The Importance Of Pneumococcal And Influenza Infection Biology Essay

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This dissertation is structured as follows. Chapter one describes the importance of pneumococcal and influenza infection and disease and the importance of studying the effect of their co-infection. It also describes previous studies regarding pneumococcal virulence factors and their role in pneumococcal pathogenecity.

Streptococcus pneumoniae (the pneumococcus) remains a major cause of morbidity and mortality in both the developing and developed worlds [1]. This important pathogen has a significant impact in public health, and result in diseases including pneumonia, meningitis, sepsis, sinusitis, arthritis, and osteomyelitis [2-5]. The rate of invasive pneumococcal diseases is very high and antibiotic resistance is increasing. It has been reported by Sahm and colleagues that the trend of S. pneumoniae drug resistance has risen against all the common antimicrobial drugs used for upper respiratory tract infections (e.g. penicillin, amoxicillin/clavulanate) in the USA [3]. Despite its importance as a pathogen, relatively little is known about the pathophysiology of pneumococcal diseases.

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There are various virulent factors which have been shown to play a key role in different stages of S. pneumoniae infection. Most important among these are the capsular polysaccharide, various surface proteins like neuraminidase (nanA, nanB), pneumococcal surface protein A (pspA), zinc metalloproteinase (ZmpC) and a pore forming cytotoxic pneumolysin. Mutants deficient in these factors have been shown to be avirulent [17, 25] and this has helped to expose their precise role in the pathogenesis of disease [41, 42, 43].

Pneumolysin (The cytotoxin)

Pneumolysin is a major virulence factor of the pneumococcus. It has been shown to contribute significantly in the invasion of bacterium in bloodstream and lungs [19, 20]. Its cytotoxic nature helps the pneumococus to invade host cells as it inhibits the ciliary motion of epithelial cells in human respiratory tract [11] and also disrupts tight junction between the epithelial cells thus providing another alternative path for invasion [12]. Through this route the pneumococcus infiltrates the blood stream [12] and pneumolysin has been shown to have a major role in development of sepsis [13-14].

nrmicro1871-f2

Fig. 1 Schematic representation of virulence factors of Streptococcus pneumoniae [17]

In recent studies of acute pneumonia it was stated that pneumolysin is an important factor for pneumococcal survial in upper and lower respiratory tracts [17, 18]. Furthermore, bacteria which express pneumolysin are found to be in higher numbers in the blood of infected mice [18, 19]. This cytolytic toxin also activates complement and it has shown both cell modulatory and complement activation in murine models [15-16].

1.1.2 The polysaccharide capsule

The polysaccharide capsule is the outermost covering of S. pneumoniae and its thickness varies between 200-400 nm [29]. The infectivity of the pneumococcus is strongly influenced by the amount of polysaccharide capsule as it has been shown to modulate the attachment of pneumococci to the host cells (mice) [26- 28]. It is not only necessary for virulence of pneumococcus, but is essential for its survival as well as it has shown to protect bacteria from opsonophagocytosis [26]. In recent studies it was stated that capsule reduces amount of complement deposited over the surface of the bacteria and acts as an inert shield from IgG [30]. Interestingly, it is believed that once the pneumococcus has adhered to human epithelial cell it loses its capsule [26-28].

1.1.3 Neuraminidase (Sialidase)

Amongst all pneumococcal surface proteins neuraminidase is regarded as one of the most important virulent factor. In pneumococcal infection it has a role in bacterial adherence by cleaving the sialic acid residue from the terminal polysaccharide of epithelial cells, this property of pneumococcus is also been targeted for producing vaccines [21,22]. There are three known genes which encode pneumococcal neuraminidase (nanA, nanB, nanC). All pneumococcal strains have gene nanA and most of them have nanB, but only half of known strains encode nanC [24]. These genes have shown to play an important part in survival of pneumococcus in the respiratory tract and bloodstream of mice [25]. In this study it was showed that two enzymes (nanA, nanB) have different roles in pneumococcal pathogenecity since nanA mutants deficient in nanA were rapidly cleared from the tract but nanB mutants were not cleared although their population did not increase [25].

1.1.4 Role of competence in virulence of pneumococcus

Pneumococcal competence (the ability of the bacterium to uptake foreign DNA) has also been shown to play a role in pneumococcal pathogenecity. It was also observed that competence also plays part in the induction of biofilm formation. In pneumococci competence is regulated by a set genes comABCDE and by an alternative sigma factor comX [44]. ComX-independent genes have been shown to have role in transformation and the expression of comX-dependent genes appears to be related to stress [44].

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The addition of a synthetic competence stimulating peptide (CSP) has been shown to influence the development of pneumococcal biofilms in vitro and pneumococci harvested from biofilms are found to be more virulent in mice [31]. Furthermore, a comD mutant showed relatively reduced virulence as it was not able to form biofilm [33].

1.1.5 The pneumococcus and the upper respiratory tract

The nasopharynx is an important environment for the pneumococcus, not only it is a common coloniser, but it also readily invades the cell lining in the nasopharynx. The cells of particular interest are those which line the surface of the nasopharynx i.e. the ciliated nasal epithelial cells, since these are the first line of defence and present the primary interaction with pneumococcus. These cells occurs throughout the nasal lining, they have hair like projections which move in a coordinated wave-like motion and propels mucus and trap objects (like bacteria, dust particles) and push them towards the back of pharynx where it is prevented from entering the lungs. Therefore, studying the ability of the pneumococcus to invade ciliated cultures could help to unravel important mechanisms of pneumococcal infection.

A B

Fig 1.2 A: SEM of ciliated nasal epithelial cells of rabbit, B: schematic representation of cilia.

1.1.6 Tissue specific virulence of Streptococcus pneumoniae

The role of each virulent factor varies according to the site of infection. Recently a study was performed to observe the pattern of gene expression of pneumococci in blood stream, lungs and brain of infected mice [31]. Oggioni and colleague studied the variation in expression of virulent genes of S. pneumoniae in three distinct in vivo models and showed two patterns of gene expression one in bloodstream of mice and other in the lung and brain. It was observed that pneumolysin and pneumococcal surface protein A (PspA) were highly expressed in the bloodstream of infected mice compare to the lung and brain, where expression was 4-5 times reduced (see table 1.2). Contrastingly expression of neuraminidase encoding genes nanA, nanB and genes like comA and comE which are involved in competence was expressed 7-8 times greater in lung and brain compared to blood (see table 1.2).

Table 1.2 Relative quantification of pneumococcal gene expression. Showing the relative fold change (standard deviation) from reference condition [31]

Virulence genes

Strain

Brain

Lung

Blood

Agar

Biofilm

ply

SP1923

0.3(0.2)

0.2(0.1)

1.3(0.3)

0.01(0.01)

0.2(0.1)

pspA

SP0117

0.3(0.1)

0.3(0.1)

1.3(0.2)

0.4(0.1)

0.2(0.1)

cps4A

SP0346

2.2(0.9)

1.6(0.8)

1.2(0.2)

7.0(1.8)

1.6(0.4)

pspC

SP2190

1.5(0.9)

1.2(0.2)

1.8(0.4)

0.7(0.1)

0.6(0.4)

nanA

SP1693

18.8(12.0)

16.6(10.2)

1.3(0.7)

0.3(0.1)

59.4(28.0)

nanB

SP1687

3.8(0.9)

3.2(0.7)

2.0(0.3)

0.3(0.2)

4.8(1.1)

IgA

SP1154

3.3(0.8)

2.5(0.7)

1.1(0.5)

0.3(0.1)

nd

zmpB

SP0664

2.2(0.9)

2.2(1.6)

1.2(0.2)

0.3(0.1)

nd

zmpC

SP0071

5.2(1.6)

4.1(0.9)

1.4(0.3)

0.3(0.1)

nd

sodA

SP0766

10.4(3.1)

13.9(10.8)

1.5(0.6)

1.7(0.9)

nd

nox

SP1469

7.4(4.8)

8.7(9.2)

1.0(0.3)

1.3(0.8)

0.07(0.02)

lytA

SP1937

2.0(1.1)

1.3(0.2)

2.3(0.4)

2.5(0.2)

nd

prtA

SP0641

0.8(0.5)

0.8(0.2)

1.3(0.2)

0.2(0.1)

0.4(0.3)

ddlA

SP1671

nd

1.8(0.3)

2.4(0.8)

nd

4.0(0.6)

comA

SP0042

10.0(1.5)

7.1(1.0)

1.3(0.4)

2.0(1.2)

7.1(0.7)

comE, tcs12

SP2235

6.0(2.1)

4.1(1.5)

1.3(0.3)

1.3(0.5)

nd

comX

SP0014

12.0(8.4)

7.3(4.2)

1.6(0.3)

1.1(0.1)

5.1(0.8)

ctsR

SP2195

nd

4.8(2.8)

1.6(0.5)

2.8(1.6)

nd

mgrA

SP1800

31.7(12.8)

18.7(9.8)

4.9(0.8)

47.4(21.9)

15.8(7.7)

regR

SP0330

4.0(2.8)

5.1(2.5)

3.4(0.7)

8.5(8.0)

3.1(0.8)

stkP

SP1732

3.9(2.1)

5.9(6.9)

5.3(3.7)

0.4(0.4)

1.4(0.1)

dprA

SP1266

12.2(5.9)

7.7(6.9)

0.9(0.7)

0.06(0.03)

nd

dtxR

SP1638

10.1(5.9)

11.7(9.1)

1.0(0.4)

0.5(0.3)

nd

msmR

SP1899

8.2(5.6)

5.6(3.1)

0.6(0.29)

0.03(0.04)

2.23(0.6)

1.2.0 Influenza

Influenza is also a major pathogen. Infection with influenza virus results in millions of deaths worldwide every year. In USA alone it has accounted for about 40000 lives per year between 1979 and 2001[34]. It has been reported to cause three pandemics in 20th century and has killed more than tens of millions of people. More recently in 2009 a novel influenza strains (H1N1) which initially emerged in Mexico later spread throughout the world.

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Influenza virus is 80-120 nm in diameter, roughly spherical in shape, unusually contains about 6-9 pieces of a negative sense single stranded RNA which encode one or two genes. It can be classified into three main groups Influenza virus A, B and C which have all been shown to cause disease in humans. Generally they are serotype is denoted as HxNx where H-Haemoglutinin and N-Neuraminidase and x for integer depending upon variation in the protein in each form (see Figure 1.3).

untitled

Fig 1.3 Structural Diagram of the influenza virus [48].

1.2.1 Influenza virus A

Influenza A is the most virulent among the three influenza and results in severe disease in humans. On the basis of antibody response it can be sub divided into various serotype and ordered according to number of deaths [36]. This virus is commonly found in aquatic birds, but when occasionally transmitted to other species may result in human flu pandemic [35].

1.2.2 Influenza virus B

This virus is less virulent then type A and mostly affects humans, its mutation rate is slower than that of type A, and results in less genetic variation therefore some immunity against this virus can be acquired at an early age. Type B has only one recognised serotype has never caused a pandemic [46].

1.2.3 Influenza virus C

This type is the least lethal among the three and is generally found to affect humans, canines and pigs. It less common and generally causes mild disease in children [47].

1.2.2 Mechanism of transmission

Influenza virus has been known to spread by three routes. Direct transmission; when a person comes in direct contact with the mucus of the infected persons. Secondly by aerosol transmission; when a person inhales a droplet of virus produced from the infected person while coughing or sneezing. Thirdly, by indirect transmission i.e. when a person come in contact with a contaminated surface.

1.2.3 Treatment

Two antiviral drugs have been used against the flu which can be classified according to their mode of action; Neuraminidase inhibitor and M2 inhibitor (admantanes). Neuraminidase inhibitors act in such a way that stops the spread of virus in the body; oseltamivir commercially known as “tamifluâ€Â works on this principle and is effective against influenza A and B [37]. M2 inhibitors work by blocking an ion channel of the M2 protein and thus prevents virus from infecting further cells [38]. This treatment is sometime ineffective against type B influenza as they do not possess an M2 protein [39]. Neuraminidase inhibitors are preferred over M2 inhibitor as they have less down stream toxicity.

The impact of influenza virus and S. pneumoniae coinfection

Importantly influenza virus and the pneumococcus are two very different pathogens which are commonly isolated together and are known to share a synergistic relationship [ref]. Although the mechanism behind this is unknown, it is thought that influenza infection alters the lungs in a way that predisposes to adherence and invasion of the pneumococcus [6].

In 2009 the WHO declared an outbreak of pandemic influenza (H1N1pdm) and the case fatality rate (CFR) increased almost eight fold from 0.6% to 4.5% in Argentina alone. During this time 3056 H1N1 infected patients were reported amongst which about 137 died. Gustavo Palacious and colleagues further examined 199 of these patients using massTag PCR for 33 bacterial species and discovered that about 56% of the severe cases of H1N1 were coinfected with S. pneumoniae [40]. Twenty five percent of those that suffered with mild disease (hospitalization) were shown to be coinfected with S. pneumoniae therefore suggesting that the severity of disease was somehow correlated with the level of coinfection [40].

1.4 Summary and Hypotheses

The two main objectives of this study are

to identify important pneumococcal genes involved in the interaction with human ciliated nasal epithelial cells and to investigate the survival of Streptococcus pneumoniae in this environment

We will also identify patterns of gene expression of Streptococcus pneumoniae and examine how the pattern of expression changes with the presence of influenza.

Hypotheses are described in Table 1.1.

Table 1.1 Genes of interest and hypothesised outcome

Name of the gene

Role of the gene

What if they are up regulated in the presence of influenza?

nanA

To produce neuraminidase A which cleave the sialic acid from cell surface and help in colonisation in upper respiratory tract

Implies that Sialic acid is not present on the surface of epithelial cell or neuraminidase A is already present

nanB

To produce neuraminidase B which cleave the sialic acid from cell surface and help in colonisation in upper respiratory tract

Implies that Sialic acid is not present on the surface of epithelial cell or neuraminidase B is already present

sodA

To produce superoxidase dimutase, converts superoxide into oxygen and hydrogen peroxide and help bacteria to survive oxygen stress.

Cells are responding to infection by attacking bacteria with superoxide and bacteria is responding by converting it into hydrogen peroxide

nox

To produce NADH oxidase, which plays role in oxygen signalling pathways

Outcome predicted to be similar to sodA

ply

To produce the toxin Pneumolysin, which is as a member of thiol-activated cytolysins (TACY), commonly bind to membrane cholesterol and form transmembrane pore

The pneumococcous requires pneumolysin to form pore across the membrane

comA

It is used to produce Competence associated protein which is member of an ATP-dependent transport protein superfamily [8].

The pneumococcous are preparing for the formation of biofilm

comX1

It is a regulatory gene, expressed during competence via a mesh of signals and may regulate structural genes.

The pneumococcous are preparing for the formation of biofilm

pspA

It is used to produce pneumococcal surface protein A and prevents killing of pneumococci by apolactoferrin; an iron depleted form of lactoferrin, which is component of innate immunity [9].

Apolactoferrin is present and pneumococcous is producing PspA protein to survive which also increases pneumococcous binding to cells.

zmpC

It is used to produce surface located zinc metalloproteinase, it is found to cleave human matrix metalloproteinase 9 (MMP-9) [10].

MMP-9 is released by cells and pneumococcous reacts by increasing expression of zmpC.