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This thesis is structured as follows. Chapter one describes the importance of pneumococcal and influenza infection and disease and the importance of analyzing the consequence of their co-infection. It besides describes old surveies sing pneumococcal virulency factors and their function in pneumococcal pathogenecity.

Streptococcus pneumoniae ( the Diplococcus pneumoniae ) remains a major cause of morbidity and mortality in both the development and developed universes [ 1 ] . This of import pathogen has a important impact in public wellness, and consequence in diseases including pneumonia, meningitis, sepsis, sinusitis, arthritis, and osteomyelitis [ 2-5 ] . The rate of invasive pneumococcal diseases is really high and antibiotic opposition is increasing. It has been reported by Sahm and co-workers that the tendency of S. pneumoniae drug opposition has risen against all the common antimicrobic drugs used for upper respiratory piece of land infections ( e.g. penicillin, amoxicillin/clavulanate ) in the USA [ 3 ] . Despite its importance as a pathogen, comparatively small is known about the pathophysiology of pneumococcal diseases.

There are assorted deadly factors which have been shown to play a cardinal function in different phases of S. pneumoniae infection. Most of import among these are the capsular polyose, assorted surface proteins like neuraminidase ( nanA, nanB ) , pneumococcal surface protein A ( pspA ) , zinc metalloproteinase ( ZmpC ) and a pore organizing cytotoxic pneumolysin. Mutants deficient in these factors have been shown to be avirulent [ 17, 25 ] and this has helped to expose their precise function in the pathogenesis of disease [ 41, 42, 43 ] .

Pneumolysin ( The cytotoxin )

Pneumolysin is a major virulency factor of the Diplococcus pneumoniae. It has been shown to lend significantly in the invasion of bacteria in blood stream and lungs [ 19, 20 ] . Its cytotoxic nature helps the pneumococus to occupy host cells as it inhibits the ciliary gesture of epithelial cells in human respiratory piece of land [ 11 ] and besides disrupts tight junction between the epithelial cells therefore supplying another alternate way for invasion [ 12 ] . Through this path the Diplococcus pneumoniae infiltrates the blood watercourse [ 12 ] and pneumolysin has been shown to hold a major function in development of sepsis [ 13-14 ] .

nrmicro1871-f2

Fig. 1 Conventional representation of virulency factors of Streptococcus pneumoniae [ 17 ]

In recent surveies of ague pneumonia it was stated that pneumolysin is an of import factor for pneumococcal survial in upper and lower respiratory piece of lands [ 17, 18 ] . Furthermore, bacterium which express pneumolysin are found to be in higher Numberss in the blood of septic mice [ 18, 19 ] . This cytolytic toxin besides activates complement and it has shown both cell modulatory and complement activation in murine theoretical accounts [ 15-16 ] .

1.1.2 The polyose capsule

The polyose capsule is the outermost covering of S. pneumoniae and its thickness varies between 200-400 nm [ 29 ] . The infectivity of the Diplococcus pneumoniae is strongly influenced by the sum of polyose capsule as it has been shown to modulate the fond regard of Diplococcus pneumoniae to the host cells ( mice ) [ 26- 28 ] . It is non merely necessary for virulency of Diplococcus pneumoniae, but is indispensable for its endurance every bit good as it has shown to protect bacteriums from opsonophagocytosis [ 26 ] . In recent surveies it was stated that capsule reduces sum of complement deposited over the surface of the bacteriums and Acts of the Apostless as an inert shield from IgG [ 30 ] . Interestingly, it is believed that one time the Diplococcus pneumoniae 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 of import virulent factor. In pneumococcal infection it has a function in bacterial attachment by spliting the sialic acid residue from the terminal polyose of epithelial cells, this belongings of Diplococcus pneumoniae is besides been targeted for bring forthing vaccinums [ 21,22 ] . There are three known cistrons which encode pneumococcal neuraminidase ( nanA, nanB, nanC ) . All pneumococcal strains have cistron nanA and most of them have nanB, but merely half of known strains encode nanC [ 24 ] . These cistrons have shown to play an of import portion in endurance of Diplococcus pneumoniae in the respiratory piece of land and blood stream of mice [ 25 ] . In this survey it was showed that two enzymes ( nanA, nanB ) have different functions in pneumococcal pathogenecity since nanA mutations deficient in nanA were quickly cleared from the piece of land but nanB mutations were non cleared although their population did non increase [ 25 ] .

1.1.4 Role of competency in virulency of Diplococcus pneumoniae

Pneumococcal competency ( the ability of the bacteria to uptake foreign DNA ) has besides been shown to play a function in pneumococcal pathogenecity. It was besides observed that competency besides plays portion in the initiation of biofilm formation. In pneumococci competency is regulated by a set cistrons comABCDE and by an alternate sigma factor comX [ 44 ] . ComX-independent cistrons have been shown to hold function in transmutation and the look of comX-dependent cistrons appears to be related to emphasize [ 44 ] .

The add-on of a man-made competency exciting peptide ( CSP ) has been shown to act upon the development of pneumococcal biofilms in vitro and Diplococcus pneumoniae harvested from biofilms are found to be more deadly in mice [ 31 ] . Furthermore, a comD mutation showed comparatively decreased virulency as it was non able to organize biofilm [ 33 ] .

1.1.5 The Diplococcus pneumoniae and the upper respiratory piece of land

The nasopharynx is an of import environment for the Diplococcus pneumoniae, non merely it is a common colonizer, but it besides readily invades the cell liner in the nasopharynx. The cells of peculiar involvement are those which line the surface of the nasopharynx i.e. the ciliated rhinal epithelial cells, since these are the first line of defense mechanism and show the primary interaction with Diplococcus pneumoniae. These cells occurs throughout the nasal liner, they have hair like projections which move in a co-ordinated wave-like gesture and propels mucous secretion and trap objects ( like bacteriums, dust atoms ) and force them towards the dorsum of throat where it is prevented from come ining the lungs. Therefore, analyzing the ability of the Diplococcus pneumoniae to occupy ciliated civilizations could assist to unknot of import mechanisms of pneumococcal infection.

A Bacillus

Fig 1.2 A: SEM of ciliated rhinal epithelial cells of coney, B: conventional representation of cilia.

1.1.6 Tissue specific virulency of Streptococcus pneumoniae

The function of each virulent factor varies harmonizing to the site of infection. Recently a survey was performed to detect the form of cistron look of Diplococcus pneumoniae in blood watercourse, lungs and encephalon of septic mice [ 31 ] . Oggioni and co-worker studied the fluctuation in look of deadly cistrons of S. pneumoniae in three distinct in vivo theoretical accounts and showed two forms of cistron look one in blood stream of mice and other in the lung and encephalon. It was observed that pneumolysin and pneumococcal surface protein A ( PspA ) were extremely expressed in the blood stream of septic mice compare to the lung and encephalon, where look was 4-5 times reduced ( see table 1.2 ) . Contrastingly look of neuraminidase encoding cistrons nanA, nanB and cistrons like coma and semen which are involved in competency was expressed 7-8 times greater in lung and encephalon compared to blood ( see table 1.2 ) .

Table 1.2 Relative quantification of pneumococcal cistron look. Showing the comparative crease alteration ( standard divergence ) from mention status [ 31 ]

Virulence cistrons

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 )

Immunoglobulin a

SP1154

3.3 ( 0.8 )

2.5 ( 0.7 )

1.1 ( 0.5 )

0.3 ( 0.1 )

neodymium

zmpB

SP0664

2.2 ( 0.9 )

2.2 ( 1.6 )

1.2 ( 0.2 )

0.3 ( 0.1 )

neodymium

zmpC

SP0071

5.2 ( 1.6 )

4.1 ( 0.9 )

1.4 ( 0.3 )

0.3 ( 0.1 )

neodymium

sodium carbonate

SP0766

10.4 ( 3.1 )

13.9 ( 10.8 )

1.5 ( 0.6 )

1.7 ( 0.9 )

neodymium

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 )

neodymium

prtA

SP0641

0.8 ( 0.5 )

0.8 ( 0.2 )

1.3 ( 0.2 )

0.2 ( 0.1 )

0.4 ( 0.3 )

ddlA

SP1671

neodymium

1.8 ( 0.3 )

2.4 ( 0.8 )

neodymium

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 )

semen, tcs12

SP2235

6.0 ( 2.1 )

4.1 ( 1.5 )

1.3 ( 0.3 )

1.3 ( 0.5 )

neodymium

comX

SP0014

12.0 ( 8.4 )

7.3 ( 4.2 )

1.6 ( 0.3 )

1.1 ( 0.1 )

5.1 ( 0.8 )

ctsR

SP2195

neodymium

4.8 ( 2.8 )

1.6 ( 0.5 )

2.8 ( 1.6 )

neodymium

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 )

neodymium

dtxR

SP1638

10.1 ( 5.9 )

11.7 ( 9.1 )

1.0 ( 0.4 )

0.5 ( 0.3 )

neodymium

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 besides a major pathogen. Infection with influenza virus consequences in 1000000s of deceases worldwide every twelvemonth. In USA entirely it has accounted for about 40000 lives per twelvemonth between 1979 and 2001 [ 34 ] . It has been reported to do three pandemics in twentieth century and has killed more than 10s of 1000000s of people. More late in 2009 a fresh grippe strains ( H1N1 ) which ab initio emerged in Mexico subsequently spread throughout the universe.

Influenza virus is 80-120 nanometer in diameter, approximately spherical in form, remarkably contains about 6-9 pieces of a negative sense individual stranded RNA which encode one or two cistrons. It can be classified into three chief groups Influenza virus A, B and C which have all been shown to do disease in worlds. By and large they are serotype is denoted as HxNx where H-Haemoglutinin and N-Neuraminidase and x for whole number depending upon fluctuation in the protein in each signifier ( see Figure 1.3 ) .

ignoble

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

1.2.1 Influenza virus Angstrom

Influenza A is the most deadly among the three grippe and consequences in terrible disease in worlds. On the footing of antibody response it can be sub divided into assorted serotype and ordered harmonizing to figure of deceases [ 36 ] . This virus is normally found in aquatic birds, but when on occasion transmitted to other species may ensue in human grippe pandemic [ 35 ] .

1.2.2 Influenza virus B

This virus is less deadly so type A and largely affects worlds, its mutant rate is slower than that of type A, and consequences in less familial fluctuation hence some unsusceptibility against this virus can be acquired at an early age. Type B has merely one recognised serotype has ne’er caused a pandemic [ 46 ] .

1.2.3 Influenza virus C

This type is the least deadly among the three and is by and large found to impact worlds, eyetooths and hogs. It less common and by and large causes mild disease in kids [ 47 ] .

1.2.2 Mechanism of transmittal

Influenza virus has been known to distribute by three paths. Direct transmittal ; when a individual comes in direct contact with the mucous secretion of the septic individuals. Second by aerosol transmittal ; when a individual inhales a droplet of virus produced from the septic individual while coughing or sneezing. Third, by indirect transmittal i.e. when a individual come in contact with a contaminated surface.

1.2.3 Treatment

Two antiviral drugs have been used against the grippe which can be classified harmonizing to their manner of action ; Neuraminidase inhibitor and M2 inhibitor ( admantanes ) . Neuraminidase inhibitors act in such a manner that stops the spread of virus in the organic structure ; oseltamivir commercially known as & A ; acirc ; ˆ?tamiflu & A ; acirc ; ˆA? works on this rule and is effectual against grippe A and B [ 37 ] . M2 inhibitors work by barricading an ion channel of the M2 protein and therefore prevents virus from infecting farther cells [ 38 ] . This intervention is sometime uneffective against type B grippe as they do non possess an M2 protein [ 39 ] . Neuraminidase inhibitors are preferred over M2 inhibitor as they have less down watercourse toxicity.

The impact of influenza virus and S. pneumoniae coinfection

Importantly influenza virus and the Diplococcus pneumoniae are two really different pathogens which are normally isolated together and are known to portion a interactive relationship [ ref ] . Although the mechanism behind this is unknown, it is thought that influenza infection alters the lungs in a manner that predisposes to adherence and invasion of the Diplococcus pneumoniae [ 6 ] .

In 2009 the WHO declared an eruption of pandemic grippe ( H1N1pdm ) and the instance human death rate ( CFR ) increased about eight creases from 0.6 % to 4.5 % in Argentina entirely. During this clip 3056 H1N1 infected patients were reported amongst which about 137 died. Gustavo Palacious and co-workers further examined 199 of these patients utilizing massTag PCR for 33 bacterial species and discovered that about 56 % of the terrible instances of H1N1 were coinfected with S. pneumoniae [ 40 ] . Twenty five per centum of those that suffered with mild disease ( hospitalization ) were shown to be coinfected with S. pneumoniae hence proposing that the badness of disease was someway correlated with the degree of coinfection [ 40 ] .

1.4 Summary and Hypotheses

The two chief aims of this survey are

to place of import pneumococcal cistrons involved in the interaction with human ciliated rhinal epithelial cells and to look into the endurance of Streptococcus pneumoniae in this environment

We will besides place forms of cistron look of Streptococcus pneumoniae and analyze how the form of look alterations with the presence of grippe.

Hypothesiss are described in Table 1.1.

Table 1.1 Genes of involvement and hypothesised result

Name of the cistron

Role of the cistron

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

nanA

To bring forth neuraminidase A which cleave the sialic acid from cell surface and aid in colonization in upper respiratory piece of land

Implies that Sialic acid is non present on the surface of epithelial cell or neuraminidase A is already present

nanB

To bring forth neuraminidase B which cleave the sialic acid from cell surface and aid in colonization in upper respiratory piece of land

Implies that Sialic acid is non present on the surface of epithelial cell or neuraminidase B is already present

sodium carbonate

To bring forth superoxidase dimutase, converts superoxide into O and H peroxide and assist bacteriums to last O emphasis.

Cells are reacting to infection by assailing bacteriums with superoxide and bacterium is reacting by change overing it into H peroxide

Nox

To bring forth NADH oxidase, which plays function in O signalling tracts

Outcome predicted to be similar to sodA

ply

To bring forth the toxin Pneumolysin, which is as a member of thiol-activated cytolysins ( TACY ) , normally bind to membrane cholesterin and signifier transmembrane pore

The pneumococcous requires pneumolysin to organize pore across the membrane

coma

It is used to bring forth Competence associated protein which is member of an ATP-dependent conveyance protein superfamily [ 8 ] .

The pneumococcous are fixing for the formation of biofilm

comX1

It is a regulative cistron, expressed during competency via a mesh of signals and may modulate structural cistrons.

The pneumococcous are fixing for the formation of biofilm

pspA

It is used to bring forth pneumococcal surface protein A and prevents killing of Diplococcus pneumoniae by apolactoferrin ; an Fe depleted signifier of lactoferrin, which is constituent of unconditioned unsusceptibility [ 9 ] .

Apolactoferrin is present and pneumococcous is bring forthing PspA protein to last which besides increases pneumococcous adhering to cells.

zmpC

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

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

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