Res. Microbiol.

151 (2000) 413–419

© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0923250800001753/REV

Virulence factors and the pathogenesis of disease caused

by Streptococcus pneumoniae

Tim J. Mitchell*
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of
Glasgow, G12-8QQ Scotland, UK

Abstract – Streptococcus pneumoniae is a major pathogen of man causing diseases such as pneumonia,
meningitis and otitis media. The mechanisms by which this organism causes these diseases are still largely
unknown. The use of molecular approaches to identifying and studying putative virulence factors in
combination with the application of animal models has allowed some of the mechanisms of the disease
process to be defined. © 2000 Éditions scientifiques et médicales Elsevier SAS
Streptococcus pneumoniae / virulence factors / pathogenesis

1. Introduction type shifts made possible by horizontal gene

Streptococcus pneumoniae (the pneumococcus)
remains a major pathogen of man despite the
advent of antibiotic therapy. The organism is the
causative agent of several important diseases
including pneumonia, meningitis and otitis
media. Even during infection with fully antibi-
otic susceptible strains of the organism there is
an underlying mortality rate of 10% for pneu-
monia and up to 30% for meningitis [1]. Fatality
rates of 5–22% reported for pneumococcal pneu-
monia have been suggested to be due to irrepa-
rable damage caused during the early phase of
the disease [2]. This situation is worsening due
to the failure of chemotherapy due to the appear-
ance of antibiotic-resistant strains [3]. There is a
vaccine available for the prevention of pneumo-
coccal disease but this has shortcomings in that
it does not protect against all serotypes of the
organism and it does not confer good protection
in individuals at the extremes of age (< 2 and
> 60 years old). New conjugate vaccines based
on capsular polysaccharides are in clinical trials
but these may have a limited life due to sero-
* Tel.: +44 (0)141 330 3749; fax: +44 (0)141 330 3727;
T.Mitchell@bio.gla.ac.uk
transfer in this organism [4]. For this reason
there is a pressing need for new therapeutic or
prophylactic measures against this organism.
One way to develop such measures is to under-
stand the mechanism by which the organism
causes disease. The availability of the (almost)
complete genome sequence of the organism
allows us to probe the role of various genes in
the pathogenesis of infection by construction of
isogenic mutants and use in animal models. The
definition of the role of these genes in the
disease process also involves an analysis of the
host response to the organism. This review will
summarise the role of some virulence factors in
experimental infections and will also mention
how these virulence factors might be regulated.
2. Virulence factors and their regulation
Prior to the release in November 1997 of the
genome sequence of the organism the number
of putative virulence factors suggested for the
pneumococcus was relatively small [5]. With the
availability of the genome sequence we are now
spoilt for choice in terms of genes to analyse
and the emphasis has shifted from gene identi-
fication to functional studies. Here I will review
some of the functional studies carried out with
414 T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419
Figure 1. Some of the virulence factors produced by S. pneumo-
niae.
well-characterised virulence factors and discuss
some of the newer data relating to host response
and the role of two-component transcriptional
regulators (TCS) in virulence. A summary of
some of the putative virulence factors of the
pneumococcus is shown in figure 1.
3. Pneumolysin
Much work has been carried out on the role
of this protein in infection models. The gene
was originally cloned in 1983 [6, 7] and the
availability of this gene allowed the construc-
tion of defined pneumolysin-negative knock-
outs and isogenic replacement mutants [8–10].
These mutants have been used in several labo-
ratories to probe the role of the toxin. The toxin
itself has been the subject of a detailed structure
function analysis and a three-dimensional struc-
ture has been proposed based on the structure
of the related toxin perfringolysin O. Functional
domains have been mapped onto this structure
[11]. By altering these domains and
re-introducing the gene into the pneumococcus
it has been possible to probe the role of indi-
vidual amino acids in the pathogenesis of infec-
tion [8]. A summary of the effects of the muta-
tions and the possible role of pneumolysin is
given in table I. Pneumolysin has at least two
biological activities that contribute to virulence
in animal models: i) lytic activity and ii) ability
to activate complement. However, introduction
of point mutations, which block these two activi-
ties, results in an organism that is still more
virulent than the pneumolysin-negative mutant
[12]. This suggests that there is another activity
of the toxin which contributes to virulence.
4. Surface proteins
The surface of the pneumococcus is decorated
with a range of surface proteins that may play a
role in pathogenesis. These proteins may be
either anchored to the cell wall via the Gram-
positive attachment motif (LPXTG) or may be
noncovalently attached via an interaction with
choline (choline-binding proteins, Cbps). One of
the neuraminidase enzymes and hyaluronidase
produced by the pneumococcus spend part of
their time as cell surface proteins as judged by
the presence of the LPXTG motif and cell locali-
sation experiments [13]. Choline-binding pro-
teins share common repeat elements associated
with this property and include PspA, LytA, and
CbpA which will be described here.
4.1. Pneumococcal surface protein A (PspA)
The mechanism of action of PspA is not fully
understood, but the protein is present on the
surface of all pneumococci and is required for
full viulence [14, 15]. It has been reported that
PspA is a lactoferrin-binding protein [16]. PspA
also inhibits complement activation by Strepto-
coccus pneumoniae [17]. The amino-terminal half
of the protein contains a highly charged coil–
coil domain and it is variations in this sequence
that generate the heterogeneity observed in this
molecule. The region still contains sufficient
conserved epitopes to allow vaccination with a
single PspA type to confer protective immunity
to a range of other PspA types [18].
4.2. Choline-binding protein (CbpA)
CbpA is present on the surface of the pneu-
mococcus and reacts with pooled convalescent
 T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419 415

Table I. Mutations of pneumolysin and their effects on the virulence of S. pneumoniae.

Mutation Biological effects Reference
Respiratory tract
Null Reduced virulence [9]
Less inflammation during pneumonia [27]
Delayed invasion of bloodstream from lungs [27]
Less tight-junction separation in human organ cultures [35]
No TNF production in alveoli Our unpublished data
Bacteraemia model
Chronic infection rather than acute [36]
Induces protection from subsequent challenge with wild type [36]
Eye infection model
Mediates inflammation [37]
Meningitis
No overall effect on inflammation [38]
Mediates hearing loss [31]
Otitis media
No role [39]
Respiratory tract
Complement activity Reduced virulence [40]
abolished Important at later times of infection (lobar pneumonia) [40]
Protects small numbers of bacteria early (broncho-pneumonia) [8]
Bacteraemia model
No effect [41]
Eye infection model
Mediates inflammation [42]
Respiratory tract
Lytic activity abolished Reduced virulence [40]
Important at early times of infection [40]
Mediates permeability change in epithelium [40]

serum from patients with bacteraemic pneumo-
coccal pneumonia [19]. If the gene for CbpA is
disrupted by insertion duplication mutagenesis
the mutant pneumococci have a reduced ability
to bind to cytokine-activated type II pneu-
mocytes and endothelial cells in vitro suggest-
ing that this protein plays a role in adherence.
This is supported by the fact that the mutant
organism also shows decreased ability to bind
to glycoconjugates containing lacto-N-
neotetraose and sialic acids, the putative recep-
tors for pneumococci on such activated cells.
Moreover, the mutant organism is less able to
colonise the nasopharynx of infant rats. Another
pneumococcal surface protein designated SpsA,
which binds to the secretory component of
secretory immunoglobulin A, has been described
[20]. Sequence analysis shows that SpsA and
CbpA are variants of the same protein. CbpA
therefore has at least two possible functions in
the pathogenesis of pneumococcal disease. Inter-
action with IgA secretory component may inter-
fere with the protective function of IgA and may
also promote adherence directly [20]. The mecha-
nism by which CbpA mediates attachment to
cytokine-activated lung cells is independent of
its ability to bind to the secretory component as
this protein was not present in the assays of
binding to these cells in vitro.
4.3. Pneumococcal surface adhesin A (PsaA)
PsaA was originally reported as a surface
adhesin based on sequence homology with other
streptococcal adhesins [21]. Pneumococci that
do not express PsaA are virtually avirulent in
animal models of disease and show reduced
ability to adhere to type II pneumocytes in vitro
[22]. The psaA gene is part of an operon consist-
ing of three genes [23]. The operon has the
features of an ATP binding cassette (ABC) trans-
416 T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419
port system that appears to be a manganese
transporter. PsaA has been crystallised and its
three-dimensional structure determined to
2-angstrom resolution [24], which showed that
PsaA has a novel structure for an ABC-type
binding protein. The PsaA protein may be an
adhesin or it may reflect a requirement for
manganese either as a growth factor in vivo or a
regulator of adhesin expression.
5. Pneumococcal enzymes involved
in pathogenesis
5.1. Autolysin
Autolysin is a cell-wall-degrading enzyme
found in the cell envelope of the pneumococcus.
The enzyme is normally inactive but under
conditions in which biosynthesis stops, such as
nutrient starvation or penicillin treatment, the
enzyme is activated and causes autolysis of the
bacterial cell. Such autolysis releases degrada-
tion products of the cell wall, including pepti-
doglycan and teichoic acid. These cell wall
degradation products can cause inflammation.
The action of autolysin also releases intracellu-
lar constituents including toxins such as pneu-
molysin.
The role of autolysin in virulence has been
demonstrated by use of autolysin-negative
mutants constructed by insertion-duplication
mutagenesis [25, 26]. Such mutants are less
virulent than their wild-type parents. When
instilled into the mouse lung the autolysin-
negative mutant was rapidly cleared from the
lung and did not cause pneumonia [27]. No
inflammatory response was seen in the lungs of
animals infected with the autolysin-negative
mutant.
Autolysin probably contributes to virulence
by allowing the release of other active compo-
nents of the pneumococcus such as pneumol-
ysin.
5.2. Neuraminidases
A variety of evidence suggests that neuramini-
dase enzymes play a role in pathogenesis. His-
tochemical examination of mice dying after
intraperitoneal administration of partially puri-
fied neuraminidase has indicated marked
decreases in the sialic acid content of the liver
and kidney when compared to control animals
[28]. Coma and bacteraemia occur significantly
more often in patients with pneumococcal men-
ingitis when the concentration of N-acetyl
neuraminic acid in the cerebrospinal fluid is
elevated [29]. The pneumococcus makes at least
two enzymes with neuraminase activity (NanA
and NanB) and the genes for these proteins
have been cloned [13, 30]. Availability of the
genes allowed the construction of an isogenic
mutant of the pneumococcus which does not
express nanA [31]. This mutant was less virulent
in an animal model of pneumonia (TJM, unpub-
lished data). The number of mutant organisms
in the lung remained the same over 3 days of
infection while the wild-type organisms
increased by 4 logs during 2 days when the
animals died. This suggests that neuraminidase
allows the pneumococcus to survive and repli-
cate in the lung. NanA does not appear to
contribute to inflammation or hearing loss in
animal models of meningitis [31]. The role of
NanB in the pathogenesis of pneumonia still
remains to be defined.
5.3. Hyaluronidase
The pneumococcus produces the enzyme
hyaluronidase, which degrades hyaluronic acid,
a component of connective tissue. It has there-
fore been suggested that hyaluronidase allows
greater access of organisms to the tissue and
may also play a role in the translocation of
pneumococci between tissues, from the lung to
the blood for example. As well as playing a role
in tissue integrity hyaluronic acid also plays a
role in the generation of the inflammatory
response. Production of hyaluronidase could
also affect the inflammatory response in the
lung.
The gene for pneumococcal hyaluronidase
has been cloned [32]. This sequence has been
used to construct a hyaluronidase-negative
mutant by insertion-duplication mutagenesis
(TJM, unpublished). Interruption of the hyalu-
 T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419 417

Table II. The two-component systems (TCS) of Streptococcus pneumoniae*.

TCS System Possible function Reference
1 Unknown
2 Unknown
3 Unknown
4 Homology suggests phosphate sensing. Recent study suggests not involved in [43]
phosphate metabolism
5 Penicillin tolerance. Competence [44]
6 Virulence. Null mutant shows reduced virulence in murine pneumonia model Our unpublished data
7 Unknown
8 Virulence. Null mutant shows reduced virulence in murine pneumonia model Our unpublished data
9 Unknown
10 Vancomycin tolerance [45]
11 Unknown
12 Competence [46]
13 Peptide (quorum?) sensing Our unpublished data

* Table is based on [34].

ronidase gene caused a reduction in virulence in
a pneumonia model. During pneumonia the
hyaluronidase-negative mutant of the pneumo-
coccus appeared in the bloodstream at the same
time as the wild-type parent but only reached
levels of 103/mL, whereas the wild type reached
108/mL. Direct instillation of pneumococci into
the blood showed no difference in the ability of
the mutant and wild type to survive. Hyalu-
ronidase is therefore involved in the invasion of
the bloodstream.
5.4. Superoxide dismutase (SOD)
The pneumococcus contains two types of
SOD (MnSOD and FeSOD). Inactivation of the
gene for MnSOD (sodA) showed that MnSOD
plays a role in the virulence of the pneumococ-
cus in animal models [33]. The pattern of inflam-
mation in lungs infected with the mutant was
different from that seen with wild-type organ-
isms. After infection with the mutant neutro-
phils were packed around bronchioles in con-
trast to the wild-type infection where
neutrophils were more diffusely localised.
6. Two-component
signal transduction systems
Analysis of the genome sequence released by
TIGR shows that there are 13 TCS systems in the
pneumococcus [34]. These are summarised in
table II. The role of these systems in virulence
has not yet been fully studied. Analysis of
mutations in an in vivo model involving sys-
temic challenge of mice suggested that none of
the 11 systems tested (two of the mutations
were lethal) had any effect on virulence. We
have introduced these mutations into a type 2
strain of the pneumococcus and used them to
evaluate the role played by these systems in a
pneumonia model. Our preliminary data sug-
gest that at least two of the systems are essential
for virulence in this model. The pathways
involved appear to be specific for pulmonary
infection routes. The signals being sensed and
the genes being regulated by these pathways
are currently being evaluated. Preliminary data
suggest the systems may regulate the expres-
sion or activity of surface proteins.
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