REVIEWS

The role of Streptococcus pneumoniae

virulence factors in host respiratory
colonization and disease
Aras Kadioglu*, Jeffrey N. Weiser, James C. Paton and Peter W. Andrew*
Abstract | Streptococcus pneumoniae is a Gram-positive bacterial pathogen that colonizes
the mucosal surfaces of the host nasopharynx and upper airway. Through a combination of
virulence-factor activity and an ability to evade the early components of the host immune
response, this organism can spread from the upper respiratory tract to the sterile regions of
the lower respiratory tract, which leads to pneumonia. In this Review, we describe how
S.pneumoniae uses its armamentarium of virulence factors to colonize the upper and lower
respiratory tracts of the host and cause disease.

Nasooropharynx
The parts of the pharynx that
are above and below the
palate, respectively.
Carrier state
The state of carrying an
organism without
manifestation of disease.
Herd immunity
Immunity induced by
vaccination that protects a
proportion of unvaccinated
individuals.
*Department of Infection,
Immunity & Inflammation,
University of Leicester,
Leicester LE1 9HN, United
Kingdom.
Streptococcus pneumoniae (often referred to as the
pneumococcus) is the most common bacterial respira-
tory pathogen in the United Kingdom, and frequently
causes community-acquired pneumonia, which can
have mortality rates of more than 20% in patients with
concurrent pneumococcal septicaemia1,2. Worldwide,
the situation is worse; pneumococcal septicaemia
is a major cause of infant mortality in developing
countries, where it causes approximately 25% of all
preventable deaths in children under the age of 5 and
more than 1.2 million infant deaths per year3,4. In
countries that have a high prevalence of HIV-1 infec-
tion, there has been a significant increase in the rate
of pneumococcal pneumonia and associated bacter-
aemia, and this increase has been most marked in
young adults.
The pneumococcus resides on the mucosal surface
of the upper respiratory tract. It can be readily cultured
from the nasooropharynx of humans and, occasion-
ally, other large mammals that live in association with
humans. Although colonization at this site seems to
be asymptomatic, if the organism gains access to the
normally sterile parts of the airway a rapid inflamma-
(occult bacteraemia). Thus, all pneumococcal disease
begins with the establishment of colonization, that is,
the creation of the carrier state5,6. None of the common
forms of pneumococcal disease, however, promote
pneumococcal transmission, which implies that the
virulence characteristics of the pneumococcus are prob-
ably adaptations that increase its persistence within a
host during colonization.
Person-to-person spread is thought to occur through
direct contact with the secretions of colonized individ-
uals. Once acquired, an individual strain can be car-
ried for weeks to months before its eventual clearance.
However, it remains unclear how effective colonization
is as an immunizing event. Colonization is most com-
mon in early childhood; most infants acquire one or
many strains sequentially or simultaneously. The rates
of carriage vary widely among the 91 known pneumo-
coccal capsular serotypes, which express structurally
and antigenically different capsular polysaccharides.
The factors that are responsible for these differences,
and the variations in serotype distribution between
regions and over time, are not understood. After the
first years of life, the rates of colonization decline to less

Department of Microbiology,
School of Medicine, University
of Pennsylvania, Pennsylvania
19104-6076, USA.
tory response ensues that results in disease. The most than 10% in adult populations. The importance of car-
common manifestations of pneumococcal disease riage by young children as the main reservoir for this
include acute otitis media, which involves infection pathogen has been demonstrated by the recent wide-

School of Molecular and
Biomedical Science,
University of Adelaide, South
Australia 5005, Australia.
Correspondence to A.K.
e-mail: ak13@leicester.ac.uk
doi:10.1038/nrmicro1871
of the middle-ear space, and pneumonia, which affects
the terminal airways. Bacteraemic infection, which is
associated with greater morbidity and mortality, typi-
cally occurs as a complication of pneumonia (bacter-
aemic pneumococcal pneumonia) or, less often, if the
organism spreads directly from its niche in the pharynx
spread use of the pneumococcal capsular polysaccha-
ride (CPS) conjugate vaccine (BOX1). Reduced carriage
of the organism in vaccinated children results in herd
immunity, which can have an impact on the frequency
of pneumococcal disease in those age groups that are
not receiving the vaccine7.

288 | april 2008 | volume 6 www.nature.com/reviews/micro

2008 Nature Publishing Group
 REVIEWS

Box 1 | New approaches to pneumococcal vaccination

Current pneumococcal vaccines are exclusively targeted at the capsular polysaccharide (CPS) of Streptococcus
pneumoniae, and these vaccines provide strictly serotype-specific protection. Polyvalent purified CPS vaccines were first
licensed in the late 1970s, and the current 23-valent formulation is effective against approximately 90% of disease-
causing serotypes in the United States and Europe. Unfortunately, CPSs are Tcell-independent antigens and are
therefore poorly immunogenic in young children, particularly for the five pneumococcal serotypes that most commonly
cause invasive disease in children145. The poor immunogenicity of CPS antigens has been overcome by conjugation to
protein carriers this converts them into Tcell-dependent antigens, which are considerably more immunogenic and a
pneumococcal CPSprotein conjugate vaccine (PCV) is now licensed in several countries. However, protection is still
serotype specific and, because of the high cost, the number of serotypes that are targeted has been reduced to seven.
Disturbingly, studies of nasopharyngeal colonization with S.pneumoniae during trials of PCV showed that, although the
carriage of vaccine serotypes was reduced, the vacated niche was promptly occupied by non-vaccine pneumococcal
serotypes that are potentially capable of causing disease146. Similar trends were also observed after the general
introduction of the conjugate vaccine in the United States147. Although PCV has dramatically reduced the incidence of
invasive disease that is caused by vaccine serotypes in the United States, increases in the incidence of bacteraemia caused
by non-included serotypes have also been reported148149. Thus, in the long term, the widespread introduction of PCV
might merely alter the serotype distribution of invasive pneumococcal disease, rather than reducing its overall burden. In
addition, the cost of PCV is high, and therefore it is unlikely that it will be used extensively in developing countries, where
the death rate of children from invasive pneumococcal disease is highest.
Clearly, there is an urgent need to develop alternative pneumococcal vaccines that do not suffer from these
shortcomings. The most promising approach to date is to develop vaccines that are based on pneumococcal proteins that
contribute to virulence and are common to all serotypes. Such vaccines need to be highly immunogenic and elicit
immunological memory even in infants, who respond well to Tcell-dependent protein antigens. Ensuring high expression
levels of antigenic proteins in recombinant bacteria will enable large-scale vaccine production at low cost, thereby
producing vaccines that are more affordable, particularly for developing countries. Several candidate protein vaccine
antigens have been identified, including non-toxic derivatives of pneumolysin, choline-binding proteins, such as PspA and
CbpA, metal-binding lipoproteins, such as PsaA and PiaA, the poly-histidine triad proteins PhtB and PhtE and the
neuraminidase NanA. Immunization with each of these proteins provided varying degrees of protection against challenge
by one or more S.pneumoniae strains in mouse models of sepsis, pneumonia or carriage146, and there is now clear evidence
that immunization with certain combinations of virulence proteins provides additive, or even synergistic, protection149151,
the most effective to date being a combination of Ply, PspA and CbpA152,153.

Mechanisms of colonization These isolates also elicited a comparable immune

Glycocalyx
Extracellular polysaccharide
material that is excreted by
epithelial cells and forms an
outer layer on epithelial
surfaces.
Experimental colonization of adults has been used to
investigate the host factors that affect susceptibility
to acquisition of S.pneumoniae and its subsequent
clearance8. These studies reveal that carriage induces
the production of both mucosal and systemic immu-
noglobulin, which is mainly strain- and type-specific.
In contrast to the high levels of serotype-specific
anti-capsular antibody that are generated following
administration of the conjugate vaccine, it is unclear
whether the relatively small amounts that are induced
by colonization are sufficient to enhance clearance. In
this regard, the decline in carriage rates after child-
hood is observed widely among the different pneu-
mococcal serotypes, which suggests that if exposure
during prior colonization events leads to immunity
it might not develop in a serotype-specific manner9.
Immunogenic major cell-surface proteins, particularly
pneumococcal surface protein A (PspA), also induce
antibody, but this response is predominately directed
at the more variable, exposed regions of PspA. The
level of pre-existing cross-reactive immunoglobulin to
PspA correlates with susceptibility to the establishment
of pneumococcal carriage8.
Animal models have also been used to define the
host and bacterial factors that contribute to pneumo-
coccal colonization (Table1). For example, isolates that
were previously examined in human experimental colo-
nization studies also colonized inbred adult mice when
a similar inoculum was used for a similar duration.
response to CPS and PspA, thereby demonstrating the
relevance of this animal model10. It should be noted,
however, that mice can also be colonized by a wide spec-
trum of pneumococcal serotypes, although their sus-
ceptibility to disease by different strains varies greatly.
Therefore, for certain pneumococcal serotypes, mice
are also useful models to study the complications that
arise from bacterial colonization, including pneumonia
and bacteraemic infection. The experimental findings
from mouse-colonization and airway-infection models
will be summarized in the following sections.
Interactions with host structures. Within minutes of
entering the nasal cavity, S.pneumoniae cells encounter
mucus secretions (FIG.1a). The expression of a capsule
reduces entrapment in the mucus, thereby allowing
the pneumococcus to access the epithelial surfaces 11.
Almost all pneumococcal CPSs are negatively charged,
which could increase their repulsion of the sialic acid-
rich mucopolysaccharides that are found in mucus12.
Initially, bacteria are detected within the glycocalyx layer
that overlies the respiratory and olfactory epithelium11
(FIG.1b). This might allow bacterial access to receptors
on the apical surface of the epithelial cells that line the
nasal spaces. Once at the epithelial surface, the expres-
sion of a thick capsule seems to be disadvantageous for
the pneumococcus, because of its inhibitory effect on
adherence. Most pneumococcal isolates that have been
investigated display phase variation between two forms

nature reviews | microbiology volume 6 | april 2008 | 289

2008 Nature Publishing Group
REVIEWS

Table 1 | Pneumococcal virulence factors and their main role in colonization and disease
Pneumococcal virulence Main role in colonization
factors and disease
Upper-airway colonization
Capsule Prevents entrapment in the nasal mucus, thereby allowing access to epithelial surfaces.
Also inhibits effective opsonophagocytosis.
ChoP Binds to rPAF on the epithelial surface of the human nasopharynx.
CbpA (also known as PspC) Binds to human secretory component on a polymeric Ig receptor during the first stage of
translocation across the epithelium.
NanA, BgaA and StrH Act sequentially to cleave terminal sugars from human glycoconjugates, which might
reveal receptors for adherence.
Hyl Breaks down hyaluronan-containing extracellular matrix components.
PavA Binds to fibronectin.
Eno Binds to plasminogen.
Competition in upper airway
Bacteriocin (pneumocin) Small antimicrobial peptide that targets members of the same species.
Respiratory-tract infection and pneumonia
Ply Cytolytic toxin that also activates complement. An important determinant of virulence in
in vivo models of disease. Wide range of effects on host immune components at sub-lytic
concentrations.
PspA Prevents binding of C3 onto pneumococcal surface. Also binds lactoferrin.
LytA Digests the cell wall, which results in the release of Ply.
PsaA Component of the ABC transport system, which is involved in resistance to oxidative stress.
PiaA and PiuA Component of the ABC transport system.
NanA and NanB Aid colonization by revealing receptors for adherence, modifying the surfaces of
competing bacteria that are within the same niche and/or modifying the function of host
clearance glycoproteins.
IgA Cleaves human IgA1.
This list of virulence factors is not exhaustive and only selected examples are shown. For detailed descriptions of virulence factors
see the main text. BgaA, -galactosidase; CbpA, choline-binding protein A; ChoP, phosphorylcholine; Eno, enolase; Hyl,
hyaluronate lyase; IgA, IgA1 protease; IgA1, immunoglobulin A1; LytA, autolysin A; Nan, neuraminidase; PavA, pneumococcal
adhesion and virulence A; PiaA, pneumococcal iron acquisition A; PiuA, pneumococcal iron uptake A; Ply, pneumolysin; PsaA,
pneumococcal surface antigen A; PspA, pneumococcal surface protein A; StrH, -N-acetylglucosaminidase.

Phase variation
A molecular mechanism that
leads to switching of the gene-
expression state; for example,
onoff expression.
Lipoteichoic acid
A teichoic acid species that is
connected to membrane
glycolipids. The
stereochemistry of LTAs and
the biosynthetic origin of the
glycerolphosphates are
different from those of wall
teichoic acids, which have
glycerol-phosphate backbones.
Human secretory
component
An epithelial glycoprotein that
is required for the active
transport of polymeric
immunoglobulins across
mucosal surfaces.
Glycoconjugate
A carbohydrate that is
covalently linked to other
chemical species.
that can be distinguished by their opaque or transparent
colony morphologies (discussed below)13. During the
initial stages of colonization, transparent variants that
express a thinner capsule and possess other character-
istics that promote binding to host tissues prevail over
opaque variants13.
By analysing human epithelial cells in culture, sev-
eral receptorligand interactions have been proposed.
The bacterial adhesins that are involved include phos-
phorylcholine (ChoP), which is a constituent of both
cell-wall-associated acids and lipoteichoic acids14. ChoP
is an unusual bacterial structural component that is a
cell-surface feature of several other microorganisms that
reside primarily in the upper respiratory tract, such as
Haemophilus influenzae, Actinobacillus actinomycetem-
comitans and commensal and pathogenic species of
the genus Neisseria. As many ChoP-expressing species
occupy a similar biological niche, and have a common set
of genes (licAD) for choline uptake and incorporation,
bacterial ChoP might be particularly important for host
airway colonization15. In H.influenzae, ChoP expression
enhances persistence in the upper respiratory tract16. In
the pneumococcus, ChoP mediates bacterial adherence
to the receptor for platelet-activating factor (rPAF) and
activates host cell signalling through this receptor. As
the natural ligand for rPAF platelet-activating fac-
tor (PAF) also contains ChoP, the pneumococcus
might mimic PAF to use its receptor, which is widely
distributed on host tissues such as the epithelial surface
of the human nasopharynx. Another adhesin is choline-
binding protein A (CbpA; also referred to as PspC or
SpsA), which is non-covalently anchored to ChoP. CbpA
binds to human secretory component, which is found on
the polymeric immunoglobulin receptor, and secretory
forms of immunoglobulin17,18. In addition, some isolates
express a pilus-like structure that adheres to an unknown
epithelial-cell receptor19,20.
Other studies have provided evidence for adhesive
interactions with host cell glycoconjugates. Pneumococcal
adherence to epithelial cells from the human pharynx is
inhibited by Nacetylglucosamine-(1,3)-galactose21.
The pneumococcus is also one of many pathogens that
have been reported to bind to Nacetylglucosamine-
(1,4)-galactose, which is a constituent of some
human glycosphingolipids22. In addition, S.pneumoniae
produces three surface-associated exoglycosidases:
a neuraminidase, NanA, a galactosidase, BgaA, and a
N-acetylglucosaminidase, StrH23. These enzymes act

290 | april 2008 | volume 6 www.nature.com/reviews/micro

2008 Nature Publishing Group
 REVIEWS

a 30 minutes b 1 day

c 3 days d 14 days

Figure 1 | Nasal colonization by Streptococcus pneumoniae. Progression of nasal colonization of BALB/c

Nature Reviewsmice with a
| Microbiology
serotype23F pneumococcal isolate. ad show the infection as it progresses from 30 minutes to 14 days after infection
(40 magnification). Bacteria (red) were detected using serotype-specific antisera. Mouse tissue was stained using DAPI
(4,6-diamidino-2-phenylindole; blue). The inset in c shows neutrophils stained (green) with an antibody to murine Ly6G.
Images courtesy of A. Roche, University of Pennsylvania, USA.

Basement membrane
A tissue structure that consists
of a network of collagen fibres
that attach the overlying
epithelial layer to the
connective tissue underneath.
sequentially to remove the terminal sugars that are found
on many human glycoconjugates and, therefore, might
unmask receptors for adherence, thereby affecting the
function of glycosylated host clearance molecules and/or
providing a nutrient source.
Binding to extracellular matrix components could
also promote bacterial adherence once the organ-
ism has gained access to basement membranes. The
expression of a surface-attached hyaluronidase could
facilitate bacterial spread through a matrix of the
hyaluronan-containing polysaccharide component
of connective tissues24. Pneumococcal adhesion and
virulence A (PavA) and enolase are two pneumococ-
cal surface adhesins that bind the extracellular-matrix
components fibronectin and plasminogen, respec-
tively 25,26. PavA is localized to the pneumococcal
outer cell surface and has been shown to be impor-
tant in virulence, as the absence of PavA significantly
reduces systemic bacterial loads and increases host
survival25. Pneumococcal enolase mutants also exhibit
attenuation in a model of respiratory infection, which
indicates that plasminogen binding has a role in
respiratorydisease26.
The pneumococcal factors that enable host epithelial
and tissue barriers to be breached during the progres-
sion from colonization to invasive infection are poorly
understood. In both mouse models and in humans,
opaque variants that express increased amounts of CPS
and are more resistant to opsonophagocytic killing are
selected for during the transition from the mucosal
surface to the bloodstream27,28.
Innate and adaptive immunity during colonization.
Between 1 and 3 days after the initiation of coloni-
zation in mice, there is an influx of neutrophils and
uptake of pneumococcal cells in the lumens of the nasal
spaces11 (FIG.1). The association of pneumococci with
neutrophils resembles the host response to infection of
normally sterile sites. However, this acute inflammatory
response (or mild suppurative rhinitis) is ineffective at
clearing the carrier state, and pneumococci persist on
the epithelial surface, even after resolution of the neu-
trophilic infiltrate (FIG.1d). Defined mutants that lack
expression of the pore-forming cytotoxin pneumolysin
show diminished neutrophil influx and persist for longer
periods in a model of nasopharyngeal colonization29.

nature reviews | microbiology volume 6 | april 2008 | 291

2008 Nature Publishing Group
REVIEWS
Complement
A part of the innate immune
system that comprises serum
proteins which can protect
against infection.
Fab fragment
The region of an antibody that
binds to an antigen.
Bacteriocin
A bacterially produced, small,
heat-stable peptide that is
active against other bacteria
and to which the producer has
a specific immunity
mechanism. Bacteriocins can
have a narrow or broad target
spectrum.
292 | april 2008 | volume 6 www.nature.com/reviews/micro
Epithelial cells in culture respond to the osmotic
stress of pneumolysin-mediated pore formation by
activating the p38 mitogen-activated protein kinase,
which results in an increase in chemokines that attract
neutrophils30. These effects of pneumolysin on the epi-
thelial surface suggest that the pneumococcus might
have evolved to promote the inflammatory response,
perhaps because the resulting secretions increase the
likelihood of transmission, even though this increased
host response could accelerate the eventual clearance
of the pathogen.
Host-mediated killing of S.pneumoniae is gener-
ally thought to require opsonization by a serotype-
specific antibody together with complement, followed
by phagocytosis. Interestingly, the course of a colo-
nization event is unaffected in mice that fail to gen-
erate a specific antibody, and neither the depletion
of neutrophils nor complement impacts on initial
colonization by the organism 10. By contrast, mice
that lack Toll-like receptor 2 (TLR2), an initiator of
the inflammatory responses that follow the recogni-
tion of lipoteichoic acid and/or lipoproteins, exhibit
delayed pneumococcal clearance29. In addition, mice
that do not express major histocompatibility complex
class II also show prolonged carriage, which indicates an
important role for CD4+ Tcells rather than humoral
immunity31 (BOX2). The CD4+ T-cell-dependent effec-
tor functions that lead to loss of established coloniza-
tion have yet to be identified. There is also evidence
that pneumolysin stimulates inflammatory responses
through TLR4 (Ref.32).
Another factor that limits the effectiveness of the
host humoral response on mucosal surfaces is bac-
terial expression of a secreted zinc metalloprotease
that specifically targets human immunoglobulin A1
(IgA1), which constitutes more than 90% of the IgA
in the human airway33. The cleavage of bound IgA1
produces bacterial surface antigens that are bound
to Fab fragments, which prevents inflammation from
being initiated through host recognition of the Fc
region of the antibody. Because of the activity of the
IgA1 protease, antibody-mediated clearance might
occur only after sufficient amounts of other classes
and subclasses of specific antibody have been gener-
ated. Indeed, the decrease in pneumococcal coloniza-
tion following the use of the CPS conjugate vaccine
could be attributable to its induction of high levels of
IgG, which is not targeted by the IgA1 protease. The
ability of S.pneumoniae to inactivate IgA1 in immu-
nocompetent hosts could account for the generally
unaltered incidence of infection that is observed in
most IgAdeficient individuals.
Creactive protein (CRP) is another important
component of the innate immune response against
S.pneumoniae. CRP binds specifically to ChoP and, after
binding, CRP interacts with complement component
C1q to activate the classical pathway of complement.
Transgenic mice that express the human crp gene are
more resistant to pneumococcal infection34. Moreover,
at the concentrations found in the airway, CRP can block
pneumococcal adherence through rPAF35.
2008 Nature Publishing Group
Impact of competition. It is estimated that more than 700
different microbial species can reside within the human
pharynx36. Successful colonization probably requires
mechanisms that counteract the presence of microbial
competitors. There is evidence that in a mouse model of
pneumococcal infection, co-colonization with another
bacterial species that resides in the same niche can result
in the clearance of S.pneumoniae through the induc-
tion of complement-mediated neutrophil killing37. This
demonstrates that one microbial species can harness the
innate immune response of its host to prevail over a com-
petitor. A further implication is that the pneumococcus
is resistant to the innate immune responses it stimulates,
but is not necessarily resistant to the responses that are
induced by its competitors.
Pneumococcal strains also compete with each other.
The increase in the prevalence of previously uncommon
serotypes in populations in which the pneumococcal CPS
conjugate vaccine is extensively used (a phenomenon that
is referred to as serotype replacement) suggests that non-
vaccine pneumococcal types are being out-competed by
the serotypes that are present in the vaccine. One mecha-
nism that could underlie this intra-species competition is
the strain-specific activity of pneumococcal bacteriocins,
which are known as pneumocins38. These small antimi-
crobial peptides, which are expressed by the blp locus and
are under the control of a quorum-sensing pheromone
(BlpC), target members of the same species that do not
produce the cognate immunity protein. In a mouse model
of colonization, differences of only a few amino acids
in the bipartite pneumocin BlpMN are sufficient to dictate
the outcome of competition between two isolates.
Another consequence of the highly populated micro-
bial environment in the human pharynx is the availability
of exogenous DNA from closely related oral strepto-
coccal species and co-colonizing pneumococci. These
nucleic acids can be taken up by S.pneumoniae because
of its natural competence and, subsequently, be used to
increase its overall fitness. The acquisition of genes that
encode altered penicillin-binding proteins, for example,
has facilitated resistance to lactam antibiotics, which
is now a common problem in the treatment of pneumo-
coccal infections39. The ability of the pneumococcus to
take up DNA fragments and incorporate homologous
sequences into its genome is only observed during aero-
bic growth. Indeed, this activity could also be a means
of compensating for the high mutation rates that result
from the oxidative lifestyle of this organism.
Protein virulence factors
Over the past 20 years, the importance of proteins for
S.pneumoniae virulence has become clear. Research
in this area has been stimulated by the realization that
pneumococcal proteins represent a promising avenue
for the development of vaccines that are common to all
pneumococcal serotypes. Consequently, the number of
potential virulence factors that have been characterized
has increased greatly in recent years, and are too numer-
ous to review in detail here. We will therefore discuss only
a selection of the pneumococcal virulence factors that are
promising candidates for use as vaccine antigens (FIG.2).
 REVIEWS

Box 2 | CD4+ Tcells and pneumococcal disease

Research into the host immune response to pneumococcal disease has focused primarily on the role of humoral
components of innate and acquired immunity and the cellular aspects of innate immunity. However, an appreciation of
the involvement of CD4+ Tcells in the clinical setting, in which HIV-1-infected patients who have lower CD4+ Tcell
counts are significantly more likely to be persistent pneumococcal carriers than non-HIV-1-infected patients and
patients with AIDS frequently develop severe pneumococcal infections, has led to more detailed investigations of the
role of these cells154,155.
Recently, the role of CD4+ Tcells in pneumococcal disease has been revisited using mouse models of pneumococcal
disease. To summarize, this work showed that in lungs of mice that are infected intranasally with Streptococcus
pneumoniae, CD4+ Tcells contribute to early host resistance to infection, as shown by an early rapid increase in Tcell
infiltration to areas that are subject to increased pneumococcal invasion. In addition, an increased host susceptibility to
infection that was due to significantly increased bacterial loads both in lungs and blood was observed in CD4+-deficient
mice44,57. Interestingly, infections that used pneumolysin-deficient isogenic pneumococcal mutants resulted in a total
lack of Tcell infiltration into infected lungs, which suggests that pneumolysin was responsible for the pattern of Tcell
infiltration57. Studies that used invitro chemotaxis assays with purified human CD4+ Tcells have shown that the CD4+
Tcell migratory process is indeed dependent on the presence of pneumolysin, as pneumolysin-deficient pneumococci
failed to stimulate CD4+ Tcell chemotaxis. Sub-lytic concentrations of purified pneumolysin or pneumolysin-positive
S.pneumoniae were able to successfully stimulate CD4+ Tcell chemotaxis44.
Protection studies in mice have recently confirmed the role of CD4+ Tcells as part of an antibody-independent immune
response to pneumococcal infection31. It was demonstrated that intranasal immunization with live pneumococci (or a
killed, non-encapsulated whole-cell vaccine) against subsequent pneumococcal intranasal challenge, protected
antibody-deficient mice, but not CD4+ T-cell-deficient mice. Other researchers have also shown that CD4+ Tcells are
required for efficient clearance of nasopharyngeal pneumococcal colonization in naive mice, as CD4+ T-cell-deficient
mice failed to clear pathogen colonization29. The inability of CD4+ T-cell-deficient mice to clear nasopharyngeal
colonization was explained by the lack of induction of a T helper 1 (TH1) response, which was previously shown to have a
protective role in the response to pneumococcal disease in humans156. In addition, it has been demonstrated that
pneumolysin promotes CD4+ T-cell-dependent clearance of colonization29.
Recent studies have also found that CD4+ T-cell proliferative responses to pneumolysin are significantly higher in
children who do not have detectable pneumococci in their nasopharynx, which suggests that natural CD4+ Tcell
immunity to pneumococcal protein antigens could modulate nasopharyngeal carriage157. Indeed, previous work had
shown that mucosal antibody production against pneumococcal proteins, such as pneumolysin and CbpA, was Tcell
dependent158. Although it is unclear whether this Tcell immunity cleared existing bacterial carriage or prevented new
colonization, it is worth considering that future vaccines should include conserved pneumococcal protein antigens that
are capable of inducing CD4+ Tcell immunity.

Pneumolysin. Many studies have shown that pneumo-
lysin is a potent, wide-ranging virulence factor. It is found
in virtually all pneumococcal isolates, and its amino acid
sequence is well conserved, although a small number of
variants have been described40,41. Pneumolysin is a mem-
ber of the family of cholesterol-dependent cytolysins that
are synthesized by Gram-positive bacteria.
Pneumolysin is produced as a 52 kDa soluble protein
that oligomerizes in the membrane of target cells to
form a large ring-shaped transmembrane pore. The pore
is 260 in diameter and is composed of approximately
40 monomer subunits. During its conversion from a
soluble monomer to a membrane-inserted oligomer,
pneumolysin undergoes a series of spectacular structural
changes42. The oligomers are thought to be responsible for
the cytolytic activity of the toxin and the plethora of cell-
modulatory activities that are evident at sub-lytic con-
centrations. These activities include: inhibition of ciliary
beating on respiratory epithelium and brain ependyma;
inhibition of the phagocyte respiratory burst; and induc-
tion of cytokine synthesis and CD4+ Tcell activation and
chemotaxis43,44. Site-directed mutagenesis has shown that
pneumolysin activates the classical complement pathway
independently of its cell-modulatory activity45.
Both the cell-modulatory and complement-activation
activities of pneumolysin have been shown to contrib-
ute to pneumococcal virulence in murine models of
pneumonia4648; however, it is noteworthy that strains
of a serotype1 clone that produce a non-cytolytic
pneumolysin have been isolated from cases of invasive
pneumococcal disease41. Interestingly, S.pneumoniae
that expressed a mutated pneumolysin protein which
lacked haemolytic and complement-activating activity
was shown to be more virulent than a pneumococcus
in which the pneumolysin gene was deleted47. This
indicated that pneumolysin has an additional, uniden-
tified function. It was suggested that this activity was an
interaction with TLR4, because a pneumococcal mutant
that produced pneumolysin without haemolytic activity
was reported to activate TLR4-dependent responses32.
In addition, Baba and colleagues 49 reported that a
non-cytolytic pneumolysin stimulated the production
of interferon. This could also be an unidentified
function, although S.pneumoniae strains that express
this form of pneumolysin were no more virulent than
the pneumococcus in which the pneumolysin gene
had been knocked out (A.K. and P.W.A., unpublished
observations).
There is substantial evidence that pneumolysin is cru-
cial for pneumococcal virulence in pneumonia4648,50,51,
although its relative importance can vary from strain
to strain. For example, Alexander etal.52 found that
mice that had been immunized with pneumolysin
were protected significantly from nine pneumococcal

nature reviews | microbiology volume 6 | april 2008 | 293

2008 Nature Publishing Group
REVIEWS
Opsonic antibody
A bacteria-binding (opsonizing)
antibody, such as IgG and IgA,
that interacts with Fc receptors
on phagocytic cells, which
leads to an increased uptake of
bacteria.
Teichoic acid
A cell-envelope glycopolymer
that is composed of many
identical sugar-phosphate
repeating units, which are
usually modified with d-alanine
and additional sugars.
Apolactoferrin
The iron-depleted form of the
glycoprotein lactoferrin.
294 | april 2008 | volume 6 www.nature.com/reviews/micro
ATP-binding
cassette
transporter
LytA
Metal-
Pneumolysin binding
PsaA
proteins
Capsule PiaA
PiuA
Cell wall
Cell membrane
PspC
Eno Sortases
PavA PspA
Choline-
Hyl binding
proteins
LPXTG-anchored
neuraminindase proteins
Nature Reviews | Microbiology
Figure 2 | Pneumococcal virulence factors.
Streptococcus pneumoniae virulence is multi-faceted.
Important pneumococcal virulence factors include: the
capsule; the cell wall; choline-binding proteins;
pneumococcal surface proteins A and C (PspA and PspC);
the LPXTG-anchored neuraminidase proteins; hyaluronate
lyase (Hyl); pneumococcal adhesion and virulence A (PavA);
enolase (Eno); pneumolysin; autolysin A (LytA); and the
metal-binding proteins pneumococcal surface antigen A
(PsaA), pneumococcal iron acquisition A (PiaA) and
pneumococcal iron uptake A (PiuA).
strains, but no protection was afforded against a
tenth strain. It is likely that the strain-specific nature
of this pneumococcal virulence factor will also hold
true for many other, and perhaps all, pneumococcal
virulence factors.
In a mouse model of acute pneumonia, pneu-
molysin was shown to be essential for the survival of
S.pneumoniae in both the upper and lower respiratory
tracts53,54. There is also good evidence that pneumolysin
is required for bacterial spread from the lungs to the
bloodstream5356. In bacteraemic infection, if pneu-
molysin is expressed, high numbers of pneumococci
are found in the bloodstream, and the host succumbs
to infection54,56; however, in the absence of pneumo-
lysin, high pneumococcal numbers are tolerated in the
blood without overt disease symptoms57, and chronic
bacteraemia can develop58.
For models of meningitis, however, there is more
debate on the importance of pneumolysin. In contrast
to a report that suggested only a limited role for pneu-
molysin in S.pneumoniae meningitis59, subsequent
studies found that pneumolysin was a key determi-
nant of pneumococcal virulence in this disease 60,61.
Furthermore, pneumolysin has also been shown to be
required for pneumococcal-induced deafness in men-
ingitis62 and for pneumococcal-induced damage to the
brain ependyma63,64. Additional evidence that supports
the role of pneumolysin in meningitis was discussed in
2008 Nature Publishing Group
a recent review of the role and action of pneumolysin in
pneumococcal diseases65.
S.pneumoniae is also a natural pathogen of horses66.
Interestingly, all equine isolates are serotype 3 (Ref.66),
and have a major deletion within the gene that encodes
pneumolysin, as well as the gene that encodes the
autolysin LytA (discussed below)67.
Pneumococcal cell-surface proteins
Cell-surface proteins have attracted considerable atten-
tion because of their potential as vaccine antigens that
can stimulate the production of opsonic antibodies. Three
major groups of pneumococcal cell-surface proteins have
been identified: choline-binding proteins, lipoproteins
and proteins that are covalently linked to the bacterial
cell wall by a carboxy (C)terminal sortase (LPXTG; in
which X denotes any amino acid) motif.
Choline-binding proteins. As mentioned previously,
pneumococci express ChoP as a component of their
cell-wall teichoic acids and membrane-bound lipoteichoic
acids. ChoP anchors a group of proteins, the choline-
binding proteins, to the cell wall. Most of these proteins
have repeat sequences of approximately 20 amino acids
that mediate attachment of the proteins to the cell surface
through phosphorylcholine. The amino (N)terminal
sequences vary widely, and are the sites of the specific
activities of the different proteins 68. S.pneumoniae
encodes 1015 choline-binding proteins69, including
PspA, PspC and LytA.
PspA.PspA has three structural domains, and its
Nterminal region is composed of repeated helices
that protrude from the bacterial cell surface70. Its highly
electronegative properties are thought to inhibit com-
plement binding70. Between the N terminus and the
Cterminal choline-binding region is a proline-rich
region of 6080 amino acids that probably confer flex-
ibility70. PspA is a highly variable molecule that, based
on the sequences of the N termini, can be grouped into
three families that, in turn, can be subdivided into six
different clades71.
PspA interferes with the fixation of complement
component C3 on the pneumococcal cell surface,
and thus inhibits complement-mediated opsoniza-
tion. PspA is also a lactoferrin-binding protein and,
through this activity, is thought to protect the bacte-
rium from the bactericidal activity of apolactoferrin.
PspA-knockout mutants are more sensitive to killing
by apolactoferrin, and anti-PspA antibodies enhance
the bactericidal activity of apolactoferrin72,73. There
are conflicting data, however, on the invivo effect of
deleting the pspA gene. Although studies of serotype 3
(Refs74,75) and 4 (Ref.20) pneumococci showed
that PspA is required for invivo growth, similar
experiments that used a loss-of-function mutant of
a serotype 2 strain failed to show an effect54,76,77. An
investigation by Abeyta and colleagues 77 suggested
that the contribution of PspA to virulence might
depend on the properties of the pneumococcal
polysaccharide capsule.

Factor H
A component of the alternative
complement pathway that is
involved in the regulation of
complement activation.
nature reviews | microbiology volume 6 | april 2008 | 295
PspC. PspC is a multifunctional cell-surface protein that
is known by several other names that reflect its different
activities. For example, it is also known as choline-binding
protein A (CbpA), because it was the predominant entity
to be purified by ChoP-affinity chromatography78. A
pspC-knockout mutant binds less well to epithelial cells
and sialic acid invitro, and shows reduced nasopharyngeal
colonization compared with the wild type78.
PspC also binds the polymeric immunoglobulin
receptor that normally transports secretory IgA; hence
it is also called SpsA (secretory pneumococcal surface
protein A, as mentioned above)79,80. This activity could
be the first stage of translocation across the respiratory
epithelium, which is consistent with the reduced viru-
lence of a pspC mutant in a mouse model of pneumonia48.
Iannelli etal.81 reported that PspC mutants of serotype2
and 3 S.pneumoniae were less virulent in a sepsis
model. Additional studies have supported this finding82.
However, Orihuela etal.54 failed to find any effect on
sepsis or pneumonia using a pspA mutant of the same
serotype2 strain, although an impairment in pathogen
survival in the nasopharynx was observed.
An additional property of PspC is its ability to bind to
factor H83,84, which has been shown to prevent formation of
C3b though the alternative complement pathway, and thus
prevent pneumococcal opsonization82. PspC proteins are
highly polymorphic54, and can be divided into two struc-
tural groups. In addition to the PspC molecule that binds
to phosphorylcholine, the variant that was first described
as Hic83 is anchored to the bacterial cell wall using the
classical LPXTG motif. However, both the Hic variant
and the classic choline-binding form of the protein are
able to sequester factor H. PspC also binds complement
component C3 (Ref.85).
LytA. LytA is an amidase that cleaves the Nacetyl
muramoyll-alanine bond of pneumococcal peptidog-
lycan86. The autolytic action of this enzyme leads to the
cell lysis that is typical of pneumococcal cells growing in
batch culture, but this enzyme also participates in cell-
wall growth and turnover. LytA-negative S. pneumoniae
mutants were shown to have reduced virulence in murine
models of pneumonia and bacteraemia51,54,76,87. The con-
tribution of LytA to virulence is thought to be mediated,
in part, by its function in the release of pneumolysin and
inflammatory peptidoglycan and teichoic acids from
lysed bacterial cells.
Divalent metal-ion-binding lipoproteins
Between 42 and 45 pneumococcal cell-surface lipopro-
teins have been described to date69. These include the
peptide isomerases PpmA and SlrA, which have been
shown to be involved in S.pneumoniae virulence88,89. The
metal-binding lipoproteins pneumococcal surface anti-
gen A (PsaA), pneumococcal iron acquisition A (PiaA)
and pneumococcal iron uptake A (PiuA) are additional
cell-surface lipoproteins that have been shown to be
essential for pneumococcal virulence.
PsaA. Deleting psaA abolishes virulence in murine
models of pneumonia, bacteraemia and colonization9092.
2008 Nature Publishing Group
REVIEWS
Originally, PsaA was proposed to be a pneumococcal
adhesin because of its sequence similarities to puta-
tive adhesins from other streptococci93. Subsequent
investigations seemed to support this assertion. psaA
pneumococcal mutants were deficient in binding to
mammalian cells invitro90,94 and, more recently, it was
reported that anti-PsaA antibodies inhibited pneu-
mococcal adherence95. However, PsaA is actually the
divalent metal-ion-binding lipoprotein component of
an ATP-binding cassette (ABC) transport system that
has specificity for manganese96,97. Structural analysis of
PsaA shows a potential divalent metal-ion-binding site
that can accommodate zinc or manganese ions98, and
psaA mutants require manganese for normal growth96.
Therefore, the observed effects on bacterial adherence
are presumably a result of the pleiotropic impact of a
deficiency in manganese transport on the expression
of other pneumococcal genes, including adhesins. This
proposal is consistent with structural studies which indi-
cate that PsaA is unlikely to protrude beyond the pneu-
mococcal cell wall68,98. Furthermore, psaA is transcribed
as part of the psaBCA operon, and if other genes of the
operon (psaB or psaC) are mutated, comparable defi-
ciencies in bacterial adherence are observed99. A more
important outcome of the psaA mutant studies, how-
ever, is that manganese uptake seems to be essential for
pneumococcal resistance to oxidative stress, which can
result from the production of hydrogen peroxide during
pneumococcal metabolism, as well as the generation of
reactive oxygen species during the host innate immune
response. It is this property that probably accounts for
the avirulence of psaA mutants in mouse models of
colonization and invasive disease97,100.
PiaA and PiuA.Three ABC transporter operons that
mediate iron uptake, pia, piu and pit, have been described
in S.pneumoniae101,102. Each operon comprises genes that
encode a metal-binding protein, a membrane permease
and an ATPase, but the pia and piu systems seem to be
particularly important102. PiaA and PiuA are the lipopro-
tein metal-binding components of these systems, and
immunization with these proteins is protective103. There
seems to be redundancy in the iron-uptake systems,
however, as only a piupia double-mutant strain had sig-
nificantly reduced growth in an iron-deficient medium
and, although a single mutation in pia or piu decreased
virulence in models of pneumonia and bacteraemia,
the double mutant was attenuated to a significantly
greater extent102.
LPXTG-anchored proteins
The best-characterized mechanism of anchoring pro-
teins to the peptidoglycan of Gram-positive bacteria is
through the sortase transpeptidase that recognizes the
amino-acid sequence LPXTG in surface-located pro-
teins. Some pneumococcal strains encode a single sortase
gene, whereas in others multiple sortase-like genes are
predicted69. Where multiple sortase genes are present,
it is proposed that StrA is responsible for anchoring
most LXPTG-containing proteins and the other sortase
proteins mediate the anchoring of a subset of surface
REVIEWS
C-type lectin
A carbohydrate-binding
protein that is found in a wide
range of animals. C-type lectins
share a highly conserved
calcium-dependent
carbohydrate-recognition
domain that is used to
distinguish them from other
animal lectins.
296 | april 2008 | volume 6 www.nature.com/reviews/micro
proteins, perhaps in response to specific environmental
cues20. StrA has been reported to have a role in pneumo-
coccal colonization, pneumonia and bacteraemia, and
in adhesion to nasopharyngeal cells104106. It is estimated
that up to 20 S.pneumoniae proteins are anchored by an
LPXTG motif 69, including the neuraminidases.
Neuraminidases. Neuraminidases, also known as siali-
dases, cleave terminal sialic acid residues from glyco-
proteins, glycolipids and cell-surface oligosaccharides.
A recent study showed that neuraminidases can remove
sialic acid from soluble proteins, such as lactoferrin, IgA2
and secretory component107. S.pneumoniae encodes at
least three neuraminidase genes: nanA, nanB and nanC.
All strains encode nanA, and most also encode nanB;
however, only approximately 50% of strains encode
nanC108. Neuraminidases are secreted from the cell,
but only NanA contains the LPXTG sequence, which
suggests that these enzymes have different functions
invivo. This hypothesis is supported by the observa-
tion that NanB has a much lower acidic pH optimum
than NanA109. Experiments that used loss-of-function
mutants in a mouse model of acute pneumonia have
shown that NanA and NanB are important for pneu-
mococcal survival in the respiratory tract and blood-
stream110. These experiments also indicated that the
two enzymes have differing roles; the NanA mutant was
rapidly cleared from the respiratory tract, but the NanB
mutant persisted, although the number of organisms
did not increase110. However, other work that used a
mouse nasopharyngeal-colonization model showed no
reduction in the ability of a NanA mutant to colonize
the nasopharynx over a longer period23. Although there
is no direct experimental evidence of a biological role
for NanC, an analysis of the distribution of nanC among
S.pneumoniae isolates suggested a tissue-specific role
nanC was more common in isolates from cerebrospinal
fluid than in carriage isolates108.
Tissue specificity of virulence
It is clear that the contribution of each virulence fac-
tor varies, individually and collectively, according to
the invivo location of the bacterium. Using reverse-
transcription PCR, a recent study observed two patterns
of invivo gene expression by S.pneumoniae: one pattern
was characteristic of pneumococci in the bloodstream
and the other of pneumococci in the lungs and brain111.
In the bloodstream, for example, the expression of ply
(which encodes pneumolysin) and pspA was increased,
whereas in lung and brain tissue the expression of nanA
and nanB, and the competence genes comA, comE and
comX, was higher. The involvement of the competence
system in virulence was confirmed by administer-
ing competence-stimulating peptide (CSP) and using
a competence-negative S.pneumoniae mutant. CSP
modulated virulence, but in a tissue-specific manner.
Thus, the administration of CSP enhanced virulence in
pneumonia models, and a comD mutant had reduced
virulence. These observations contrast directly with
those of bacteraemia, in which the administration of
CSP decreases virulence. Finally, CSP was also shown
2008 Nature Publishing Group
to induce pneumococcal biofilm formation invitro,
and the pattern of pneumococcal-gene expression in
biofilms paralleled that observed in the lungs, whereas
the gene-expression profile in the bloodstream echoed
that observed in planktonic culture invitro111.
The pneumococcal capsule
Structure, function and role in virulence. The polysaccha-
ride capsule forms the outermost layer of S.pneumoniae
cells, and is approximately 200400 nm thick112. With the
exception of serotype 3, and possibly others, the capsule
is covalently attached to the outer surface of the cell-wall
peptidoglycan113. A total of 90 structurally and serologi-
cally distinct CPS types have been recognized to date114.
Capsule production is indispensable for pneumococcal
virulence and is strongly anti-phagocytic in non-immune
hosts115 Although non-encapsulated strains have been
associated with superficial infections, such as conjunctivi-
tis116,117, clinical isolates from other sterile sites are encap-
sulated, and spontaneous non-encapsulated derivatives of
these strains are largely avirulent. Most CPS serotypes are
highly charged at physiological pH, and this can directly
interfere with interactions with phagocytes118. The capsule
also forms an inert shield that seems to prevent either the
Fc region of IgG or complement component iC3b, which
is bound to deeper cell-surface structures (for example,
teichoic acids and cell-surface proteins), from interact-
ing with their relevant receptors on phagocytic cells119,120.
More recent data suggest that the capsule can also reduce
the total amount of complement that is deposited on the
bacterial surface 121. It also reduces the trapping of
pneumococcal cells in neutrophil extracellular traps122.
The virulence of S.pneumoniae is related to the
thickness of the capsule in a particular strain and
serotype123. However, pneumococci from the differ-
ent CPS serotypes differ markedly in their capacity to
cause disease115. This presumably reflects their relative
capacity to resist phagocytosis, as well as differences in
their ability to elicit a humoral immune response. For
example, Hostetter124 reported serotype-dependent
differences in both the amount and site of covalently
bound C3b on the surface of opsonized pneumococcal
cells, as well as differences in the degradative processing
of bound C3b to iC3b and C3d. Recent studies in mice
also suggest an additional potential contributing factor.
SIGNR1, a Ctype lectin that is expressed on macrophages
in the marginal zone of the spleen was shown to mediate
the uptake of both purified CPS and S.pneumoniae125.
The spleen is known to play a crucial part in the clear-
ance of blood-borne pneumococci, and mice that are
deficient in SIGNR1 are hypersensitive to invasive
pneumococcal disease 126. Ctype lectins, such as
SIGNR1 and its human homologues, exhibit oligosac-
charide specificity, and differences in their affinity for
individual CPS pneumococcal types would undoubtedly
influence phagocytosis and bacterial clearance. SIGN-R1
could also influence the presentation of CPS antigens to
the immune system, thereby affecting the hosts capacity
to mount an antibody response. Finally, Fernebro etal.127
reported that the capsule provides a degree of resistance
to spontaneous or antibiotic-induced autolysis, thereby

Clonal type contributing to antibiotic tolerance in clinical isolates.
A pneumococcal clonal lineage Interestingly, this capacity also varied significantly
is determined by sequence between capsular serotypes.
analysis of a set of Although isogenic S.pneumoniae expresses differ-
housekeeping genes, as
opposed to its capsular
ent CPS serotypes that exhibit marked differences in
serotype. murine virulence, non-capsular factors are also clearly
important128,129. Molecular epidemiological analysis
has demonstrated that properties which are associated
with a particular clonal type, in addition to capsular
serotype, influence the potential of S.pneumoniae to
cause invasive disease in humans. The contribution
of host factors was demonstrated in a subsequent
study in which isolates that had high human-invasion
potential exhibited significantly different virulence
and disease kinetics in BALB/c mice compared with
C57BL/6 mice130.
Regulation of CPS production. The transition from
nasopharyngeal colonization to invasive disease is clearly
a watershed in the relationship between S.pneumoniae
and its host, and involves a major switch in the expres-
sion of important virulence determinants as the pathogen
adapts to its altered microenvironment. Maximal expres-
sion of capsule is essential for systemic virulence, but the
extent of exposure of other important pneumococcal
surface structures, such as adhesins, is also influenced
by capsular thickness. Non-encapsulated pneumococci
exhibit greater adherence to human respiratory epithe-
lial cells (A549) invitro compared with isogenic deriva-
tives that express either serotype 3 or 19F capsules131.
Furthermore, previously encapsulated pneumococci
Ligase?
+
Wzx Wzy capsular polysaccharide
flippase polymerase
Figure 3 | Steps in Streptococcus pneumoniae capsule biosynthesis.
Nature ReviewsCapsule
| Microbiology
biosynthesis is regulated by the cps locus, which encodes specific glycosyltransferases
that assemble the oligosaccharide repeats on the cytoplasmic face of the membrane, and
a flippase (Wzx), which transports the repeat units to the external surface of the
membrane. At this location, the repeat units are polymerized by Wzy to form high-
molecular-weight capsular polysaccharides, which are then, presumably, ligated to the
cell wall. The cps locus also encodes enzymes for the synthesis of activated sugar
precursors, as well as conserved genes that are involved in regulation.
nature reviews | microbiology volume 6 | april 2008 | 297
2008 Nature Publishing Group
REVIEWS
seem to express little CPS during intimate contact with
respiratory epithelial cells invitro or invivo132.
The capacity to regulate CPS production at the tran-
scriptional, translational or post-translational level is
important for the survival of S.pneumoniae in different
host environments. To date, no transcriptional-control
elements have been identified in association with the
pneumococcal 70 cps promoter133. However, there is
some evidence to suggest that the level of expression of
the cps locus differs between the transparent and opaque
phase variants, as more CpsD was detectable by western
immunoblotting in the opaque phase variants28. The level
of S.pneumoniae cps mRNA, relative to 16S ribosomal
RNA, that was isolated from the blood of infected mice
was shown to be approximately fourfold higher compared
with the levels in S.pneumoniae that had been grown
invitro134. However, significant differences in cps mRNA
levels could not be detected between pneumococci that
were isolated from the nasopharynx, blood or lungs of
infected mice135.
The first four genes of the cps locus (cpsAD) are com-
mon to all pneumococcal serotypes, with the exception
of serotypes 3 and 37. The proteins that are encoded by
these genes, CpsAD, have been shown to affect the level
of CPS expression136. The central region of the cps locus
comprises genes that encode specific glycosyl transferases
that assemble the serotype-specific oligosaccharide-
repeat unit on a lipid carrier. This region also includes
a flippase (Wzx) that transports the repeat unit to the
external face of the membrane and a polymerase (Wzy)
that links the units together. The final region of the locus
comprises genes that encode the synthesis of activated
sugar precursors (FIG.3).
Interestingly, a CpsA homologue in group B strep-
tococci seems to function as a transcriptional activa-
tor137; however, to date, the cpsA gene has not been
shown to impact on cps transcription in S.pneumoniae.
Nevertheless, cpsA-deletion mutants produced reduced
levels of CPS138140. CPS biosynthesis of all but two pneu-
mococcal serotypes has been shown to be dependent
upon a regulatory system that is determined by CpsB,
CpsC and CpsD. CpsB is a manganese-dependent phos-
photyrosine-protein phosphatase, CpsC is a membrane
protein that is related to polysaccharide co-polymerases
and CpsD is an autophosphorylating protein-tyrosine
kinase136,138,139. CpsC is required for CpsD tyrosine auto-
phosphorylation; a cpsC-deletion mutant is rough, and
CpsD does not become phosphorylated. Mutation of the
cpsD gene to inactivate the ATP-binding site eliminated
CPS production. It has also been shown that CpsB is
required to dephosphorylate CpsD; in cpsB-deletion
mutants, the proportion of CpsD that is phosphor-
ylated increases dramatically, and there is a significant
decrease in the amount of CPS that is produced. These
observations suggest that the non-phosphorylated form
of CpsD is active in CPS biosynthesis136,138,139. Recently,
a novel role for CpsC in the attachment of CPS to the
pneumococcal cell wall was also identified141. Therefore,
CpsB, CpsC and CpsD function together to regulate
CPS assembly, export and attachment to the cell wall
by tyrosine phosphorylation of CpsD (FIG.4).
REVIEWS

production by controlling the availability of precursors or

a b
3
1 The pneumococcus has proven to be a particularly for-
Cell wall Cell wall
CpsC
P P CpsC CpsC P P
P P Ligase P P
ATP ATP
Wzx Wzy CpsD CpsD Wzx Wzy CpsD
2 4
P P
P P CpsB
Figure 4 | Model showing the regulation of capsular polysaccharide (CPS)
production by tyrosine phosphorylation of CpsD. a | CpsC, CpsD
Nature and|ATP
Reviews interact to
Microbiology
promote CPS biosynthesis by the polysaccharide polymerase (step 1). CpsD
autophosphorylates, which causes a change in protein interactions and slows CPS
biosynthesis (step 2). b | The CPS polymer is then transferred to the putative CPS cell-wall
ligase, and is ligated to the cell wall (step 3). Finally, CpsB dephosphorylates CpsD,
thereby allowing the cycle to be repeated (step 4).
Interestingly, RegM, a homologue of the staphylococcal
catabolite control protein CcpA, which is involved in the
regulation of sugar-metabolism pathways, has been shown
to affect transcription of the cps locus, which suggests that
a carbon source might also influence capsular expres-
sion142. Indeed, two proteins that are involved in sugar
metabolism have been shown to affect CPS production.
Pgm is the phosphoglucomutase that catalyses the con-
version of glucose6-phosphate to glucose1-phosphate,
and GalU is a glucose1-phosphate uridylyltransferase
that catalyses the formation of uridine diphosphate-
glucose (UDP-Glc) from glucose1-phosphate. Mutants
of S.pneumoniae in which either the galU or pgm gene was
disrupted produced almost no CPS and exhibited growth
defects137,143. Additionally, pneumococcal strains in which
the pgm gene had defined point mutations that signifi-
cantly reduced but did not eliminate enzymatic activity
still produced reduced amounts of CPS, even though
the mutants no longer exhibited growth defects144. Both
Pgm and GalU are required for the synthesis of UDP-Glc,
which is a precursor for the biosynthesis of all 90 pneumo-
coccal CPS types, as well as other cellular structures, such
as teichoic acid. Thus, limiting the supply of this precursor
would be expected to impact heavily upon CPS produc-
tion in the pneumococcus. Indirect modulation of CPS
co-factors could be one of the regulatory mechanisms that
is used by S.pneumoniae.
Conclusions
midable foe, and can evade elimination by serum therapy,
chemotherapy, multiple classes of antibiotic and, more
recently, conjugate polysaccharide vaccines. For each of
CpsC
these approaches, there was considerable initial confi-
Ligase dence that Sir William Oslers captain of the men of death
had finally been dealt a fatal blow. The adaptability of the
organism in response to an array of selective pressures,
CpsD however, has allowed it to retain its capacity to colonize
its human host without decreasing its fitness to the point
CpsB
of diminished virulence. The ability of S.pneumoniae to
adapt stems from the remarkable plasticity and heteroge-
neity of its genome. Whole-genome sequencing of large
numbers of pneumococcal isolates is now revealing both
the extent of strain-to-strain variation in gene content
and also the size of the S.pneumoniae pan-genome, as
well as other streptococci that provide an additional
reservoir of genetic information.
For the most part, the initial focus of investigations
of S.pneumoniae was limited to the roles of its various
capsule serotypes and the mechanisms that underpin its
natural competence for the uptake of DNA. As outlined
in this Review, advances in molecular biology and bacte-
rial genetics have not only facilitated a fuller understand-
ing of the biology of its capsule and competence, but have
also allowed characterization of the many attributes that
contribute to bacteriahost interactions. In addition, rep-
resentative models of pneumococcal infection are now
being applied in genetically modified mice to define the
host factors and other aspects of the immune response
that contribute to colonization and disease. These studies
have, for example, emphasized the importance of CD4+
Tcells, rather than antibody responses, in the mediation
of immunity to S.pneumoniae (BOX2). The complexity of
the bacteria and host factors that have been illuminated
by these studies has also provided insight into novel
strategies for antimicrobials and a next generation of
more broadly effective pneumococcal vaccines that will
use combinations of protein antigens (BOX1). Despite our
progress, key aspects of pneumococcal biology, such as
the bacteria and host factors that impact on pathogen
transmission, remain largely unexplored. It is still pre-
mature, therefore, to assume that the pneumococcus has
yielded all of its most important secrets.

1. Balakrishnan, I., Crook, P., Morris, R. & Gillespie, S.H.
Early predictors of mortality in pneumococcal
bacteraemia. J.Infect. Dis. 40, 256261 (2000).
2. Lim, W.S. etal. Study of community acquired
pneumonia aetiology (SCAPA) in adults admitted to
hospital: implications for management guidelines.
Thorax 50, 296301 (2001).
3. Denny, F.W. & Loda, F.A. Acute respiratory infections
are the leading cause of death in children in developing
countries. Am. J.Trop. Med. Hyg. 35, 12 (1986).
4. Berkley, J.A. etal. Bacteremia among children
admitted to a rural hospital in Kenya. N.Engl. J.Med.
352, 3947 (2005).
5. Bogaert, D., de Groot, R. & Hermans, P.
Streptococcus pneumoniae colonisation: the key to
pneumococcal disease. Lancet Infect. Dis. 4, 144154
(2004).
6. Regev-Yochay, G. etal. Association between carriage
of Streptococcus pneumoniae and Staphylococcus
aureus in children. JAMA 292, 716720 (2004).
7. Lexau, C. etal. Changing epidemiology of invasive
pneumococcal disease among older adults in the era
of pediatric pneumococcal conjugate vaccine. JAMA
294, 20432051 (2005).
8. McCool, T. L., Cate, T. R., Moy, G. & Weiser, J. N. The
immune response to pneumococcal proteins during
experimental human carriage. J.Exp. Med. 195,
359365 (2002).
One of the few studies of pneumococcal
pathogenesis in humans. This paper characterizes
pneumococcal colonization and the mucosal
immune response in human volunteers. It also
provides strong circumstantial evidence for the
importance of PspA in the colonization of the
human nasopharynx by pneumococci.
9. Lipsitch, M. etal. Are anticapsular antibodies
the primary mechanism of protection against
invasive pneumococcal disease? PLoS Med. 2, e15
(2005).

298 | april 2008 | volume 6 www.nature.com/reviews/micro

2008 Nature Publishing Group

10. McCool, T. & Weiser, J. Limited role of antibody in
clearance of Streptococcus pneumoniae in a murine
model of colonisation. Infect. Immun. 72, 58075813
(2004).
First paper to clearly show that antibodies are not
required for the clearance of pneumococcal
colonization based on studies that used mice with
genetic defects in humoral immunity.
11. Nelson, A. etal. Capsule enhances pneumococcal
colonisation by limiting mucus-mediated clearance.
Infect. Immun. 75, 8390 (2007).
12. Kamerling, J. in Streptococcus pneumoniae: Molecular
Biology & Mechanisms of Disease (ed. Tomasz, A.)
81114 (Mary Ann Liebert, New York, 2000).
13. Weiser, J.N., Austrian, R., Sreenivasan, P.K. &
Masure, H.R. Phase variation in pneumococcal
opacity: relationship between colonial morphology
and nasopharyngeal colonisation. Infect. Immun. 62,
25822589 (1994).
Describes pneumococcal phase variation and its
important role in nasopharyngeal colonization and
invasive disease, thereby providing an interesting
insight into the interaction of the pneumococcus
with its host.
14. Cundell, D.R., Gerard, N.P., Gerard, C., Idanpaan-
Heikkila, I. & Tuomanen, E.I. Streptococcus
pneumoniae anchor to activated human cells by the
receptor for platelet-activating factor. Nature 377,
435438 (1995).
Shows that the pneumococcus binds to a receptor
that is now recognized as being used by several
other important pathogens that reside in the airway.
15. Weiser, J. in Colonisation of Mucosal Surfaces
(ed. Nataro, J.) 6172 (ASM, Washington DC, 2005).
16. Weiser, J.N. etal. Phosphorylcholine on the
lipopolysaccharide of Haemophilus influenzae
contributes to persistence in the respiratory tract and
sensitivity to serum killing mediated by Creactive
protein. J.Exp. Med. 187, 631640 (1998).
17. Rosenow, C. etal. Contribution of novel choline-
binding proteins to adherence, colonisation and
immunogenicity of Streptococcus pneumoniae. Mol.
Microbiol. 25, 819829 (1997).
18. Hammerschmidt, S., Talay, S.R., Brandtzaeg, P. &
Chhatwal, G.S. SpsA, a novel pneumococcal surface
protein with specific binding to secretory
immunoglobulin A and secretory component. Mol.
Microbiol. 25, 11131124 (1997).
19. Barocchi, M. etal. A pneumococcal pilus influences
virulence and host inflammatory responses. Proc. Natl
Acad. Sci. USA 103, 28572862 (2006).
20. Hava, D. & Camilli, A. Large-scale identification of
serotype 4 Streptococcus pneumoniae virulence
factors. Mol. Microbiol. 45, 13891406 (2002).
Important study that used signature-tagged
mutagenesis to identify 387 pneumococcal mutants
that were attenuated in murine models of pneumonia.
21. Andersson, B. etal. Identification of an active
dissaccharide unit of a glycoconjugate receptor for
pneumococci attaching to human pharyngeal epithelial
cells. J.Exp. Med. 158, 559570 (1983).
22. Krivan, H.C., Roberts, D.D. & Ginsberg, V. Many
pulmonary pathogenic bacteria bind specifically to the
carbohydrate sequence GalNAc14Gal found in
some glycolipids. Proc. Natl Acad. Sci. USA 85,
61576161 (1988).
23. King, S., Hippe, K. & Weiser, J. Deglycosylation of
human glycoconjugates by the sequential activities of
exoglycosidases expressed by Streptococcus
pneumoniae. Mol. Microbiol. 59, 961974 (2006).
24. Jedrzejas, M., Mello, L., de Groot, B. & Li, S.
Mechanism of hyaluronan degradation by
Streptococcus pneumoniae hyaluronate lyase.
Structures of complexes with the substrate. J.Biol.
Chem. 277, 2828728297 (2002).
25. Holmes, A. etal. The pavA gene of Streptococcus
pneumoniae encodes a fibronectin-binding protein
that is essential for virulence. Mol. Microbiol. 41,
13951408 (2001).
26. Bergmann, S., Rohde, M., Chhatwal, G. &
Hammerschmidt, S. -Enolase of Streptococcus
pneumoniae is a plasmin(ogen)-binding protein
displayed on the bacterial cell surface. Mol. Microbiol.
40, 12731287 (2001).
27. Kim, J.O. etal. Relationship between cell-surface
carbohydrates and intrastrain variation on
opsonophagocytosis of Streptococcus pneumoniae.
Infect. Immun. 67, 23272333 (1999).
28. Weiser, J. etal. Changes in availability of oxygen
accentuate differences in capsular polysaccharide
expression by phenotypic variants and clinical isolates
nature reviews | microbiology volume 6 | april 2008 | 299
of Streptococcus pneumoniae. Infect. Immun. 69,
54305439 (2001).
29. van Rossum, A., Lysenko, E. & Weiser, J. Host and
bacterial factors contributing to the clearance of
colonisation by Streptococcus pneumoniae in a murine
model. Infect. Immun. 73, 77187726 (2005).
30. Ratner, A. etal. Epithelial cells are sensitive detectors
of bacterial pore-forming toxins. J.Biol. Chem. 281,
1299412998 (2006).
31. Malley, R. etal. CD4+ Tcells mediate antibody-
independent acquired immunity to pneumococcal
colonisation. Proc. Natl Acad. Sci. USA 102,
48484853 (2005).
32. Malley, R. etal. Recognition of pneumolysin by Toll-
like receptor 4 confers resistance to pneumococcal
infection. Proc. Natl Acad. Sci. USA 100, 19661971
(2003).
Demonstrates that the pneumococcal toxin
pneumolysin triggers inflammatory responses in
host macrophages by interacting with TLR4. Such
signalling is crucial for the innate immune response
to the pneumococcus, and is a paradigm of the fine
balance between the protective and deleterious
effects of innate inflammatory responses to
mucosal pathogens.
33. Wani, J., Gilbert, J., Plaut, A. & Weiser, J.
Identification, cloning and sequencing of the
immunoglobulin A1 protease gene of Streptococcus
pneumoniae. Infect. Immun. 64, 39673974 (1996).
34. Szalai, A.J., Briles, D.E. & Volanakis, J.E. Human
Creactive protein is protective against fatal
Streptococcus pneumoniae infection in transgenic
mice. J.Immunol. 155, 25572563 (1995).
35. Gould, J. & Weiser, J. The inhibitory effect of Creactive
protein on bacterial phosphorylcholine-platelet
activating factor receptor mediated adherence is blocked
by surfactant. J.Infect. Dis. 186, 361371 (2002).
36. Aas, J.A. etal. Defining the normal bacterial flora of
the oral cavity. J.Clin. Microbiol. 43, 57215732
(2005).
37. Lysenko, E.S., Ratner, A.J., Nelson, A.L. & Weiser,
J.N. The role of innate immune responses in the
outcome of interspecies competition for colonisation
of mucosal surfaces. PLoS Pathog. 1, 19 (2005).
38. Dawid, S., Roche, A. & Weiser, J. The blp bacteriocins
of Streptococcus pneumoniae mediate intraspecies
competition both invitro and invivo. Infect. Immun.
75, 443451 (2007).
39. Dowson, C., Coffey, T. & Spratt, B. Origin and
molecular epidemiology of penicillinbindingprotein-
mediated resistance to -lactam antibiotics. Trends
Microbiol. 2, 361366 (1994).
40. Lock, R.A., Zhang, Q.Y., Berry, A.M. & Paton, J.C.
Sequence variation in the Streptococcus pneumoniae
pneumolysin gene affecting haemolytic activity and
electrophoretic mobility of the toxin. Infect. Immun.
21, 7183 (1996).
41. Kirkham, L.A.S. etal. Identification of invasive
serotype1 pneumococcal isolates that express
nonhemolytic pneumolysin. J.Clin. Microbiol. 44,
151159 (2006).
42. Tilley, S., Orlova, E., Gilbert, R., Andrew, P. & Saibil, H.
Structural basis of pore formation by the bacterial
toxin pneumolysin. Cell 121, 247256 (2005).
43. Hirst, R., Kadioglu, A., OCallaghan, C. & Andrew, P.
The role of pneumolysin in pneumococcal pneumonia
and meningitis. Clin. Exp. Immunol. 138, 195201
(2004).
44. Kadioglu, A., Coward, W., Colston, M., Hewitt, C. &
Andrew, P. CD4Tlymphocyte interactions with
pneumolysin and pneumococci suggest a crucial
protective role in the host response to pneumococcal
infection. Infect. Immun. 72, 26892697 (2004).
The first study to demonstrate an early protective
role for CD4+ T cells during pneumococcal
pneumonia in vivo.
45. Mitchell, T.J., Andrew, P.W., Saunders, F.K., Smith,
A.N. & Boulnois, G.J. Complement activation and
antibody binding by pneumolysin via a region
homologous to a human acute phase protein. Mol.
Microbiol. 5, 18831888 (1991).
46. Rubins, J. etal. Distinct roles for pneumolysins
cytotoxic and complement activities in the
pathogenesis of pneumococcal pneumonia. Am.
J.Respir. Crit. Care Med. 153, 13391346 (1996).
47. Alexander, J.E. etal. Amino acid changes affecting
the behaviour of pneumococci in pneumonia. Microb.
Pathog. 24, 167174 (1998).
48. Jounblat, R., Kadioglu, A., Mitchell, T. & Andrew, P.
Pneumococcal behavior and host responses during
bronchopneumonia are affected differently by the
2008 Nature Publishing Group
REVIEWS
cytolytic and complement-activating activities of
pneumolysin. Infect. Immun. 71, 18131819 (2003).
49. Baba, H. etal. Induction of gamma interferon and
nitric oxide by truncated pneumolysin that lacks pore-
forming activity. Infect. Immun. 70, 107113 (2002).
50. Berry, A.M. etal. Effect of defined point mutations in
the pneumolysin gene on the virulence of
Streptococcus pneumoniae. Infect. Immun. 63,
19691974 (1995).
51. Canvin, J.R. etal. The role of pneumolysin and
autolysin in the pathology of pneumonia and
septicaemia in mice infected with a type2
pneumococcus. J.Infect. Dis. 172, 119123 (1995).
52. Alexander, J.E. etal. Immunization of mice with
pneumolysin toxoid confers a significant degree of
protection against at least nine serotypes of
Streptococcus pneumoniae. Infect. Immun. 62,
56835688 (1994).
53. Kadioglu, A. etal. Upper and lower respiratory tract
infection by Streptococcus pneumoniae is affected by
pneumolysin deficiency and differences in capsule
type. Infect. Immun. 70, 28862890 (2002).
54. Orihuela, C.J., Gao, G.L., Francis, K.P., Yu, J. &
Tuomanen, E.I. Tissue-specific contributions of
pneumococcal virulence factors to pathogenesis.
J.Infect. Dis. 190, 16611669 (2004).
55. Berry, A.M., Yother, J., Briles, D.E., Hansman, D. &
Paton, J.C. Reduced virulence of a defined
pneumolysin-negative mutant of Streptococcus
pneumoniae. Infect. Immun. 57, 20372042 (1989).
56. Berry, A.M., Ogunniyi, A.D., Miller, D.C. & Paton,
J.C. Comparative virulence of Streptococcus
pneumoniae strains with insertion-duplication, point,
and deletion mutations in the pneumolysin gene.
Infect. Immun. 67, 981985 (1999).
57. Kadioglu, A. etal. Host cellular immune response to
pneumococcal lung infection in mice. Infect. Immun.
68, 15571562 (2000).
Demonstrated the central role of pneumolysin in
driving the pattern of inflammation and cellular
infiltration into the lungs in vivo. Also the first to
show an early, pneumolysin-dependent involvement
of T cells in respiratory infection.
58. Benton, K.A., Everson, M.P. & Briles, D.E.
Apneumolysin negative mutant of Streptococcus
pneumoniae causes chronic bacteremia rather than
acute sepsis in mice. Infect. Immun. 63, 448455
(1995).
59. Friedland, I.R. etal. The limited role of pneumolysin
in the pathogenesis of pneumococcal meningitis.
J.Infect. Dis. 172, 805809 (1995).
60. Braun, J. etal. Pneumococcal pneumolysin and H2O2
mediate brain cell apoptosis during meningitis.
J.Clin. Invest. 109, 1927 (2002).
61. Wellmer, A. etal. Decreased virulence of a
pneumolysin deficient strain of Streptococcus
pneumoniae in murine meningitis. Infect. Immun. 70,
65046508 (2002).
62. Winter, A.J. etal. in Proc. 7th Intern. Cong. Infect.
Dis. Abstr. 73.004 (International Society for Infectious
Diseases, Brookline,1996).
63. Hirst, R.A. etal. Relative roles of pneumolysin and
hydrogen peroxide from Streptococcus pneumoniae in
inhibition of ependymal ciliary beat frequency. Infect.
Immun. 68, 15571562 (2000).
64. Hirst, R., Mohammed, B., Mitchell, T., Andrew, P. &
OCallaghan, C. Streptococcus pneumoniae-induced
inhibition of rat ependymal cilia is attenuated by
antipneumolysin antibody. Infect. Immun. 72,
66946698 (2004).
65. Hirst, R.A., Kadioglu, A., OCallaghan, C. & Andrew,
P.W. The role of pneumolysin in pneumococcal
pneumonia and meningitis. Clin. Exp. Immunol. 138,
195201 (2004).
66. Chanter, N. Streptococcus pneumoniae and equine
disease. Equine Vet. J. 26, 56 (1994).
67. Whatmore, A.M. etal. Molecular characterization of
equine isolates of Streptococcus pneumoniae: natural
disruption of genes encoding the virulence factors
pneumolysin and autolysin. Infect. Immun. 67,
27762782 (1999).
68. Jedrzejas, M.J. Pneumococcal virulence factors:
structure and function. Microbiol. Mol. Biol. Rev. 65,
187207 (2001).
69. Bergmann, S. & Hammerschmidt, S. Versatility of
pneumococcal surface proteins. Microbiology 152,
295303 (2006).
70. Jedrzejas, M.J., Lamani, E. & Becker, R.S.
Characterization of selected strains of pneumococcal
surface protein A.J.Biol. Chem. 276, 3312133128
(2001).
REVIEWS
71. Hollingshead, S., Becker, R. & Briles, D. Diversity of
PSDPA: mosaic genes and evidence for past
recombination in Streptococcus pneumoniae. Infect.
Immun. 68, 58895900 (2000).
72. Shaper, M., Hollingshead, S.K., Benjamin, W.H. &
Briles, D.E. PspA protects Streptococcus pneumoniae
from killing by apolactoferrin, and antibody to PspA
enhances killing of pneumococci by apolactoferrin.
Infect. Immun. 72, 50315040 (2004).
73. Briles, D.E. & Mirza, S. PspA inhibits the antibacterial
effect of lactoferrin on Streptococcus pneumoniae.
Biochem. Cell Biol. 84, 401 (2006).
74. McDaniel, L.S. etal. Use of insertional inactivation to
facilitate studies of biological properties of
pneumococcal protein A (PspA). J.Exp. Med. 165,
381394 (1987).
75. Ren, B., Szalai, A.J., Hollingshead, S.K. & Briles, D.E.
Effects of PspA and antibodies to PspA on activation
and deposition of complement on the pneumococcal
surface. Infect. Immun. 72, 114122 (2004).
76. Berry, A. & Paton, J. Additive attenuation of virulence
of Streptococcus pneumoniae by mutation of genes
encoding pneumolysin and other putative
pneumococcal virulence proteins. Infect. Immun. 68,
133140 (2000).
77. Abeyta, M., Hardy, G.G. & Yother, J. Genetic
alteration of capsule type but not PspA type affects
accessibility of surface-bound complement and surface
antigens of Streptococcus pneumoniae. Infect. Immun.
71, 218225 (2003).
78. Rosenow, C. etal. Contribution of novel choline-
binding proteins to adherence, colonisation and
immunogenicity of Streptococcus pneumoniae. Mol.
Microbiol. 25, 819829 (1997).
79. Hammerschmidt, S., Tillig, M., Wolff, S. & Chaatwal, J.
Species specific binding of human secretory
component to SpsA protein of Streptococcus
pneumoniae via a hexapeptide motif. Mol. Microbiol.
36, 726736 (2000).
80. Zhang, J. etal. The polymeric immunoglobulin
receptor translocates pneumococci across human
nasopharyngeal epithelial cells. Cell 102, 827837
(2000).
81. Iannelli, F., Chiavolini, D., Ricci, S., Oggioni, M.R. &
Pozzi, G. Pneumococcal surface protein C contributes
to sepsis caused by Streptococcus pneumoniae in
mice. Infect. Immun. 72, 30773080 (2004).
82. Quin, L.R. etal. Invivo binding of complement
regulator factor H by Streptococcus pneumoniae.
J.Infect. Dis. 192, 19962003 (2005).
83. Janulczyk, R., Iannelli, F., Sjoholm, A.G., Pozzi, G. &
Bjorck, L. Hic, a novel surface protein of Streptococcus
pneumoniae that interferes with complement function.
J.Biol. Chem. 275, 3725737263 (2000).
84. Dave, S., Carmicle, S., Hammerschmidt, S.,
Pangburn, M. & McDonald, L. Dual roles of PspC, a
surface protein of Streptococcus pneumoniae, in
binding human secretory IgA and factor H.J.Immunol.
173, 471477 (2004).
85. Cheng, Q., Finkel, D. & Hostetter, M.K. Novel
purification scheme and functions for a C3-binding
protein from Streptococcus pneumoniae. Biochemistry
39, 54505457 (2000).
86. Howard, L.V. & Gooder, H. Specificity of autolysin of
Streptococcus (Diplococcus) pneumoniae. J. Bacteriol.
117, 796804 (1974).
87. Berry, A.M., Lock, R.A., Hansman, D. & Paton, J.C.
Contribution of autolysin to virulence of Streptococcus
pneumoniae. Infect. Immun. 57, 23242330 (1989).
88. Overweg, K. etal. The putative proteinase maturation
protein A of Streptococcus pneumoniae is a conserved
surface protein with potential to elicit protective immune
responses. Infect. Immun. 68, 41804188 (2000).
89. Hermans, P.W.M. etal. The streptococcal lipoprotein
rotamase A (SlrA) is a functional peptidyl-prolyl
isomerase involved in pneumococcal colonisation.
J.Biol. Chem. 281, 968976 (2006).
90. Berry, A.M. & Paton, J.C. Sequence heterogeneity of
PsaA, a 37-kilodalton putative adhesin essential for
virulence of Streptococcus pneumoniae. Infect.
Immun. 64, 52555262 (1996).
91. Marra, A., Lawson, S., Asundi, J.S., Brigham, D. &
Hromockyj, A.E. Invivo characterization of the psa
genes from Streptococcus pneumoniae in multiple
models of infection. Microbiology 148, 14831491
(2002).
92. Johnson, S.E. etal. Inhibition of pneumococcal
carriage in mice by subcutaneous immunization with
peptides from the common surface protein
pneumococcal surface adhesin A. Infect. Immun. 185,
489496 (2002).
300 | april 2008 | volume 6 www.nature.com/reviews/micro
93. Sampson, J.S., OConnor, S.P., Stinson, A.R.,
Tharpe, J.A. & Russell, H. Cloning and nucleotide-
sequence analysis of psaA, the Streptococcus
pneumoniae gene encoding a 37-kilodalton protein
homologous to previously reported Streptococcus sp.
adhesins. Infect. Immun. 62, 319324 (1994).
94. Briles, D.E. etal. Intranasal immunization of mice
with a mixture of the pneumococcal proteins PsaA and
PspA is highly protective against nasopharyngeal
carriage of Streptococcus pneumoniae. Infect. Immun.
68, 796800 (2000).
95. Romero-Steiner, S. etal. Inhibition of pneumococcal
adherence to human nasopharyngeal epithelial cells
by anti-PsaA antibodies. Clin. Diagn. Lab. Immunol.
10, 246251 (2003).
96. Dintilhac, A., Alloing, G., Granadel, C. & Claverys, J.P.
Competence and virulence of Streptococcus
pneumoniae: Adc and PsaA mutants exhibit a
requirement for Zn and Mn resulting from inactivation
of putative ABC metal permeases. Mol. Microbiol. 25,
727739 (1997).
97. McAllister, L.J. etal. Molecular analysis of the psa
permease complex of Streptococcus pneumoniae.
Mol. Microbiol. 53, 889901 (2004).
98. Lawrence, M.C. etal. The crystal structure of
pneumococcal surface antigen PsaA reveals a metal-
binding site and a novel structure for a putative ABC-
type binding protein. Structure 15, 15531561
(1998).
99. Johnston, J.W. etal. Lipoprotein PsaA in virulence of
Streptococcus pneumoniae: surface accessibility and
role in protection from superoxide. Infect. Immun. 72,
58585867 (2004).
100. Tseng, H.J., McEwan, A.G., Paton, J.C. & Jennings,
M.P. Virulence of Streptococcus pneumoniae: PsaA
mutants are hypersensitive to oxidative stress. Infect.
Immun. 70, 16351639 (2002).
101. Brown, J.S., Gilliland, S.M. & Holden, D.W.
AStreptococcus pneumoniae pathogenicity island
encoding an ABC transporter involved in iron uptake
and virulence. Mol. Microbiol. 40, 572585 (2001).
102. Brown, J.S., Gilliland, S.M., Ruiz-Albert, J. & Holden,
D.W. Characterization of Pit, a Streptococcus
pneumoniae iron uptake ABC transporter. Infect.
Immun. 70, 43894398 (2002).
103. Brown, J.S., Ogunniyi, A.D., Woodrow, M.C., Holden,
D.W. & Paton, J.C. Immunization with components of
two iron uptake ABC transporters protects mice
against systemic Streptococcus pneumoniae infection.
Infect. Immun. 69, 67026706 (2001).
104. Chen, S., Paterson, G.K., Tong, F.H., Mitchell, T.J. &
DeMaria, T.F. Sortase A contributes to pneumococcal
nasopharyngeal colonisation in the chinchilla model.
FEMS Microbiol. Lett. 253, 151154 (2005).
105. Paterson, G.K. & Mitchell, T.J. The role of
Streptococcus pneumoniae sortase A in colonisation
and pathogenesis. Microbes Infect. 8, 145153
(2006).
106. Kharat, A.S. & Tomasz, A. Inactivation of the srtA
gene affects localization of surface proteins and
decreases adhesion of Streptococcus pneumoniae to
human pharyngeal cells invitro. Infect. Immun. 71,
27582765 (2003).
107. King, S.J. etal. Phase variable desialylation of host
proteins that bind to Streptococcus pneumoniae
invivo and protect the airway. Mol. Microbiol. 54,
159171 (2004).
108. Pettigrew, M.M., Fennie, K.P., York, M.P., Daniels, J.
& Ghaffar, F. Variation in the presence of
neuraminidase genes among Streptococcus
pneumoniae isolates with identical sequence types.
Infect. Immun. 74, 33603365 (2006).
109. Berry, A.M., Lock, R.A. & Paton, J.C. Cloning and
characterization of nanB, a second Streptococcus
pneumoniae neuraminidase gene, and purification of
the NanB enzyme from recombinant Escherichia coli.
J.Bacteriol. 178, 48544860 (1996).
110. Manco, S. etal. Pneumococcal neuraminidases A and
B both have essential roles during infection of the
respiratory tract and sepsis. Infect. Immun. 74,
40144020 (2006).
111. Oggioni, M.R. etal. Switch from planktonic to sessile
life: a major event in pneumococcal pathogenesis.
Mol. Microbiol. 61, 11961210 (2006).
Describes the interesting role of CSP in
pneumococcal virulence and biofilm formation.
Also demonstrates different patterns of
pneumococcal-gene expression during infection in
the host: one that is typical of bacteria in blood
and one that is typical of bacteria in tissue, such as
brain and lung.
2008 Nature Publishing Group
112. Sorensen, U.B.S., Blom, J., Birch-Andersen, A. &
Henrichsen, J. Ultrastructural localization of capsules,
cell wall polysaccharide, cell wall proteins, and F
antigen in pneumococci. Infect. Immun. 56,
18901896 (1988).
113. Sorensen, U.B.S., Henrichsen, J., Chen, H. C. & Szu,
S.C. Covalent linkage between the capsular
polysaccharide and the cell wall peptidoglycan of
Streptococcus pneumoniae revealed by
immunochemical methods. Microb. Pathog. 8,
325334 (1990).
114. Henrichsen, J. Six newly recognized types of
Streptococcus pneumoniae. J.Clin. Microbiol. 33,
27592762 (1995).
115. Austrian, R. Some observations on the pneumococcus
and on the current status of pneumococcal disease
and its prevention. Rev. Infect. Dis. 3, S1S17
(1981).
116. Martin, M. etal. An outbreak of conjunctivitis due to
atypical Streptococcus pneumoniae. N.Engl. J.Med.
348, 11121121 (2003).
117. Crum, N.F., Barrozo, C.P., Chapman, F.A., Ryan,
M.A. & Russell, K.L. An outbreak of conjunctivitis
due to a novel unencapsulated Streptococcus
pneumoniae among military trainees. Clin. Infect. Dis.
39, 11481154 (2004).
118. Lee, C. J., Banks, S.D. & Li, J.P. Virulence, immunity
and vaccine related to Streptococcus pneumoniae.
Crit. Rev. Microbiol. 18, 89114 (1991).
119. Winkelstein, J.A. The role of complement in the hosts
defense against Streptococcus pneumoniae. Rev.
Infect. Dis. 3, 289298 (1981).
120. Musher, D.M. Infections caused by Streptococcus
pneumoniae: clinical spectrum, pathogenesis,
immunity and treatment. Clin. Infect. Dis. 14,
801807 (1992).
121. Abeyta, M., Hardy, G.G. & Yother, Y. Genetic
alteration of capsule type but not PspA type affects
accessibility of surface-bound complement and surface
antigens of Streptococcus pneumoniae. Infect. Immun.
71, 218225 (2003).
122. Wartha, F. et al. Capsule and dalanylated lipoteichoic
acids protect Streptococcus pneumoniae against
neutrophil extracellular traps. Cell. Microbiol. 9,
11621171 (2007).
123. MacLeod, C.M. & Krauss, M.R. Relation of virulence
of pneumococcal strains for mice to the quantity of
capsular polysaccharide formed invitro. J.Exp. Med.
92, 19 (1950).
Important early paper that demonstrates the in
vivo role of the capsule in pneumococcal virulence.
124. Hostetter, M.K. Serotypic variations among virulent
pneumococci in deposition and degradation of
covalently bound C3b: implications for phagocytosis
and antibody production. J.Infect. Dis. 153,
682693 (1986).
125. Kang, Y. S. etal. The Ctype lectin SIGNR1 mediates
uptake of the capsular polysaccharide of
Streptococcus pneumoniae in the marginal zone of the
mouse spleen. Proc. Natl Acad. Sci. USA 101,
215220 (2004).
126. Lanoue, A. etal. SIGNR1 contributes to protection
against lethal pneumococcal infection in mice. J.Exp.
Med. 200, 13831393 (2004).
127. Fernebro. J. etal. Capsular expression in
Streptococcus pneumoniae negatively affects
spontaneous and antibiotic-induced lysis and
contributes to antibiotic tolerance. J.Infect. Dis. 189,
328338 (2004).
128. Kelly, T., Dillard, J.P. & Yother, J. Effect of genetic
switching of capsular type on virulence of
Streptococcus pneumoniae. Infect. Immun. 62,
18131819 (1994).
129. Nesin, M., Ramirez, M. & Tomasz, A. Capsular
transformation of a multidrug-resistant Streptococcus
pneumoniae invivo. J.Infect. Dis. 177, 707713
(1998).
130. Sandgren, A. etal. Virulence in mice of pneumococcal
clonal types with known invasive disease potential in
humans. J. Infect. Dis. 192, 791800 (2005).
131. Talbot, U., Paton, A.W. & Paton, J.C. Uptake of
Streptococcus pneumoniae by respiratory epithelial
cells. Infect. Immun. 64, 37723777 (1996).
132. Hammerschmidt, S. etal. Illustration of pneumococcal
polysaccharide capsule during adherence and invasion
of epithelial cells. Infect. Immun. 73, 46534667
(2005).
133. Muoz, R., Mollerach, M., Lpez, R. & Garca, E.
Molecular organization of the genes required for the
synthesis of type1 capsular polysaccharide of
Streptococcus pneumoniae: formation of binary

encapsulated pneumococci and identification of
cryptic dTDP-rhamnose biosynthesis genes. Mol.
Microbiol. 25, 7992 (1997).
134. Ogunniyi, A.D., Giammarinaro, P. & Paton, J.C. The
genes encoding virulence-associated proteins and the
capsule of Streptococcus pneumoniae are upregulated
and differentially expressed invivo. Microbiology
148, 20452053 (2002).
135. LeMessurier, K.S., Ogunniyi, A.D. & Paton, J.C.
Differential expression of key pneumococcal virulence
genes invivo. Microbiology 152, 305311 (2006).
136. Morona, J.K., Paton, J.C., Miller, D.C. & Morona, R.
Tyrosine phosphorylation of CpsD negatively regulates
capsular polysaccharide biosynthesis in Streptococcus
pneumoniae. Mol. Microbiol. 35, 14311442
(2000).
Provides the first evidence for the post-
translational regulation of capsule expression
through tyrosine phosphorylation of a conserved
capsule-biosynthesis protein. The mechanism
probably operates in diverse encapsulated
bacteria, and is a potential drug target.
137. Cieslewicz, M.J., Kasper, D.L., Wang, Y. & Wessels,
M.R. Functional analysis in typeIa group B
Streptococcus of a cluster of genes involved in
extracellular polysaccharide production by diverse
species of streptococci. J.Biol. Chem. 276, 139146
(2001).
138. Morona, J.K., Morona, R., Miller, D.C. & Paton, J.C.
Mutational analysis of the carboxy-terminal (YGX)4
repeat domain of CpsD, an autophosphorylating
tyrosine kinase required for capsule biosynthesis in
Streptococcus pneumoniae. J.Bacteriol. 185,
30093019 (2003).
139. Morona, J.K., Miller, D.C., Morona, R. & Paton,
J.C. The effect that mutations in the conserved
capsular polysaccharide biosynthesis genes cpsA,
cpsB and cpsD have on virulence of Streptococcus
pneumoniae. J.Infect. Dis. 189, 19051913
(2004).
140. Bender, M.H., Cartee, R.T. & Yother, J. Positive
correlation between tyrosine phosphorylation of CpsD
and capsular polysaccharide production in
Streptococcus pneumoniae. J.Bacteriol. 185,
60576066 (2003).
141. Morona, J.K., Morona, R. & Paton, J.C. Attachment
of capsular polysaccharide to the cell wall of
Streptococcus pneumoniae type2 is required for
invasive disease. Proc. Natl Acad. Sci. USA 103,
85058510 (2006).
Demonstrates the involvement of conserved
capsule-biosynthesis genes in the attachment of
polysaccharide to the cell wall and the importance
of this participation for progression from
pneumonia to bacteraemia.
nature reviews | microbiology volume 6 | april 2008 | 301
142. Giammarinaro, P. & Paton, J.C. Role of RegM, a
homologue of the catabolite repressor protein CcpA,
in the virulence of Streptococcus pneumoniae. Infect.
Immun. 70, 54545461 (2002).
143. Mollerach, M., Lpez, R. & Garca, E. Characterization
of the galU gene of Streptococcus pneumoniae
encoding a uridine diphosphoglucose
pyrophosphorylase: a gene essential for capsular
polysaccharide biosynthesis. J.Exp. Med. 188,
20472056 (1998).
144. Hardy, G.G., Magee, A.D., Ventura, C.L., Caimano,
M.J. & Yother, J. Essential role for cellular
phosphoglucomutase in virulence of type 3
Streptococcus pneumoniae. Infect. Immun. 69,
23092317 (2001).
145. Douglas, R.M., Paton, J.C., Duncan, S.J. &
Hansman, D. Antibody response to pneumococcal
vaccination in children younger than five years of age.
J.Infect. Dis. l48, 131137 (l983).
146. Paton, J.C. in The Pneumococcus (eds Tuomanen,
E.I., Mitchell, T.J., Morrison, D.A. & Spratt, B.G.)
382402 (ASM, Washington DC, 2004).
147. Huang, S.S. etal. Post-PCV7 changes in colonizing
pneumococcal serotypes in 16 Massachusetts
communities, 2001 and 2004. Pediatrics 116,
408413 (2005).
148. Reingold, A. et al. Direct and indirect effects of routine
vaccination of children with 7valent pneumococcal
conjugate vaccine on incidence of invasive
pneumococcal disease United States, 19982003.
MMWR 54, 893897 (2005).
149. Steenhoff, A.P., Shah, S.S., Ratner, A.J., Patil, S.M.
& McGowan, K.L. Emergence of vaccine-related
pneumococcal serotypes as a cause of bacteremia.
Clin. Infect. Dis. 42, 907914 (2006).
150. Ogunniyi, A.D., Folland, R.L., Hollingshead, S., Briles,
D.E. & Paton, J.C. Immunization of mice with
combinations of pneumococcal virulence proteins
elicits enhanced protection against challenge with
Streptococcus pneumoniae. Infect. Immun. 68,
30283033 (2000).
The first paper to demonstrate that immunization
with combinations of pneumococcal virulence
proteins elicits greater levels of protection
against challenge than if the same antigens are
used individually. Thus, combinations of protein
antigens may be capable of eliciting robust
protection against diverse pneumococcal strains.
151. Briles, D.E. etal. Intranasal immunization of mice
with a mixture of the pneumococcal proteins PsaA and
PspA is highly protective against nasopharyngeal
carriage of Streptococcus pneumoniae. Infect. Immun.
68, 796800 (2000).
152. Briles, D.E. etal. Immunizations with pneumococcal
surface protein A and pneumolysin are protective
2008 Nature Publishing Group
REVIEWS
against pneumonia in a murine model of pulmonary
infection with Streptococcus pneumoniae. J.Infect.
Dis. 188, 339348 (2003).
153. Ogunniyi, A.D., Grabowicz, M., Briles, D.E., Cook, J.
& Paton, J.C. Development of a vaccine against
invasive pneumococcal disease based on combinations
of virulence proteins of Streptococcus pneumoniae.
Infect. Immun. 75, 350357 (2007).
154. Redd, S.C. etal. The role of human immunodeficiency
virus infection in pneumococcal bacteremia in San
Francisco residents. J.Infect. Dis. 162, 10121017
(1990).
155. Rodriguez-Barradas, M.C. etal. Colonisation by
Streptococcus pneumoniae among human
immunodeficiency virus-infected adults prevalence
of antibiotic resistance, impact of immunization, and
characterization by polymerase chain reaction with
BOX primers of isolates from persistent
S.pneumoniae carriers. J.Infect. Dis. 175,
590597 (1997).
156. Kemp, K., Bruunsgaard, H., Skinhoj, P. & Klarlund
Pedersen, B. Pneumococcal infections in humans are
associated with increased apoptosis and trafficking of
type1 cytokine-producing Tcells. Infect. Immun. 70,
50195025 (2002).
157. Zhang, Q. etal. Low CD4 Tcell immunity to
pneumolysin is associated with nasopharyngeal
carriage of pneumococci in children. J.Infect. Dis.
195, 11941202 (2007).
158. Zhang, Q. etal. Regulation of production of mucosal
antibody to pneumococcal protein antigens by
Tcellderived gamma interferon and interleukin10
in children. Infect. Immun. 74, 47354743 (2006).
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
comA | comD | comE | cpsB | galU | nanA | nanB | pgm | psaA |
psaB | psaC | pspA | pspC
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Actinobacillus actinomycetemcomitans | Haemophilus
influenzae | Streptococcus pneumoniae
Entrez Protein: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=protein
BgaA | CcpA | CpsC | CpsD | CRP | LytA | PiaA | StrH
FURTHER INFORMATION
Aras Kadioglus homepage: http://www.le.ac.uk/iii/staff/
ak13.html
Jeffrey N. Weisers homepage: http://www.med.upenn.edu/
micro/faculty/weiser.html
All links are active in the online pdf