Little This suggests that FN binding to

Little evidence has been found pertaining to FN inhibitory effect on Mycobacterial
growth. Evidently this
confirms that FN function as an adhesive protein that enhances M. smegmatis
growth as figures 16, 18 and 20 (Pasula, et al., 2002) yet it does have the potential to inhibit M. smegmatis growth.
FN directly enhances M. smegmatis
growth but its exact mechanism towards this action in vitro has not fully been
established.

 

The use of M. smegmatis as a
model for M. tb demonstrated the
direct effect of FN on M. smegmatis
bacterial growth. Three inhibition assays were carried out under the same
experimental conditions for strengthening the reliability and the reproducibility
of the results obtained. The graph depicts a decrease in bacterial colony
growth when treated with 2µg/mL FN
average colony growth valued at 190, compared to the negative control with an
average of 300, thus showing the inhibitory properties of FN. M. smegmatis cells treated with 10µg/mL FN, showed significant growth of M. smegmatis colonies. However even with increased growth the value
obtained was less than the negative control. The second and third inhibition
assay saw a similar outcome to the first. A concentration of 2µg/mL FN produced a lower M.
smegmatis colony count when added to culture compared to the negative
control. Although the second assay did not produce significant results. With a
treatment of 10µg/mL FN to culture the bacterial count
increased compared to the negative control in the second assay not the third. A
reason for this may be due to a random error in that the investigator (I)
miscalculated the number of bacterial colonies present in culture. Cross
contamination occurred during the inhibition assay. When counting the colonies,
present on the culture were colonies of fungi species, thus impacting my
results. The decrease in growth can also account for the loss of
viable mycobacterium cells in culture as there was limited nutrients, growth
factors and surface area for growth. Hence there is a possibility that the
single colonies chosen may have contained dead bacilli, this could explain the
results I had obtained. In future experiments stricter lab conditions shall be
maintained to obtain accurate data. M.
smegmatis is non-pathogenic and so does not share the same virulent properties
as M. tb such as the Antigen 85
Complex proteins, therefore repetition of this experiment shall use pathogenic M. tb to develop understanding of the
inhibitory properties of FN.

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FN
was tested at 2µg/mL and 10µg/mL, binding was highest at 10µg/mL hence a higher
value in M. smegmatis colony growth.
This suggests that FN binding to M.
smegmatis was saturable as all Mycobacterium binding sites had been
occupied. Hall-Stoodley conducted a similar experiment, the results showed that
at 10µg/mL the binding of FN to M. tb
was completely saturable with little difference in an increase to FN
concentration (Hall-Stoodley, et al., 2006) (Pasula, et al., 2002).
Due to the smaller concentration of FN, one could argue there was not enough
protein present to stimulate significant bacterial colony growth as 10µg/mL FN.

 

The
results of the experiment suggest that FN does have a direct effect on M. smegmatis growth. Hall-Stoodley et al
conducted a study on the effect of FN on M.
tb growth under shear conditions, the results suggested that FN contributed
to increased numbers of M. tb bacilli
adhering to cells in the alveolar by direct invasion. FN was also shown to
enhance the binding of M. tb strain H37Rs to mouse alveolar macrophages (Hall-Stoodley,
et al., 2006).
Another study’s results showed that M. bovis BCG binds to FN via the C-terminal
region adjacent to the HBD of the FN molecule, this suggests that the proteins
binding to the HBD are proteoglycans, inferring that FN binding proteins
present on the surface of M. tb are also proteoglycans (Pasula, et al., 2002).

 

FN is present at the sites of infection; a classic sign of infection is
inflammation. Pulmonary epithelial cells secrete FN when tissue damage exists,
a characteristic of active M. tb
pathogenesis. FN is also known to act as a opsonin of bacteria facilitating the
migration of host immune effector molecules to the site of infection such as T
lymphocytes, B lymphocytes, macrophages, NK cells, DCs, which activate
inflammatory cytokines to regulate and enhance the immune response, this
positive feedback will cause the secretion of more FN protein to adhere to M. tb bacilli. 

 

FN contains 5% carbohydrate linked via asparagine residues (Pande, et
al., 1978) (Yamada &
Olden, 1978). This accounts for FN
lectin behaviour in figure, as it had the ability to bind to maltose. In the
presence of CaCl2 FN bound to Maltose, there had been a decrease in
binding between FN with M. smegmatis
by 29% as the absorbance reading measured 1.1 OD compared to 1.6 OD without
Maltose. FN II contains a CBD (Napper, et al., 2006). FN CBD possess a mannose
receptor that binds to carbohydrates. M.
smegmatis outer cell wall is made up of proteins, lipids and
oligosaccharides (Draper, 1998), FN recognises
Maltose as a carbohydrate and so forms a ligand with this molecule instead of
other molecules on M. smegmatis
surface.

 

4.7 Maltose

EDTA was added to specific wells
in the ELISA experiment. The result of this showed that a decrease in FN
binding to M. smegmatis, 46% decrease
in binding. EDTA is a chelating agent that reacts with calcium ions and forms
metal ion complexes in solution (Mohamadi, et al., 2013) (Nielsen,
2010).

 

In addition to FN binding with M.
smegmatis, calcium was also added to specific wells in ELISA. This was done to
test if calcium ions had any significance on the functionality of FN. Figure 13
shows increased binding when CaCl2 was added to 10µg/Ml FN. This had
an absorbance reading of 1.6 OD. Without the presence of calcium ions the
binding of FN to M. smegmatis decreased with an absorbance reading of 0.9 OD.
An important concept in FN integrin function is their ability to shift between
active and inactive ligand binding states, this is done by altering the
conformation of their extracellular domain. FN is in circulation within the
host blood vessels in its inactive conformation, but activates when exposed to
certain factors such as infection. This stimulates an intracellular reaction
leading to the activation of FN (Weber, et al., 1996). My experiment showed a
positive result with the addition of Ca2+ ions, however research
showed the opposite effect, divalent ions such as Ca2+ have been
shown to modulate ligand binding inhibiting and enhancing FN binding dependent
upon the concentration of the ion. At low concentrations Ca2+ ions are
found to stimulate ligand binding of FN, whereas high concentrations of Ca2+
inhibit ligand binding (Johansson, et al., 1997) (Mould, et al., 1995). This
shows that Ca2+ aids FN binding to M. smegmatis. The outcome of this
result supports the view that Ca2+ is significant in the functionality
of Fibronectin.

 

4.6 Calcium Chloride

 

A similar reading observed to the previous
1.25µg/mL FN
concentration was obtained at 10µg/mL FN with an absorbance reading of 1.78 OD. This suggests that optimum binding
does not need a high concentration of FN. Nevertheless, if this experiment were
to be repeated under the same experimental conditions, the future data may
identify whether binding with M. smegmatis improved or remained unchanged particularly
at concentrations 1.25µg/mL FN and 10µg/mL FN. 

 

Antigen 85 Complex proteins
consist of Ag85A, Ag85B, and Ag85C proteins, they are secreted and retained in
the Mycobacterium cell wall (Wiker & Harboe, 1992). These complex
proteins catalyse the last stages of the synthesis of glycolipids for the
Mycobacterial cell wall surface that is essential for survival and growth of M.
tb (Belisle, et al., 1997). Research has shown that Ag85 Complex proteins bind
to both plasma and cellular FN, this interaction takes place at a specific
FN-binding motif, (Naito, et al., 1998) (Godfrey, et al., 1992). Past in vitro
studies identified a key sequence in the binding of recombinant Ag85 Complex
Proteins to FN. FEWYYQSGLSV is a negatively charged peptide sequence that is
the binding region of Ag85 B protein at which FN binds to M. tb (Naito, et al.,
1998). FAP binding region does not share homology with Ag85 B protein and so
contains a separate and distinct binding motif with FN (Zhao, et al., 1999).
These results are based up on in vitro experiments, as no conclusive evidence
had been acquired using viable bacteria cells for their attachment to FN.

 

The ELISA experiment was conducted to see whether FN bound to M.
smegmatis. This experiment also led to the establishment of the optimum working
concentration of FN that bound to the bacteria in the wells. Figure 12 shows
that optimum binding was achieved when M. smegmatis was treated with 1.25µg/mL obtaining a reading of 1.73 OD.
Mycobacteria possess two separate proteins that have the ability to bind FN to
its cell surface. These are FAPs and Antigen 85 Complex Proteins (Abou-Zeid, et al., 1991) (Zhao, et al., 1999).
A study conducted by Verbelen & Dufrene measured the binding strength of
FN-tips used to detect FAPs on Mycobacterial surfaces. The results of the study
suggest that FN possess receptors on their surfaces that form a receptor-ligand
interaction with Mycobacteria FAPs on the surface of the cell wall, a direct
interaction (Verbelen & Dufrene, 2009) (Henderson, et al., 2011).

 

4.5 ELISA

 

The bacteria was smeared onto the optical slide and placed over a lit
Bunsen burner. The heat from the flame of the Bunsen burner acts as a mordant enabling
the primary dye to penetrate the mycolic acid component of the cell wall (Lahiri &
Chatterjee, 1994).
Carbol-Fuschin is made up of Fuschin and Carbolic Acid. The Carbolic Acid is a
phenol. Phenol is soluble in lipids (Lamanna, 1946). The slide was
washed with a decolourising staining solution that is alcohol based. The
bacteria’s cell wall is resistant to the decolourising solution because the
cell wall is impermeable to alcohol. The counter stain Methylene blue was used,
the bacteria that retain the counter stain dye are known as non-acid fast
bacteria as they do not share the same properties as acid fast bacteria and so
appear blue. In figure 11 the clumped and isolated rod-shaped bacilli appear to
retain the primary dye colour pink/red. 
As the mycobacteria grow the cells start to stick together forming small
clumps (Draper, 1998). Tween- 80 was used
when producing a pure culture, this ensured a minimisation of large clumping. Carbencillin
(1mgml-1) is a broad spectrum antibiotic used to treat bacterial
infections (Nathwani & Wood, 1993). This reduced the
chance of contamination of other bacterial organisms during growth of the pure
M. smegmatis culture. This ensured in a positive AFS result.

 

M. smegmatis belongs to the genus Mycobacterium. This species has a unique cell wall
structure that differs from other micro-organisms. Mycobacteria cell wall
contains a large amount of mycolic acids

 

Once M. smegmatis was cultured
and the OD had reached a maximum value. The bacteria had to be confirmed before
further use of it in experiments. This also ensured that the pure culture of M. smegmatis was not contaminated with
fungal or bacterial strains.

 

As the bacteria double in numbers at a consistent rate they use up the
finite nutrients in the medium and exploit the conditions of their environment,
nonetheless not all of the bacteria will be able to survive this phase. The
stationary phase is defined as the state of no bacterial net growth (Mainer, 2008). Figure 10 shows that from 73 to 76 hours the bacteria
entered the stationary phase, the OD measured 2.0 which was a consistent
reading for the three-hour period. This is represented in the graph by a
horizontal line. Reasons for the stationary phase could be that the nutrients
became completely used up. The bacteria produced metabolic waste products that
built up to the point where they began to inhibit cell growth, hence the rate
of cell death is in equilibrium with the rate of cell division (Kolter, et al.,
1993) (Mainer, 2008). The fourth phase of
bacterial growth that was not observed is known as the death phase. This is
when viable cells are lost rather than gained. This is primarily due to the
complete absence of nutrients and the build up of metabolic waste causing a
loss of viable cells (Mainer, 2008). Since this phase
was not observed it would help to repeat this experiment to determine a viable
window when working with M. smegmatis
for future experiments.

 

M. smegmatis was needed for future experiments of my project so a pure culture was
created. There are four phases to bacterial growth, in this experiment three of
the four phases were observed. The lag phase occurred between 0 to 48 hours,
here the bacterial cells did not divide. The lag phase is defined as a delayed
response in growth by bacterium, it is the adjustment period whereby the
bacterial cells adapt in order to take advantage of the environment which in
turn will lead to increased growth (Buchanan &
Klawitter, 1991) (Rolfe, et al., 2012). At 49 hours the
bacteria began to multiply slowly, the bacteria had entered the log phase.
Figure 10 shows that at 50 hours the OD reading increased to 0.2. The steepness
of the graph continued to increase as the hours passed, at 63 hours the OD
measured 1.48. This suggests M. smegmatis
cells were growing exponentially, by exploiting their environment within the LB
medium.

 

4.3 Growth of M. smegmatis

 

I needed to further confirm the protein used in my project was FN, in
order to do this I carried out the Dot Blot experiment. Sheep anti-Fibronectin
antibody was added to the membrane paper conjugated with HRP. HRP was
conjugated to the antibody, allowing for oxidation to take place yielding a
coloured product (Krainer &
Glieder, 2015)
figure 9 showed a distinct brown pigment within circles A and B confirming the
presence of the protein FN. The negative control was the protein Transferrin in
circles C and D that remained colourless. The milk
solution was used as it acts as a blocker buffer for all the unreacted sites,
reducing non-specific binding of proteins (Mahmood & Yang, 2012). 

After the molecular weight of Fibronectin was characterised through
SDS-PAGE, I needed to further confirm this protein before use in future
experiments.

 

In order to gain results for this experiment, the molecular weight
of FN was characterised using SDS-PAGE. The overall weight of Fibronectin is
440kDa, it has two polypeptide chains bound by a disulphide bond (Vincent, et al., 1988) (Verma, 2017) seen in figure 5  (Henderson, et al., 2011). The loading dye
contains the denaturant SDS, this coated the protein to give it a uniform
negative charge. With the application of heat to SDS, the hydrogen bonds of the
protein, linearising linearizing the protein at a molecular level (Schmid, et
al., 2016).
Figure 8 showed two bands in lane 3, this is the two subunits of FN. The weight
of the two chains vary as they are seen at different positions in figure 8. This
is because the molecular weight of the chains vary due to splice variation (White, et al., 2008) (Hynes, 2004) that takes place during post translational modifications of the
protein at glycosylation sites (Kosmehl, et al., 1996). Non-distinct bands
appeared in the background of the gel in the different lanes, this is as a
result of complete protein degradation of Fibronectin.