain epithelial cells and macrophages3437. CylE also
stimulates the production of nitric oxide from
macrophages37, can trigger macrophage apoptosis (programmed cell death)38, promotes GBS invasion of
human lung epithelial cells and the release of interleukin-8 (IL-8) (REF. 39), and has been shown to contribute to virulence in animal models4042. This
haemolysin is therefore important in the pathogenesis
of GBS infection.

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Table 2 | Distribution of selected virulence factors


Organism

Virulence factor

Gene number of virulence factor, or closest match


S. mutans
S. agalactiae
S. pyogenes
S. pneumoniae
(2603V/R)
(SF370)
(TIGR4)

Streptococcus
mutans

Antigen I/II (SpaP)


GbpA
GbpC
WapA
SloC
PavA-like protein

SMU0610
SMU2112
SMU1936
SMU0987
SMU0184
SMU1449

SAG1283

SAG1283

SAG1533
SAG1190

mtsA
fbp

SP1650
SP0966

Streptococcus
agalactiae

Lmb
-protein
-proteins
FbsA
PavA-like protein
C5a-peptidase
Haemolysin (CylE)

SMU1302

SMU1449

SAG1234
SAG0032
SAG0433
SAG1052
SAG1190
SAG0416 (truncated)
SAG0669

lmb
SPy0469

fbp
scpA

SP1002
SP1772

SP0966

Streptococcus
pyogenes

SfbI

SfbII

Fbp54 (PavA-like)
SMU1149
C5a-peptidase

Mac

SIC

Haemolysins:
SLO

SLS

Pyogenic exotoxins:
SpeA, SpeC, SpeG,
SpeH, SpeJ, SpeK,
SpeL, SSA, SMEZ,
SMEZ2
GRAB

DNase

Streptokinase

Hyaluronidase

SpeB (cysteine protease)

SAG1190
SAG0416

SpyM3_0098*
SpyM3_0104*
fbp
scpA
SPy0863
sic

SP0966

slo
sagA

SP1923

Superantigens
only present in
S. pyogenes

SAG0788

grab
mf2
ska
hylA
speB

SP0314

Pneumolysin
PspA
PspC
Autolysin
PavA
PsaA
Hydrogen peroxide
NAD oxidase
Superoxide dismutase
Hyaluronidase

SAG1190
SAG1533

SAG0958
SAG0788
SAG1197

slo

fbp
mtsA

nox
sodA
hylA

SP1923
SP0117
SP2190
SP1937
SP0966
SP1650

SP1469
SP0766
SP0314

Streptococcus
pneumoniae

SMU1449
SMU0184

SMU1117
SMU0629

*Best matches are in strain MGA5315, so the gene numbers for this strain are given. FbsA, fibronectin-binding protein from S. agalactiae;
fbp, fibronectin-binding protein; Gbp, glucan-binding protein; GRAB, G-related 2-macroglobulin-binding protein; Lmb, laminin-binding
protein; Mac, Mac1-like protein; PavA, pneumococcal adhesion and virulence A; PFBP, pyogenes fibronectin-binding protein; PsaA,
pneumococcal surface antigen A; Psp, pneumococcal surface protein; Sfb, streptococcal fibronectin-binding protein; SIC, streptococcal
inhibitor of complement; SLO, streptolysin O; SloC, S. mutans LraI operon C; SLS, streptolysin S; SMEZ, streptococcal mitogenic exotoxin Z;
SpaP, streptococcal protein antigen P; Spe, streptococcal pyogenic exotoxin; SSA, streptococcal superantigen A; WapA, wall-associated
protein A.

The pathogenesis of neonatal disease caused by


GBS is therefore related to three main factors: first, the
ability to colonize the vaginas and recta of pregnant
women; second, the ability to avoid host defence
mechanisms once inside a susceptible host; and third,
the production of virulence factors, such as the
haemolysin CylE, that damage the host.
Streptococcus pyogenes

The group A streptococcus (GAS), S. pyogenes, is among


the most versatile of human pathogens. It can be carried
asymptomatically, and can cause common illnesses such
as pharyngitis, impetigo and scarlet fever. Under certain
circumstances it can also cause life-threatening diseases
such as sepsis, necrotizing fasciitis and toxic shock.As well
as causing these acute diseases, GAS is also associated with

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the post-infection sequelae of acute rheumatic fever


(swelling of the heart) and acute glomerulonephritis
(damage to the kidney). The virulence of GAS has been
reviewed recently43. The virulence factors of GAS are summarized in TABLE 1 and FIG. 1.
GAS produces a range of molecules that might
function in adhesion 44, including the lipoteichoic
acid that is present in the GAS cell wall 45 and protein adhesins such as M-protein and FBPs. M-protein
mediates attachment to CD46, which is present on
the surface of keratinocytes 46. FBPs are important
for adhesion to both respiratory epithelium and
skin cells. The FBPs include (streptococcal
fibronectin-binding protein I) SfbI47, which mediates attachment to respiratory epithelial cells 48,
SfbII 50, fibronectin-binding protein 54 (FBP54) 50,

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a
Bacterial
antigens

Antibody

C3

C4
C1
C4a

C4b

C5
C5 convertase

C3 convertase
C2a C4b

Inflammation

C2a C4b C3b

C3a

C2
Bacterium

Bacterium

C5a

C5b

C3b
Bacterium

C1
C3b
C4b

C2b

C2a
C2a
Macrophage

b
C3 convertase

C3

C3b Bb

Late events in pathway,


including formation of
membrane-attack complex

C5 convertase
Inflammation

C5

C3b Bb C3b

Factor B
C3a
C5b

C3b

Factor D

Bacterium

C5a

Bacterium

Bacterium

Ba
C3b
C3b
Bb

Bb
Macrophage

Late events in pathway,


including formation of
membrane-attack complex

Figure 2 | The complement system. The complement system can be activated by both the innate and adaptive immune
systems. a | The classical pathway of complement activation, which involves the recognition of bacterial antigens by antibodies.
b | The alternative pathway, which is activated by the binding of complement component C3b to the bacterial surface. In the
classical pathway, the early steps culminate in the formation of the C3 convertase (comprising components C4b and C2a), which
rapidly cleaves C3 into C3a (a chemoattractant) and C3b the main opsonic fragment of the early activation steps which binds
to the bacterial surface. Some C3b also binds to the C3 convertase itself, forming the C5 convertase, which cleaves the C5 into
C5a (which can diffuse away) and C5b, which initiates formation of the membrane-attack complex. In the alternative pathway, a C5
convertase is generated in an antibody-independent manner.

ENDOGLYCOSIDASE

An enzyme that hydrolyses


non-terminal glycosidic linkages
in oligosaccharides or
polysaccharides.

224

protein F2 (REF. 51) and PFBP 52. It is interesting to


note that S. pneumoniae produces a protein known
as PavA 16, which has a high level of similarity to
FBP54 and is involved in fibronectin binding and
virulence. The hyaluronic-acid capsule of GAS can
bind to CD44 on human epithelial cells 53, and this
binding leads to cytoskeletal rearrangements and the
opening of intercellular junctions, thereby facilitating access to deeper tissues54.
One of the virulence factors that GAS uses to avoid
the process of opsonophagocytosis is M-protein,
which can bind complement-control proteins, such as
factor H and a factor-H-like protein, to reduce
opsonization of GAS by the alternative complement
pathway55,56. C4b-binding protein can also be bound
by M-protein, and this results in reduced phagocytosis

| DECEMBER 2003 | VOLUME 1

of the organism. Phagocytosis can also be reduced by


the binding of host fibronectin to the M-protein57.
The binding of fibrinogen and albumin to the bacterial cell effectively disguises the antigenic surface of the
bacterium and protects it from attack by complement58. GAS can also interfere with antibody-mediated activation of the classical complement pathway
by producing proteins, including an ENDOGLYCOSIDASE
(EndoS), that interact with bound immunoglobulin
to prevent binding of the first component of the classical pathway (C1) and initiation of the complement
cascade59,60 (FIG. 2). Like GBS, GAS also produces a C5a
peptidase on the surface of the bacterium, which
destroys the chemotactic peptide C5a that is generated by complement activation and is responsible
for neutrophil recruitment to the site of infection61.

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MEMBRANE-ATTACK COMPLEX

A collection of late complement


components that damage the cell
membrane.
PYOGENIC EXOTOXINS

Toxins that induce fever.


SUPERANTIGEN

A protein that activates T cells


non-specifically.
STREPTOCOCCAL TOXIC-SHOCK
SYNDROME

A disease associated with


decreased blood pressure and
multi-organ failure.

The presence of the hyaluronic-acid capsule acts as a


physical barrier to prevent the interaction of opsonins
with their appropriate receptors on phagocytes62.
GAS also produces a protein known as Mac
(Mac1-like protein), which has homology to CD11b
the alpha chain of complement receptor 3 (CR3)
which is the receptor on phagocytes that recognizes
C3b64. The Mac protein can bind to and block CD11b
and can also block the activity of FcIII (CD16),
which is one of the receptors that recognize IgG. Some
of the CR3 receptor molecules in the membrane are
linked to CD16, and it has been shown that blocking
CR3 not only blocks phagocytosis mediated by C3b,
but can also reduce antibody-mediated phagocytosis64.
Therefore, GAS produces a protein Mac that can
block phagocytosis by two important opsonins in
serum (complement and antibody).
M1 strains of GAS also synthesize a protein known
as streptococcal inhibitor of complement (SIC)65. This
protein is highly polymorphic (one of the most polymorphic bacterial proteins known) and inhibits complement-mediated lysis by preventing the uptake of
C5b57 complexes of complement onto cell membranes66. It is unlikely that inhibition of the complement
MEMBRANE-ATTACK COMPLEX (MAC) is the primary role of
SIC in vivo, as GAS is already resistant to MAC by virtue
of its thick cell wall. However, mutants that lack SIC colonized the throats of mice less effectively than wild-type
organisms, indicating that the protein does have a role
in GAS virulence. The role of SIC is probably linked to
its ability to inhibit secretory proteinase inhibitor,
lysozyme67 and antimicrobial peptides68.
Streptolysin O (SLO) is a member of the cholesteroldependent cytolysin family. It is related to pneumolysin,
the equivalent toxin from S. pneumoniae (discussed
below). SLO is lytic to all cells that contain cholesterol in
their plasma membrane and also has a range of activities at sublytic concentrations, including interference
with phagocyte function, increased cytokine secretion
and induction of apoptosis. SLO has been proposed to
mediate the translocation of another toxin, NAD glycohydrolase, into keratinocytes through the SLO pore69.
The use of isogenic mutants has confirmed that SLO has
a role in the virulence of GAS70,71.
Streptolysin S (SLS) is one of the most potent cytotoxins known and is delivered to target cells most
effectively by direct contact with the bacterium72. The
contribution of SLS to GAS virulence has been examined
in a murine model of necrotizing soft-tissue infection73.
In this model, wild-type bacteria elicit ulceration, with
bacterial proliferation, neutrophilic inflammation and
histopathological evidence of vascular injury and tissue
necrosis. Isogenic SLS-negative versions of GAS do not
produce ulcers, and bacteria are cleared with minimal
tissue injury and inflammation. These results indicate
that SLS is an important virulence factor in necrotizing
soft-tissue infection and also indicate that SLO is not
required for ulcer formation70.
The streptococcal PYOGENIC EXOTOXINS (Spes) are a
family of SUPERANTIGENIC proteins that includes the
bacteriophage-encoded SpeA and SpeC, as well as SpeG,

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SpeH, SpeJ, SpeK, SpeL, streptococcal superantigen A


(SSA), streptococcal mitogenic exotoxin Z (SMEZ) and
SMEZ2. These superantigenic toxins are thought to be
associated with STREPTOCOCCAL TOXIC-SHOCK SYNDROME
(STSS). Superantigens are able to bind simultaneously
to major histocompatibility complex (MHC) class II
molecules on antigen-presenting cells and to the T-cell
receptor74 (FIG. 1). This binding leads to the activation of a
large number of T cells that express specific V- subsets,
and leads to secretion of cytokines such as tumournecrosis-factor- (TNF-), IL-1, IL-2 and interferon-.
The release of these cytokines results in the drop in
blood pressure and multiorgan failure that is characteristic of STSS.
GAS interacts directly with human host components such as plasminogen and acquires a plasmin-like
enzymatic activity75. Owing to the production of active
proteases, GAS has developed methods of protection
against proteolytic degradation. The recruitment of the
human protease inhibitor 2-macroglobulin (2-M) to
the surface of GAS has been proposed as such a mechanism76. The interaction between GAS and 2-M is
mediated mainly by the streptococcal surface protein
G-related 2-macroglobulin-binding protein (GRAB)76.
Mutant strains that lack GRAB have been shown to be
less virulent after intraperitoneal infection of mice76 and
in a mouse-skin model of infection77. GAS synthesizes a
range of other proteins that might be involved in the
disease process. These include DNases A, B, C and D,
streptokinase, C5a-peptidase, hyaluronidase and
streptococcal pyrogenic exotoxin B (SpeB). SpeB was
originally thought to be a superantigen, but is now
known to be a potent cysteine protease. SpeB has a role
in virulence, and studies using isogenic mutants show
that loss of SpeB is associated with decreased virulence in animal models of invasive disease78,79. This
protease can cleave human immunoglobulins80, vitronectin, fibronectin and other host proteins. SpeB can
also generate biologically active molecules such as IL-1,
kinins and histamine. These factors might aid the
spread and dissemination of the organism in the host.
Streptococcus pneumoniae: the pneumococcus

The pneumococcus is carried asymptomatically in the


nasopharynx in up to 60% of the population. However,
under appropriate conditions, it can cause diseases such
as otitis media (middle-ear infection), pneumonia,
bacteraemia and meningitis. One of the main virulence
factors of the pneumococcus is the polysaccharide
capsule, which is believed to be anti-phagocytic81.
As well as the capsule, the pneumococcus produces a
range of other molecules that are associated with pathogenesis (FIG. 1). These include components of the cell wall,
a haemolysin (pneumolysin), cell-surface proteins
(choline-binding proteins and LPXTG-anchored proteins) and hydrogen peroxide.At the time of writing, three
signature-tagged-mutagenesis screens have been carried
out on the pneumococcus, which have identified extensive lists of putative virulence factors8284. This review will
focus on selected virulence factors, the roles of which
have been partially defined using model systems.

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EPENDYMAL CILIA

Specialized ciliated cells that line


the ventricles of the brain.

226

The cell wall of the pneumococcus is important in


mediating attachment of the bacterium to activated
lung cells85. The phosphorylcholine of the cell wall binds
to the receptor for platelet-activating factor (PAF). The
PAF receptor is upregulated during the inflammatory
response and during viral infection, which might
explain the increased occurrence of pneumococcal
pneumonia following viral infection. The cell wall is also
involved in initiating the inflammation that is associated
with pneumococcal infection86.
The haemolysin that is produced by the pneumococcus pneumolysin is a pore-forming protein that
belongs to the same family as SLO (described above). This
toxin does not have a typical secretion signal, is released
by the action of the cell-bound autolysin and has been
shown to function in pathogenesis in several animal
models of disease8793. Pneumolysin has a range of
activities that have a role in these virulence models,
including the ability to stimulate the production of
inflammatory mediators, such as TNF-, IL-194, nitric
oxide95, IL-8 (REF. 96), and prostaglandins and
leukotrienes97. This toxin also activates phospholipases in
endothelial cells98 and is toxic to pulmonary endothelial
and epithelial cells99,100. In addition, pneumolysin can
inhibit nonspecific defences such as respiratory cilial
beat101 and has a role in evasion of the immune system
as it can inhibit phagocyte and lymphocyte function102,103
and interferes with the complement pathway104.
Pneumolysin has at least two main activities that are
important in its role in pathogenesis: the ability to form
pores and the ability to activate the complement pathway.
The functional regions for these activities have been
located within the molecule, and both activities have been
shown to be important in the causation of disease105. This
toxin is important in the pathogenesis of meningitis as it
can cause damage to the EPENDYMAL CILIA of the brain106 and
can induce apoptosis of brain cells91. The neurotoxicity of
pneumolysin has been shown to be due to alterations in
calcium flux into cells and in signalling through the
activation of the p38 mitogen-activated protein
kinase91,107. Pneumolysin has recently been shown to
bind to Toll-like receptor 4 (TLR4)108. This interaction
with TLR4 was essential for the protection of mice
against invasive disease caused by the pneumococcus.
Pneumolysin therefore has a diverse and important role
in the pathogenesis of pneumoccocal infections.
The surface proteins of the pneumococcus can be
broadly divided into three families according to how they
are linked to the cell surface: choline-binding proteins,
LPXTG-anchored proteins and lipoproteins. Cholinebinding proteins are anchored to the cell surface by a
non-covalent interaction of a repeat region at the carboxy-terminal end of the protein with the phosphorylcholine of the pneumococcal cell wall. The cholinebinding protein family contains several proteins that are
known to be important for virulence, including the
autolysin LytA, pneumococcal surface protein A (PspA)
and pneumococal surface protein C (PspC). PspA is a
highly variable protein that is expressed by all clinically
important pneumococcal serotypes109. Mutant bacteria
that are unable to produce PspA are less virulent in

| DECEMBER 2003 | VOLUME 1

models of systemic disease because they are more easily


cleared from the bloodstream110. PspA inhibits complement activation and reduces the effectiveness of complement receptor-mediated clearance mechanisms111, and
might function by preventing the deposition of C3b on
the cell surface or by inhibiting the formation of the
alternative pathway component C3 convertase112. It has
also been proposed that PspA binds lactoferrin and that
it is therefore involved in iron acquisition113. PspC (also
known as CbpA or SpsA) is involved in the adhesion of
bacteria to the nasopharynx; PspC-deficient mutants colonize the nasopharynx of rats less effectively and show
reduced binding to human cells114. PspC has several
activities that might be important in the disease
process, including binding to complement component
C3 (REF. 115). Some forms of PspC can also bind to the
complement-control protein factor H116,117. PspC also
stimulates the production of IL-8 from pulmonary
epithelial cells and might therefore be involved in
immune-cell recruitment and chemotaxis118. It also
mediates attachment to the polymeric immunoglobulin
receptor (pIgR) and translocation of pneumococci
across human nasopharyngeal epithelial cells119. Other
choline-binding proteins that are known to be involved
in virulence include the major autolysin, LytA120, which is
thought to mediate its effect through the release of other
virulence factors and cell-wall components. CbpD,
CbpE, CbpG, LytB and LytC have a role in the colonization of the nasopharynx121, and mutations in cbpE and
cbpG reduce adherence to human cells. CbpG, which
might be a serine protease, also has a role in sepsis121.
Members of another family of pneumococcal surface
proteins are anchored to the cell wall by covalent linkage
to peptidoglycan through a carboxy-terminal motif
LPXTG. This motif is recognized by a sortase enzyme,
which links the threonine residue of the motif to the cell
wall (BOX 1). Analysis of the pneumococal genome
sequence122 reveals a family of these proteins, including
hyaluronidase and neuraminidase enzymes. Hyaluronidase breaks down the hyaluronic-acid component
of mammalian connective tissue and extracellular
matrix and is secreted by 99% of pneumococcal clinical
isolates123. Deletion of the hyaluronidase gene alone does
not affect virulence in a mouse model of infection, but
deletion of hyaluronidase in a pneumolysin-negative
background reduces the virulence of the pneumolysinnegative mutant124. Neuramindase cleaves N-acetylneuraminic acid from glycolipids, lipoproteins and
oligosaccharides on cell surfaces and in body fluids125.
The pneumococcus has genes for the production of
at least three neuraminidases, and the role of neuraminidase A in infection has been investigated. It has a
role in nasopharyngeal colonization and development
of otitis media in the chinchilla model126, but does not
have a role in meningitis-associated deafness127. Analysis
of the pneumococcal genome sequence122 reveals the
presence of four putative sortases. The major sortase
(sortase A) has been deleted128 and was shown to release
neuraminidase into the growth medium rather than
anchoring it to the cell surface. The srtA mutant did not
show defects in virulence in mice, but had a reduced

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REVIEWS

Table 3 | Features of streptococcal genomes*


Organism and strain

Genome size (bp)

Number of genes

References

2,030,921

2,241

21

2603V/R

2,160,267

2,169

146

NEM316

2,211,485

2,387

31

MGAS315

1,900,521

2,085

147

MGAS8232

1,895,017

2,097

140

SF370

1,852,441

1,964

148

SSI-1

1,894,245

1,973

149

TIGR4

2,160,837

2,236

122

R6

2,038,615

2,219

150

S. mutans
UA159
S. agalactiae

S. pyogenes

S. pneumoniae

*Data taken from the Comprehensive Microbial Resource (see Online links).

ability to bind to pharyngeal cells128. This suggests that


anchoring of LPXTG proteins to the cell surface might
have a role in attachment and colonization rather than
in gross virulence of the pneumococcus.
Several lipoproteins have been shown to be important in the adhesion of the pneumococcus to cells and
to the virulence of the organism. Pneumococcal surface
antigen A (PsaA) is part of an ABC transporter system
that transports manganese129. psaA mutants show
reduced binding to cells, reduced virulence and
increased susceptibility to oxidative damage130. It is still
unclear whether the decreased binding of psaA mutants
is because PsaA itself acts as an adhesin or whether it
acts indirectly, perhaps by modulating the expression
of other adhesins by altering the concentration of
manganese within the cell. The final adhesin to be discussed here is PavA16. This protein is present on the
surface of the pneumococcus, despite the absence of a
typical secretion signal, and has high sequence similarity
to an FBP from other streptococci. Inactivation of the
pavA gene in the pneumococcus reduces the binding of
bacteria to fibronectin and markedly reduces the virulence of the organism.
Other proven and potential virulence factors produced by the pneumococcus include a surface-bound
131
ENOLASE that can bind plasmin and plasminogen , a
superoxide dismutase132, NADH oxidase133, hydrogen
peroxide134 and components of iron-uptake systems135.
Genomics and gene-expression analysis

ENOLASE

Also known as phosphopyruvate


hydratase. An enzyme that
catalyses the conversion of
2-phospho-D-glycerate to
phosphoenolpyruvate and water.

The availability of genome sequences has allowed comparisons to be made between species and between strains
at the genomic level. Several strains of the streptococci
described here have been sequenced (TABLE 3). There are
substantial differences between the genome sizes and
total numbers of genes, even for different strains of the
same species. Variation between strains can be large,
especially in a naturally transformable organism such as
S. pneumoniae, and there is a need to understand how
the gene content of a particular strain affects its virulence.
A comparison of the genomes of the four strains of

NATURE REVIEWS | MICROBIOLOGY

streptococci described here (using the nine genome


sequences mentioned in TABLE 3) shows there are several
hundred genes that are common to the four species.
Using S. pneumoniae as the reference genome, it has 308
genes in common with GBS (2603V/R), 312 with
S. mutans and 266 with GAS (SF370). These genomic
comparisons were made using the Comprehensive
Microbial Resource136. Using a stringent cut-off (90%
similarity), 190 genes are common to all four genomes.
Not surprisingly, most of these genes are concerned with
conserved functions such as nucleic-acid metabolism,
transport processes and protein synthesis.
The conservation of virulence-associated genes is
shown in TABLE 2. Each organism has a unique set of virulence-associated genes, which presumably reflects the
ability to cause the different ranges of disease that are
associated with each. Only a relatively limited number of
virulence-associated genes are conserved across all four
species (TABLE 3). As well as comparing total gene content,
it is also possible to compare gene organization within
the genomes of these pathogens. Such a comparison of
the genomes is shown in FIG. 3, in which each dot represents the position in the genome of a conserved sequence.
It can be seen from the comparison of two strains of the
same species that when gene order is conserved (FIG. 3d),
most genes lie on an oblique line which shows that the
genes are in the same position in both chromosomes. In
comparisons of S. pneumoniae with the other streptococcal species, there is less relationship between the positions
of the genes in the genomes, resulting in a more random
distribution of points. Gene position is not, therefore,
highly conserved between the streptococcal species.
Genome comparisons also highlight the differences in
gene content between different strains of the same organism, and show the importance of gene transfer by transformation and by phages in determining the potential of
any strain to cause disease139140.
The use of microarrays has also allowed a comparison
of expression levels for the entire transcriptome. Several
elegant experiments have been done with GAS using
both microarrays and Taqman real-time RTPCR (polymerase chain reaction after reverse transcription of RNA)
to determine which genes show increased expression
during interaction with the host, including interaction
with polymorphonuclear leukocytes (PMNLs)141, mouse
soft-tissue infection142 and acute pharyngitis143. During
phagocytosis of GAS, 276 genes (representing 16% of the
genes in the genome) were differentially regulated. At
least 11 genes that are known to be associated with virulence were upregulated, including mac, speH, speB, smeZ
and endoglycolase S (see above for a discussion of these
virulence factors). It is interesting to note that several of
the factors that are upregulated on interaction with
human PMNLs are responsible for resistance to phagocytosis. In addition, genes encoding proteins that are
responsible for detoxifying cell-damaging reactive
oxygen species were also upregulated. Analysis of
genes involved in transcription and transcriptional
regulation showed an upregulation of mga, which is
known to regulate virulence factor production144, and
of ihk/rr, a two-component system with homology to

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REVIEWS

S. agalactiae NEM316 (position kb1)

S. mutans UA159 (position kb1)

a system from Staphylococcus aureus that regulates


virulence-factor production145. Voyich and co-workers
were able to show that the ihk/rr system regulates
genes that are important in the evasion of killing of
GAS by PMNLs and, importantly, increased expression of these genes was detected in samples taken from
infected humans. Studies such as these will allow both
genomic and expression differences to be associated
with the ability of bacteria to cause disease.

b
2,022
1,820
1,618
1,416
1,214
1,013
811
609
407
205
4

432

861

1,290

1,719

2,204
1,984
1,764
1,544
1,324
1,104
884
664
444

2,149

Summary

224
3

S. pneumoniae TIGR4 (position kb1)

861

1,290

1,719

2,149

S. pneumoniae TIGR4 (position kb1)

d
1,886

2,038

S. pneumoniae R6 (position kb1)

S. pyogenes MGAS8232 (position kb1)

432

1,697
1,509
1,321
1,133
945
756
568
380
192
4

432

861

1,290

1,719

2,149

1,834
1,630
1,426
1,222
1,019
815
611
407
203
0

S. pneumoniae TIGR4 (position kb1)

432

864

1,296

1,728

2,160

S. pneumoniae TIGR4 (position kb1)

Figure 3 | Genomic comparisons between streptococcal species. All proteins with a


similarity of more than 90% were compared using the Comprehensive Microbial Resource137.
Each point on the graph represents a common gene, and the distance along the x and y
axes represents the position of the gene within the genome. Streptococcus pneumoniae
was used as the reference organism and was compared against (a) Streptococcus mutans,
(b) Streptococcus agalactiae (NEM316), (c) Streptococcus pyogenes (MGAS8232) and
(d) a laboratory strain of S. pneumoniae (R6). This allows the arrangement of similar genes in
different genomes to be visualized. Where the arrangement of genes in different genomes is
similar, the points on the graph can be joined to form an oblique line (d). Where the genes in
different genomes are not in the same positions, a more scattered plot is observed (ac).
Gene position is not highly conserved in these streptococcal species.

1.
2.

3.

4.

5.

6.

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The four species of streptococcus described here cause


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GBS cause disease in hosts that can be considered to
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Competing interests statement


The author declares that he has 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
CylE | EndoS | FBP54 | FbsA | GRAB | Lmb | M-protein |
pneumolysin | PsaA | PspA | PspC | SLO | SloC | SpaP | SpeB
FURTHER INFORMATION
Comprehensive Microbial Resource:
http://www.tigr.org/tigr-scripts/CMR2/CMRHomePage.spl
Infectious disease information
GAS:
http://www.cdc.gov/ncidod/dbmd/diseaseinfo/groupastreptococ
cal_g.htm
GBS:
http://www.cdc.gov/ncidod/dbmd/diseaseinfo/groupbstrep_g.htm
Streptococcus pneumoniae:
http://www.cdc.gov/ncidod/dbmd/diseaseinfo/streppneum_a.htm
Tim Mitchells web site:
http://www.gla.ac.uk:443/ibls/staff/staff.php?who=159525
Access to this interactive links box is free online.

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