THIS IS THE REAL ONE.

Report project work Infection and Immunity

The relation between S. Pneumoniae, cytotoxic T cells and Toll like receptor

By Lotte van Doeselaar, Elin Edsbäcker, Sanne Jonsson, René Niehus and
Robin Ramsurrun

October, 21 2010
1. Introduction

This is a report of the project work Infection and Immunity. The aim of this project work is to
integrate the disciplines immunology and microbiology by combining three topics. This
reports describes relationship between S. pneumonia, cytotoxic T cells and toll like receptor.
This was done by first describing Streptococcus Pneumonia (S. Pneumonia), the disease it
causes, and the immune response to S. Pneumonia. And then the relationship between the
different topics is discussed. (do I have to name the specific questions? )

2.1 Streptococcus pneumonia

Streptococcus pneumoniae, or pneumococcus, is a common bacterium in the oral cavity and

the throat of healthy people, especially in children. S. Pneumonia is a gram-positive
encapsulated coccus and is about 0.5 to 1.2 μm in diameter. The cells are arranged in pairs
(diplococcus) which gives them a bullet shape. S. pneumoniae is α-hemolytic, [what is this?
Explain shortly…] and a facultative anaerobe. This means that the bacterium does not rely on
the use of oxygen but is able to utilize it once it finds itself in a oxygen rich environment.
Virulent strains of S. pneumoniae are covered with a capsule that consists of a complex
mixture of monosaccharides, oligosaccharides and other components. Antigenic differences
within these polysaccharides have made it possible to identify 90 different serotypes.
The cell wall structure of pneumococcus is typical for gram-positive cocci, containing a
large peptidoglycan layer. Attached to this peptidoglycan layer are teichoic acid, LPA and
phosphocholine that provide binding domains for surface proteins.
Unique for these species is the carbohydrate structure of the cell wall called the C
polysaccharide. This structure contains phosphocholine, which the bacteria needs during cell
division. C-reactive protein, which is present in low concentrations in the blood, interacts with
the phosphocholine, causing C polysaccharide to precipitate. This is can be used by the innate
immunity to recognize identify S. pneumoniae and separate it from other streptococcal
groups.1,2,3

For Streptococcus pneumonia to colonizes the human nasopharynx and to cause disease,
certain abilities are needed . That is why several virulence factors are involved in the disease
process of S. pneumonia.
The most important virulence factor of S. pneumonia is probably the polysaccharide
capsule. First of all the capsule plays an important role in the adherence to the host cell.
Pneumococcal antibodies, found on the surface of the capsule, bind to receptors on the host
cell causing it to adhere to the host cell and making S. Pneumonia able to infect it. Another
very important function of the capsule is the antiphagocytic activity. The capsule can prevents
binding of antibodies of the bacterium to the receptors on the phagocytotic cells by blocking
the interaction between C3 receptors and C3b2. On top of that the capsule plays a crucial role
in colonization, preventing mechanical removal by mucus and it can restrict autolysis and
reduce exposure to antibiotics.
Another important virulence factor is PLY, a protein virulence factor, belonging to the
family of pore forming toxins. PLY is a 53kDa protein and is made by almost all
pneumococci. The PLY toxin binds to the membrane cholesterol where it forms large pores,
causing cell membranes to leak. The toxin also activates the complement pathway, even
though specific antibodies are absent. At inflamed areas PLY also delays neutrophil responses
and delays and reduces the redistribution of T- and B-lymphocytes. Besides this PLY induces
proinflammatory response and produces reactive oxygen intermediates and other mediators.
These effects seem to be mediated by the fact that PLY physically interacts with the
lipopolysaccharide receptor, Toll like receptor 4 (TLR4). Research showed that protection
from colonization with pneumococcal bacteria both requires TLR44 and interleukine-17 (IL-
17)5, which activates regulatory T cells. Both of these effects appear to be dependent on PLY.
Pneumococci have so called LPXTG-anchored surface proteins. The N-terminal
sequence of this protein provides a mechanism for surface location of proteases, while the C-
terminus is a recognition sequence for enzymes to recognize the LPXTG protein and to
anchore it to the cell surface. Hyaluronidase and neuraminidase are LPXTG proteins that play
a role in the virulence of S. Pneumonia. Hyaluronidase breaks down the hyaluronic acid
component of mammalian connective tissue and extracellular matrix and in this way helps
bacterial spread and colonization. Besides that hyaluronidase interacts with proinflammatory
cytokines and chemokines and can promote more cytokine secretion by binding to CD44 on
host cells, causing further inflammation.
By cleaving N-acetylneuraminic acid from glycolipids, lipoproteins and oligosaccharides on
cell surfaces neuraminidase can cause direct damage to cells and decrease recognition by
removing potential binding sites for organisms.
The Pneumococcal pilus is also involved in the virulence of S. Pneunomia . The pilus
helps the binding of pneumococci to cell surfaces and also stimulates proinflammatory
cytokine production.
Choline-binding proteins (CBP’s) are proteins, anchored to the the cell surface via
interaction with choline present in the pneumococcal cell wall, and several of these CPB’s
have shown to be a virulence factor for S. Pneumonia. LytA is one of these CPB’s and plays a
role in virulence by releasing highly inflammatory cell wall degradation products and PLY
from the cytoplasm. LytB, LytC and CpbE are important for the pneumococcal colonization.
Pneumococcal surface protein A (PspA) is a major virulence factor since it interferes with the
complement system and binds to Lactoferrin, which plays an important role in innate
immunity. Pneumococcal surface protein C (PspC) is a multifunctional virulence factor,
because it can vary in function in different pneumococcal strains. It can act as an adhesin,
binding the immunoglobulin receptor, or it can provide resistance against the complement by
binding complement regulatory proteins. Besides the CBP’s there are various other surface
binding proteins that play a role in virulence for pneumococci.
S. pneumonia can also reside inside a biofilm. By forming a layer of bacteria and
surrounding itself with a mucuslayer which is attached to a surface, the bacteria protect
themselves from antibiotics and host defense6
At last, genetic variation at different levels is a very important way of contributing to
the disease causing ability of S. pneumonia. This genetic variation may be caused by instant
mutations or by transformation, in which pneumococci obtain DNA, coding for additional or
adapted virulence, from each other via plasmids. 7

Except for S. pneumonia, the genus Streptococcus contains two other important human
pathogens; Streptococcus pyogenes and Streptococcus Agalactiae.
S. Pyogenes is a gram-positive coccus, 1-2 μm in diameter, and often arranged in chains
of 4 to 10 cells. Unlike S. pneumoniae, S. pyogenes is β-hemolytic, this shows as a clear zone
around colonies when grown on blood agar, caused by the complete lysis of red blood cells,
[still not sure what functions of being alpha or beta hemolytic are] and produces two
hemolysins; streptolysin S and O. These toxins can lyse erythrocytes, leukocytes and platelets.
The cell wall structure of S. Pyogenes is similar to the one of S. pneumonia, but all serotypes
of S. Pyogenes contain a group of a specific carbohydrate structure called the A antigen. This
antigen is used to separate S. pyogenes from other streptococcal groups. There are more than
80 serotypes which are separated by antigenic differences in proteins in the cell wall. Some
strains have a nonantigenic capsule. Despite their similarities S. Pyogenes and S. pneumoniea
do not cause the same diseases. For example, S. Pyogenes can cause pharyngitis, impetigo,
toxic shock syndrome and rheumatic fever, while S. Pneumonia mainly causes upper and
lower respiratory tract infections.
S. Agalactiae is also a gram-positive coccus. The cells are about 0.6-1.2 μm in diameter
and form short chains. Most strains of S. Agalactiae are β-hemolytic but some are
nonhemolytic [should we give the explanation, maybe not if we can’t give a short clear
definition of beta hemolytic too?]. The cell wall contains the group specific polysaccharide B
antigen, used to identify the bacterium. S. Agalactiae has a capsule which can express nine
different type-specific polysaccharides. These polysaccharides and differences in surface
proteins make it possible to identify different serotypes. 1,2

2.2 Disease caused by S. pneumonia

S. pneumonia is a natural inhabitant of the upper respiratory tract in nearly all humans and is
the leading cause for pneumonia, causing about 1-2 million infant deaths worldwide.6 Other
common diseases caused by S. pneumonia are sinusitis, otitis media, meningitis and
bacteremia, presence of bacteria in the bloodstream. Extensive research has been performed to
understand why it is one of the most successful infectious pathogens in the world but much
remains to be revealed when it comes to the pathogenesis of pneumococcal disease.
Symptoms arising from a pneumococcal infection are mostly caused by the
immunological response, while reactions from bacterial toxins play a minor roll. Pneumonia
occurs when S. pneumonia, inhabiting the oropharynx, are aspirated down to the lungs where
they colonize in the alveolar space. The patient suffering from the disease often gets sick
rather quickly and gets febrile with temperatures between 39° and 41° C, a productive cough
with blood-tinged sputum and chest pain. Abscesses do not usually form in the lungs during a
pneumococcal pneumonia except when the infection is caused by a certain serotype,
type 3.2
As mentioned earlier the capsule of S. pneumonia has a antiphagocytic function,
causing mucus and phagocytic cells to not be able to remove the bacteria. There are
antibodies directed against the capsular polysaccharides and people with low concentrations
of these antibodies have a great risk of infection. Since children and elderly often have
reduced levels of these antibodies, the disease is most common in those two risk groups2.
Bacteria living inside biofilms are better able to avoid host defenses and have a greater chance
of causing pneumonia and meningitis, while unattached bacteria are more likely to induce
bacteremia.6
S. pneumonia bacteria are spread from person to person in droplets from respiratory
secretions. This way of spreading is called air bore. Under normal and healthy conditions the
defense mechanisms of the body would prevent clinical infection by pneumococci. Though,
these defense mechanisms might be disrupted by chronical (as smoking, allergies or
respiratory diseases) or acute (as viral infections) factors. When disrupted, the body won’t be
able to protect itself from the pneumococci, which can lead to infection. Once the conditions
able the bacteria to spread, colonization by hematogenous spread takes place. Bacteria spread
from the nasopharynx to normally sterile places such as blood, peritoneum, cerebrospinal
fluid, or joint fluid. Here the pneumococcus can start colonizing and cause infection.

Another microbe that causes pneumonia is Staphylococcus aureus. By infection with S.

aureus, the disease develops after aspiration of oral secretion or by hematogenous spread.8 A
viral infection often precedes pneumonia, caused by S. aureus. The viral infection damages
the mucosa which makes the host more susceptible for developing the pneumonia. The
clinical manifestations of pneumonia caused by S. aureus are similar to those of
pneumococcal infection, but in an infection with S. Aureus abscesses can sometimes be
present. The abscesses are caused by cytotoxic toxins that are produced by the organism. One
of the virulence factors of S. aureus is panton-valentine leukocidin (PVL), which has been
associated with a necrotizing pneumonia, caused by Methicillin-resistant Staphylococcus
aureus. PVL is a toxin that consists of two components and causes pores in the cell walls of
neutrophils.9 This leads to release of neutrophilic chemotactic factors such as interleukine-8,
leukotriene B4 and several cytokines which induce cell death.9
The microbe Acinetobacter baumannii is also capable of causing pneumonia. A.
baumannii is a aerobic gram-negative coccobacillus and not as common as S. aureus.9 In
most cases this bacteria causes nosocomial pneumonia, which is acquired by patients during
hospitalization, but community-acquired A. baumannii pneumonia (CAP-AB) is becoming
more frequent. CAP-AB is also known for having a higher mortality rate than the hospital
acquired variety.10 Little is known about the virulence factors concerning this microbe but just
like S. pneumoniae and S. aureus it has the ability to create a protecting biofilm.10

2.3 The immune response to S. Pneumoniae

To combat the S. pneumoniae bacteria, a functional innate immune system is indispensable.

More precisely the lung macrophages and their activation are a key step in the fight against S.
pneumoniae. The activation of macrophages in turn cannot happen without pattern
recognition receptors (PRR).
There are six pattern recognition receptors including three Toll-like receptors shown to be
important in the initiation of an effective, innate immune response. These are:
1. C-reactive protein (an acute phase protein): is a soluble PRR binding to phosphorylcholine
in the cell wall of S. pneumonia.
2. SIGNR1 (a DC specific C-type lectin): binds to capsular polysaccharides. The activation
also results in the production of IgM antibodies assisting in clearance of pneumococci.
3. MARCO (a scavenger receptor on alveolar macrophages): binds and internalizes S.
pneumonia.
4. TLR2: is generally the most important TLR for GRAM+ bacteria but its role in the fight
against S. pneumonia is only partially understood. TLR2 was shown to recognize certain
wall components in the microbe.
5. TLR 4: is responsible for the pro-inflammatory effect of pneumolysin.
6. TLR 9: Is known to be an intracellular TLR which is specific against bacterial DNA. Still it
seems to be essential for the effective phagocytosis and killing by lung macrophages.
[Additions from Rene]

The high prevalence of Streptococcus pneumonia related diseases and the progressive
resistance to antimicrobial drugs evokes the urgent need of effective vaccines against S.
pneumonia.11
There are two vaccine forms available on the market. The first one is the Pneumococcus
polysaccharide vaccine and the second one the Pneumococcal conjugate vaccine. The used
vaccines are given depending on age and risk group and have their advantages and
disadvantages.
The Pneumococcal polysaccharide vaccine consists of the 23 most common capsular
serotypes that cause invasive pneumococcal disease in the developed world.6 By exposing the
patient to capsular serotypes of S. pneumonia, T-cell-independent B-cell responses will be
evoked. This response includes the production of antibodies specific against the virulent
capsule of the microbe.
This vaccine is highly effective and inexpensive which covers a broad spectrum
of different S. pneumonia serotypes and is thereby in widespread use. On the other hand this
vaccine is not very immunogenic since it is T-cell independent. It does not evoke
immunological memory and the antibodies decline after 3-5 years.11 There are also weak
vaccine responses shown in children and immunodeficient patients which are at greatest risk
for severe pneumococcal disease.6
The Pneumococcal conjugate vaccine is a covalently linked polysaccharide-protein conjugate.
The principle of this vaccine is to covalently attach a poor antigen (the polysaccharide) to a
carrier protein, which will be taken up by specific pre-B-cells and digested to T-cell-epitopes
resulting in T-cell activation and aid, while the polysaccharide itself would be a poor vaccine
with no anamnestic response.
The advantages of this vaccine is the better immunogenicity compared to the
polysaccharide vaccine which is also true for high risk individuals such as children under the
age of 2 years. However the vaccine is expensive and has low serotype coverage, which is a
great problem.11 There are no data about the vaccine’s efficiency in adults available.
The Pneumococcal conjugate vaccine has been in use for 10 years now and was very

successful in reducing the prevalence of pneumococcal disease in children.

This vaccine being able to induce immunity in very young children showed direct and indirect
positive effects which may explain the drastic decline of pneumococcal diseases. A study
demonstrated that invasive pneumococcal disease rates in the vaccine era have also decreased
among unvaccinated older children, adults, and elderly persons.12 This phenomena is called
herd immunity meaning that due to the vaccination of a special part of the society
unvaccinated individuals are also protected due to reduced exposure and reduced
transmission. In the case of S. pneumonia carriage rates are highest among children aged less
than 2 years old, and young children are primarily responsible for introducing new serotypes
into a household.13
New vaccines are on the way. Different immune serotypes have expanded perhaps due to the
introduction of the vaccines. These serotypes have for example a different antigenic
polysaccharide coat to avoid vaccine-induced immune recognition probably arisen through
transformation of capsular genes from one serotype to another (capsular switching).6
New vaccines may expand the spectrum of the Pneumococcal conjugate vaccine. Other
vaccine approaches include development of vaccines against many, highly conserved,
immunogenic protein antigens of the microbe—e.g. adhesins, pneumolysin, invasion proteins,
and transport proteins.14This would serve to ensure the body attacks those features of the
bacterium that are fairly consistent between different strains.

2.4 Relationship between S. pneumonia, Cytotoxic T cells and Toll like

receptor

The former paragraphs showed the basics about S. Pneumonia, how it causes disease and how
the body defends itself against an infection with the bacterium. Based on this, this paragraph
tries to describe a relationship between the tree topics; S. Pneumoniae, Cytotoxic T cells and
T cell receptor.

The primary method for the detection of Streptococcus Pneumoniae by the immune system is
through toll-like receptor 2 (TLR2). This toll-like receptor detects the presence of the
lipotechoic acid present on the surface of the gram-positive Streptococcus Pneumoniae, with
lipotechoic acid being a PAMP – pathogen associated molecular pattern. This is specifically
displayed in a study by Echannaoui H et al.15 which showed that mice who lacked the TLR2-
encoding gene were at a significantly higher risk of death from the actions of streptococcus
pneumonia when compared against wild-type mice.
Toll-like receptors are scattered across the surface of cytotoxic T cells, with TLR2
often lying in heterodimers with TLR1, and then TLR’s 4,6,8 and 9. The mere presence of
these TLR’s on the cell surface suggests that they have a role in influencing the actions and
behaviours of the cytotoxic T cells. This theory has been tested and arguably proved by
several studies, specifically it has been shown that TLR2’s can have important implications on
the actions of cytotoxic T cells, behaving as co-stimulatory molecules to enhance the cell
function in mice. One such study conducted by Asprodites et al.16 showed that both in vivo and
in vitro the addition of both cytotoxic T cells and TLR1/2 ligands synergistically increased
anti-tumour activity, directly from the activity of the CD8+ T cells. Another example is by S.
Lee et al.17 which showed that the action of TLR2 ligands increased both the survival and
proliferation of CD8+ T cells. Cottalorda A. et al.18 showed that an effect of TLR2 ligands on
the CD8+ T cells was to lower the activation threshold to antigen-presenting cells’
interactions.18 This would serve to increase survival and proliferation of the cytotoxic T cells
and so bears a resemblance to the aforementioned study.
Interestingly the presence of Streptococcus Pneumoniae would stimulate the TLR2
ligands and create the effects described above, however the actions of cytotoxic T cells would
be unlikely to have an influence on the lifestyle of the bacterium, as its actions as CD8+ cells
are mostly concerned with virally infected cells, or those which have become mutated.
Therefore the best suggestion for the existence of this feature could simply be that in the event
of infection with Streptococcus Pneumoniae or any other gram-positive bacteria, a broad scale
immune response- although not specifically tailored towards combating the bacterium –
would serve the body well. This is because Streptococcus Pneumoniae often flourishes in
combination with viral infections, and an evolutionary advantage would be to associate high
gram-positive bacterial loads with a general lack of immune system activity.
Streptococcus Pneumoniae and the TLR2 stimulatory effects could also have basic
homeostatic advantages. Streptococcus Pneumoniae has been seen in commensal flora, and so
perhaps it’s existence also serves to provide a constant stimulation to the body’s immune
system, preventing a weakened system forming when there are fewer bacteria in the
environment.
As mentioned earlier TLR4 also serves a purpose by reacting to the PLY protein
expressed by S. Pneumoniae and helps aid T helper cell activation. This suggests that it would
also serve to stimulate TLR4 expressed on the CD8+ T cells. Research has shown that the T
cells when combined with active TLR4 illicit a greater response to viral vaccines.19 This bears
resemblance to the actions of the TLR2 and T cell activation, so perhaps it is also an
evolutionary development to increase immune activity during streptococcal infections.
Considering this, one could postulate methods of exploiting these effects to increase
the efficiency of the immune defenses through application of lysed Streptococcus
Pneumoniae. Exposure to lysed Streptococcus Pneumoniae allows the use of the TLR2\TLR4
stimulating – and hence the CD8+ T cell stimulating – effects without the danger of the
virulent factors (assuming any bacterial toxins were removed). This could serve to enhance
the function of viral vaccines as they require high CD8+ T cell proliferation rates and antigen-
presentation activity; TLR2 stimulation has been shown to aid both of these factors and TLR4
stimulation has been shown to increase CD8+ T cell activity.
The TLR2 stimulating factor – lipotechoic acid – does not present a major virulent
threat as it mostly exists as a structural feature to the bacteria. Hence the addition of only
lipotechoic acid is unlikely to cause health issues, assuming it isn’t added in doses high
enough to cause too great an immune response that could harm the body. However the TLR4
stimulating factor derived from S. Pneumoniae – PLY protein- has some more serious side-
effects aside from TLR4 activation, as listen earlier. The addition of the PLY could cause too
much damage to the body and so is not really viable as a treatment option; a better choice
would be to utilize another TLR4-stimulating protein that is selected both to avoid any
possibly virulent factors, and to ensure it doesn’t cause an excessive immune response that
would have negative effects on those vaccinated.

3. Sources

1. [http://emedicine.medscape.com/article/225811-overview], consulted at October,

19 2010
2. Medical microbiology. Murrey P, Rosenthal K, Pfaller M. 6th edition. 2009.
3. Sherris Medical Microbiology; an introduction to infectious disease. Ryan KJ, Ray CG.
4th edition. 2004.

4. Malley R, Henneke P, Morse SC et al. Recognition of pneumolysin by Toll-like

receptor 4 confers resistance to pneumococcal infection. Proc Natl Acad Sci USA
2003; 100: 1966–1971.
5. Malley R, Srivastava A, Lipsitch M et al. Antibody-independent, interleukin-17A-
mediated, cross-serotype immunity to pneumococci in mice immunized intranasally
with the cell wall polysaccharide. Infect Immun 2006; 74: 2187–2195.
6. Van der Poll T, Opal SM. Pathogenesis, treatment, and prevention of pneumococcal
Pneumonia. Lancet 2009; 374: 1543–56
7. A.M. Mitchell, T.J. Mitchell Streptococcus pneumoniae: virulence factors and
variation. Clin Microbiol Infect 2010; 16: 411–418.
8. Liu GY. Molecular Pathogenesis of Staphylococcus aureus Infection. International
Pediatric Research Foundation, Inc 2009; 65;5.
9. Ho PK, Chi-Chung Cheng V and Chu CM. Antibiotic Resistance in Community-Acquired
Pneumonia Caused by Streptococcus pneumonia, Methicillin-Resistant
Staphylococcus aureus and Acinetobacter baumannii. Chest 2009; 136; 1119-1127
10. Smith MG, Gianoulis TA, Pukatzki S, et.al. New insights into Acinetobacter baumannii
pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis.
Genes Dev. 2007 21: 601-614.
11. Vila-Córcoles. Vaccinate your child and save its grandparents from a heart attack?
Current perspectives in antipneumococcal vaccination. J Intern Med (2009) vol. 266
(5) pp. 432-44
12. Flannery B, Schrag S, Bennett NM, et al. Impact of childhood vacci- nation on racial
disparities in invasive Streptococcus pneumoniae infections. JAMA 2004;291:2197–
203.
13. Leino et al. Pneumococcal carriage in children during their first two years: important
role of family exposure. Pediatr Infect Dis J (2001) vol. 20 (11) pp. 1022-7
14. Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumoniae
virulence factors in host respiratory colonization and disease. Nature Rev Microbiol
2008; 6: 288–301.
15. ‘Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus
pneumoniae meningitis because of reduced bacterial clearing and enhanced
inflammation.’
16. ‘Engagement of Toll-like receptor-2 on cytotoxic T-lymphocytes occurs in vivo and
augments antitumor activity.’
17. ‘Expression and Function of TLR2 on CD4 Versus CD8 T Cells.’
18. ‘TLR2 engagement on CD8 T cells lowers the threshold for optimal antigen-induced T
cell activation.’
19. ‘TLR4 Ligands Augment Antigen-Specific CD8+ T Lymphocyte Responses Elicited by a
Viral Vaccine Vector’