45

CHAPTER-2.1

Proteomic analysis of Cronobacter sakazakii planktonic,

biofilm and agar-surface associated cells

46

2.1.1 INTRODUCTION

“Biofilm is the surface-attached community of microorganisms which consists

of either a single or multiple microbial species embedded in a matrix of

exopolysaccharide” (Huq et al., 2008). “Biofilm mode of growth can be divided into

the different categories, viz., initial reversible cell attachment, irreversible attachment,

microcolonies formation, macrocolony formation and finally detachment and dispersal

of cells from the surface of the biofilm into the medium” (Hall-Stoodley and Stoodley,

2002). The following figure given by Monds and O’Toole (2009) represents biofilm

formation and dispersal.

Figure 2.1 Biofilm formation and dispersal (Monds and O’Toole, 2009).

Antonie van Leeuwenhoek had first identified the bacterial biofilm, while

investigating the microbial aggregates on tooth surface (Costerton et al., 1987).

Formation of biofilms allows the bacteria to stay alive in adverse environmental

conditions (Hall-Stoodley and Stoodley, 2002) and is different in nutrient utilization,

virulence factors (Pearson et al., 2006) and antimicrobial treatments (Zhang and Mah,

2008).

47

Formation of biofilm is an ancient prokaryotic adaptation (Hall-Stoodley and

Stoodley, 2002). Biofilms are morphologically, physiologically and metabolically

different from their free living ‘planktonic’ counterpart (Landini, 2009).

The genotypic and phenotypic expression changes takes place in the biofilm

mode of growth. The phenotypic change includes the production of macromolecules

which are essential for the biofilm formation. “Biofilms have increased resistance to a

variety of environmental factors” (Hall-Stoodley et al., 2004) “in comparison to

planktonic counterpart, making their abolition more difficult” (Hall-Stoodley et al.,

2004). “Microbial biofilms are of special anxiety to the food processing environment,

as biofilms formed on food material or food contact surfaces represent possible causes

of food contamination with pathogenic microorganisms or spoilage”(Sharma and

Anand, 2002). Cells of the biofilm also coordinate themselves by secreting specific

chemicals (quorum sensing). Jensen et al., (2007) have studied that quorum sensing is

connected with formation of biofilms.

“Previous studies have revealed that there are changes in the proteome profile

of planktonic and biofilm cells of Escherichia coli O 157:H7” (Tremoulet et al., 2002).

Sauer and Camper (2001) have studied that a different gene and protein expression

regulated proteins in the biofilm cells, include those involved in motility, transport,

outer membrane proteins and polysaccharide biosynthesis proteins. “Later, they

analysed the mechanism of quorum sensing involved in these changes” (Sauer and

Camper, 2001). “The biofilm proteome of Bacillus cereus is distinct from its

planktonic cells and it has been shown that biofilm phenotype has up and down

regulation of differential proteins rather than expression of new proteins” (Vilian and

Brozel, 2006). Recently Mukherjee et al., (2011) have compared the proteome of

planktonic cells and biofilms of E. coli MG 1655 and have found that the differences

representing significant increased or decreased proteins which are involved in acid

resistance, DNA protection and binding, and function of ABC transporters. The outer

membrane protein A (Omp A) has been found to be over expressed in biofilms of E.

coli than planktonic cells (Orme et al., 2006). Similar study by Pham et al., (2010) has

revealed that there is an increase in the outer membrane proteins of the biofilm cells. It

48

has been hypothesized that changes in gene expression and proteome in biofilm may be

the cause for cell adherence, virulence and drug resistance.

Giaouris et al., (2013) have studied the proteome of S. enterica serovar

Enteritidis PT4 planktonic and biofilm cells and found the proteins expressed in

biofilm cells are related to stress response, murein synthesis, DNA metabolism,

nutrient transport, degradation and energy metabolism, and detoxification. Several

hypotheses are put forward to explain the reason for biofilm formation by microbes.

The first is that, they provide stability in the growth environmental conditions.

Microbes living as biofilm might give catalytic properties by way of confining the cells

living in close proximity. “The second is that the biofilm formation provides protection

against UV light”, (Espeland and Wetzel, 2001), “acids” (McNeill and Hamilton,

2003), “metal toxicity” (Teitzel and Parsek, 2003), “dehydration and salinity” (Le

Magrex-Debar et al., 2000) and “antimicrobial agents” (Gilbert, 2002).

“Proteome analysis includes a combination of 2-D gel electrophoresis and mass

spectrometry as shown in Figure” 2.2 (Graves and Haystead, 2002). “In the first step,

the protein samples are dissolved in dithiothreitol, which helps in linearization of

proteins and decreases the formation of sulfhydryl groups, and then the samples are

separated on an immobilized pH gradient polyacrylamide gel strip in an isoelectric

focusing (IEF) cell. Later, one-dimensional polyacrylamide gel electrophoresis (PAGE)

is carried out to separate the proteins according to their molecular weight, which is the

second dimension of electrophoresis. An electrophoregram is obtained with an array of

protein spots from which the proteins of interest or the uniquely expressed proteins can

be recognized. Following this, protein spots of interest are selected and excised from

the gel peptide fingerprints which can be matched with fingerprints and/or sequences of

known proteins in electronic protein sequence databases and the selected proteins are

accordingly identified. Protein mass spectra can be generated using Matrix assisted

laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The

first step in the identification process of a novel protein with unknown function

involves the characterization of its primary structure. When used in combination, 2-D

gel electrophoresis and mass spectrometry form a powerful and provide a fairly

49

sensitive and reproducible strategy for protein identification and detection” (Flemming,

2010).

Figure 2.2 Differential 2-D gel electrophoresis proteomic analysis (Graves and

Haystead, 2002).

“Studies on biofilm formation of C. sakazakii carried out so far show that C.

sakazakii has an ability to form biofilm on glass, stainless steel, silicon and

polyvinylchloride” (Lehner et al., 2005). “Such materials are frequently used for infant

feeding utensils and in the preparation areas. Exopolysaccharide production increases

the attachment and adherence” (Scheepe-Leberkuhne and Wagner, 1986) to the utensils

and subsequently increases the risk of infection to infants and neonates. “Colonization

of C. sakazakii on the surface of blenders, spoons and brushes have been associated

with neonatal infections” (Bar-Oz et al., 2001). Kim et al., (2006) “have studied the

biofilm formation of C. sakazakii on enteral feeding tubes and stainless steel”. Grimm

50

et al., (2008) “have described the presence of cellulose as a constituent of C. sakazakii

extracellular matrix”. Later, Dancer et al., (2009b) studied the biofilm formation ability

of C. sakazakii and their results indicate that milk components (whey protein and

casein) are the main determinant in the biofilm formation. “Biofilm formation of C.

sakazakii on foods and food contact surfaces will encourage the risk of food-borne

diseases” (Iversen and Forsythe, 2003). Beuchat et al., (2009) have reported that

biofilm formation in C. sakazakii imparts protection against disinfectants. “Biofilms

are of special concern to the food processing environment” (Iversen and Forsythe,

2003) as biofilm formation allows the pathogenic microbes to persist on food material

or food contact surfaces which can be the source of spoilage or contamination of food

products such as milk products.

In order to find a remedy to biofilm formation in the food processing

environment, it is required to understand the physiology of bacterial biofilm cells

which will help in formulating efficient biofilm control strategies. “C. sakazakii has

also been reported to form biofilms on glass and stainless steel, thus increasing the risk

to infants, neonates and immuno compromised individuals” (Iversen and Forsythe,

2004a). Hence the study of biofilm cells of C. sakazakii is required to limit the

occurrence of infection in infants, neonates and immuno compromised individuals. The

in-vitro study of the biofilm is essential since it allows the examination of biofilm

phenotype that is not readily interpreted in an in-vivo system. Though the in-vitro

studies rarely demonstrate the biofilm phenotype in natural in-vivo state, still they

remain important in the examination of biofilm infections in the perspective of human

infections. Scarce information is available regarding the molecular basis of the biofilm

proteome of C. sakazakii. Therefore, the aim of the present work was to study C.

sakazakii grown as planktonic, agar-surface associated and biofilm cells. Differential

expression of individual proteins was investigated in order to characterize the

planktonic, biofilm and agar-surface associated phenotype cells using the following

techniques.

1. “One-dimenstional sodium dodecyl sulphate polyacrylamide gel electrophoresis

(1D-SDS-PAGE)” (Sambrook et al., 1989).

2. “Two-dimensional gel electrophoresis” (Sambrook et al., 1989).

51

3. “Matrix assisted laser desorption ionization-time of flight mass spectrometry

(MALDI-TOF MS)” (Flemming, 2010).

The initial part (Section 2.1.2.7) of this chapter deals with the “differences in

protein expression between the planktonic, agar-surface associated (AS) and biofilm

forms of ten C. sakazakii isolates” (Sharma and Prakash, 2013a). The whole cell

protein of the above three forms were analysed using 1-D SDS-PAGE. The latter part

(Section 2.1.2.8) of the chapter includes the 2-D gel electrophoresis technique (2.1.2.8)

and protein identification with MALDI-TOF MS (2.1.2.9) of MTCC-2958.

2.1.2 MATERIALS AND METHODS

2.1.2.1 Chemicals and reagents, software and instruments

Chemicals and reagents used in the study were procured from Sigma Aldrich

(USA), Bangalore Genei (India), Hi-Media (India), Merck (India), Difco (USA), Bio-

Rad (India), Qualigens (India), Bio-Rad (India), Axygen (India), Galaxo (India), Gel

Doc System (Zenith, India), PD Quest Version Advanced software program (Bio-Rad,

Hercules, CA,USA), Chemidoc [Bio-Rad, Segrate (Milan) Italy] and MALDI-

TOF/TOF 50 instrument (Bruker Daltonics, GmbH, Leipzig Germany).

2.1.2.2 Bacteria used in the study

The bacteria used in this study include MTCC-2958 and 9 isolates from milk

and milk products of Agra city (as described in section 1.2.4).

2.1.2.3 Reviving of the cultures

Cultures used in this study were revived in EE broth (as described in section

1.2.4b).

2.1.2.4 Harvesting of planktonic cells

“A loop full of each confirmed C. sakazakii isolates (Jal 1, Jal 2, Jal 3, Jal 4, Jal

5, Jal 6, Jal 7, Jal 8, Jal 9 and MTCC-2958) from the overnight TSA culture plates were

inoculated in 3ml of EE broth” (Sharma and Prakash, 2013a). “The EE broths were

further incubated for 24 hours at 37°C. Cell enumeration was done by CFU plating

method. The concentration of cells was adjusted to ~ 106 CFU/ml with 0.85% sodium

chloride solution. This culture was centrifuged at room temperature for 10 minutes at

52

4000 g to harvest the bacterial cells. The media components with bacterial metabolites

were removed by re-suspending the pellet in 0.85 % sodium chloride solution and by

centrifuging again for 10 minutes at 4000 g to harvest the planktonic cells. This step

was repeated twice. The supernatant was discarded and the pellets were collected in

their respective tubes” (Sharma and Prakash, 2013a).

2.1.2.5 Harvesting of the agar-surface associated (AS) cells

“For the AS growth, the revived MTCC-2958 and C. sakazakii isolates (Jal 1,

Jal 2, Jal 3, Jal 4, Jal 5, Jal 6, Jal 7, Jal 8, Jal 9) were streaked on sterile tryptic soya

agar (TSA) plates and incubated for 24 hours at 37°C. From the TSA plates, cells were

harvested using a wire loop and were suspended in 1 ml of 0.85% sodium chloride

solution. Cell enumeration was done by CFU plating method. The concentration of the

cells was adjusted to ~ 106 CFU/ml with 0.85% sodium chloride solution. The culture

was centrifuged at room temperature for 10 minutes at 4000g to harvest the bacterial

cells. The media components with bacterial metabolites were removed by re-

suspending the pellet in 0.85 % sodium chloride solution and by centrifuging again for

10 minutes at 4000 g to harvest the AS cells. This step was repeated twice. The

supernatant was discarded and the pellets were collected in their respective tubes”

(Sharma and Prakash, 2013a).

2.1.2.6 Harvesting of the biofilm cells

“MTCC-2958 and C. sakazakii isolates (Jal 1-Jal 9) were inoculated in 3ml of

EE broth with a loop full of confirmed C. sakazakii cells from overnight culture plates

and incubated for 24 hours at 37°C. After incubation the contents of the glass test tubes

were decanted and the tubes were washed twice with 0.85 % sodium chloride solution

to remove the planktonic cells. 1 ml of 0.85 % sodium chloride solution was added to

the test tube and vortexed to dislodge the biofilm cells. Biofilm cells were centrifuged

at room temperature for 10 minutes at 4000g. Cell enumeration was done by serial

dilution followed by plating on TSA plates. The concentration of the cells was adjusted

to ~ 106 CFU/ml with 0.85% sodium chloride solution. Biofilm cells of ~ 10

6 CFU/ml

were centrifuged at room temperature for 10 minutes at 4000g. The supernatant was

discarded and the pellets were collected in their respective tubes (Sharma and Prakash,

2013a).

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2.1.2.7 1-D Gel Electrophoresis

(a) Extraction of whole cell protein

“Whole cell proteins were extracted, following the procedure of” Du Toit et al.,

(2003). “Pellets of planktonic, AS cells and biofilm cells were resuspended in 300µl of

TEGL buffer (25mM Tris, 10mM EDTA pH 8, 0.9% glucose (w/v), 10mg/ml

lysozyme) and incubated at 37°C for 3 hours. Subsequently the pellets were obtained

by centrifugation at 4000g for 7 minutes and the supernatant was discarded. 200µl of

sample reducing buffer was added to each tube with the pellet obtained and mixed well

and further incubated at 100°C for 10 minutes over a boiling water bath” Du Toit et al.,

(2003). This was cooled immediately in ice and stored at -200

C for further use.

Test reagents

TEGL buffer

25 mM Tris 0.30 g

10 mM EDTA 0.37g

0.9% Glucose (w/v) 0.9 g

10 mg/ml Lysozyme 1.0 g

Distilled water 100 ml

“Sample buffer (SDS reducing buffer)” (Sambrook et al., 1989)

Distilled water 3.6 ml

“50mM Tris-HCl, pH 6.8” Sambrook et al., 1989) 1.2 ml

Glycerol 2.5 ml

“10% (w/v) SDS” (Sambrook et al., 1989) 2.0 ml

5% (w/v) Bromophenol blue 0.2 ml

“β-mercaptoethannol(added just before use)” 0.5 ml

(b) “SDS-PAGE of the whole cell protein” (Sambrook et al., 1989)

The protein profile was obtained, using a 12% gel, by SDS-PAGE. Molecular

weight marker ranging from 14.3-100 kDa was used.

Test reagents

Stock A (pH 8.8)

1.5M Tris base 18.17g

54

Distilled water 50 ml

“pH was adjusted by using 1N HCl and the volume was made up to 100 ml with

distilled water” (Sambrook et al.,1989).

Stock C

Acrylamide 29.2 g

N, N, N, N Bisacrylamide 8 g

Distilled water 100 ml

“10% (w/v) Sodium dodecyl sulphate (SDS)” (Sambrook et al., 1989)

SDS 10 g

Distilled water 100 ml

“10% ammonium persulphate (APS), freshly prepared” Sambrook et al., 1989)

APS 10 g

Distilled water 100 ml

“All the solutions were prepared using double distilled water and were stored at 4°C in

amber colored glass bottles” (Sambrook et al.,1989).

“10X Electrode (running) buffer, pH 8.3 (Stock solution)” (Sambrook et al., 1989)

Tris base 3.03 g

Glycine 14.4 g

SDS 1.0 g

Distilled water 100 ml

“1X Electrode (running) buffer, pH 8.3 (working solution)” (Sambrook et al., 1989)

10X Electrode running buffer 20 ml

Distilled water 180 ml

Protein molecular weight marker

Marker 3-5µl

5% bromophenol blue 2.5µl

Glycerol 2.5µl

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(c) Preparation of SDS polyacrylamide gel

Separation or resolving gel 12 %

Components 12% gel

1. Distilled water 3.3 ml

2. Stock C 4 ml

3. Stock A 2.5 ml

4. 10% SDS 100 µl

5. 10% APS 100 µl

6. TEMED 4 µl

“APS and N,N,N’,N’-tetramethyl ethylene diamine (TEMED) were added to the

solution just before use” (Sambrook et al.,1989) .

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Preparation of a 5% stacking gel

Components 5% gel

1. Distilled water 3.05 ml

2. Stock C 1.25 ml

3. Stock A 0.65 ml

4. 10% SDS 50 µl

5. 10% APS 25 µl

6. TEMED 5 µl

Procedure

The gel was prepared in between the two glass slabs, whose sides were sealed

by 1% agar and the slabs were held together by clips. The gel components were mixed

and slowly poured in between the two glass slabs. A comb was inserted immediately

between the two slabs to prepare the wells. This was left to polymerize for about 30

minutes. The comb was carefully removed and the slab was fixed vertically in the

electrophoretic chamber. Both the upper and the lower reservoir were filled with 1X

Tris–glycine buffer (Electrode running buffer, pH 8.3). About 30µl of the denatured

protein samples was loaded in each well. The molecular weight protein marker was

loaded in adjoining lane. This apparatus was kept at room temperature and the two

electrodes were connected to the electrophoresis power supply. Initially the gel was run

at 45V for 15 min and then a constant supply of 7V/cm was applied to the apparatus.

“Once the tracking dye, bromophenol blue, reached the bottom of the gel the power

supply was cut and the gel was taken out carefully” (Sambrook et al., 1989). “It was

immediately kept in fixing solution (methanol 20 ml, acetic acid 10 ml, distilled water

70 ml) , until the blue line of bromophenol blue turned yellow” (Sambrook et al.,1989).

The gel was rinsed with water and immersed in the staining solution of Commassie

brilliant blue for 4-5 hrs with constant shaking at room temperature. Destaining was

done in 7% acetic acid till the bands became visible. “Quick destaining was done in a

solution containing methanol, water and acetic acid in the ratio 9:9:2 respectively”

(Sambrook et al., 1989). Gels were analysed by using Total Lab Quant v 11.4 gel

57

analysis software. WCP profiles were analysed for the differences in number and

molecular weights of protein bands.

2.1.2.8 2-D Gel Electrophoresis

Principle

“The 2-D gel electrophoretic technique is used to separate and visualize

proteins and relies on the separation of proteins according to their pI (neutral charge at

a certain pH), termed as isoelectric focusing (IEF), followed by one-dimensional gel

electrophoresis separating proteins according to their physical properties (size and

molecular weight)” (Sambrook et al.,1989).

2.1.2.8.1 Reviving of the culture

Culture was revived in EE broth as described in Section 1.2.4b.

2.1.2.7.2 Harvesting of planktonic, AS and biofilm cells of MTCC-2958

Growth was harvested as described in Section 2.1.2.4, 2.1.2.5 and 2.1.2.6.

2.1.2.8.3 Preparation of cell lysate

“Planktonic, AS and biofilm harvested cells were washed in 0.85% sodium

chloride solution at least three times and were suspended in sonication buffer at

concentration of 1g wet cells per 5 ml of buffer and then broken by intermittent

sonication. Pellets were sonicated eight times for 2 minutes at power level 5 and 50%

of the active cycle. After sonication the soluble protein fractions were separated from

cell debris by centrifugation at 12,000 g for 10 min at 4°C. The supernatant was

collected and stored at -20°C” (Flemming, 2010).

Test reagents

Sonication buffer (pH 7.4)

Tris HCl 50 mM

MgCl2 10 mM

PMSF (phenylmethylsulfonyl fluoride) 1 mM

EGTA (ethylene glycol tetracetic acid) 1 mM

Sodium azide 0.1%

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PIC (protease inhibitor cocktail, 1ml of the cocktail solution is added in 20 ml

of cell lysate). PMSF was added just before use.

All the above contents were weighed (except PMSF) and kept in a bottle. “The pH was

adjusted to 7.4 using 4 N HCl and the volume was made up to100 ml” (Sambrook et

al., 1989).

2.1.2.8.4 Precipitation of protein with trichloroacetic acid (TCA)-acetone

“Cell lysates were treated with 1% SDS and then processed via the TCA-

acetone precipitation method” (Sharma et al., 2010).

Procedure

“About 0.5 ml of the cell lysate and 5µl of 1% SDS solution was mixed. The

mixture was boiled for 5-6 minutes. It was cooled down. Then 10% TCA (v/v) was

added to the cell extract and the mixture was incubated at -20°C overnight for

precipitation of proteins. The precipitated proteins were collected by centrifugation at

18,000 g for 15 minutes at 4°C. The supernatant was discarded and the pellet was

washed thrice with 100 % ice cold acetone and then allowed to air dry. The protein

pellet was suspended in appropriate volume of rehydration buffer. The mixture was

vortexed 2-3 times after every 10 minutes and left overnight to dissolve the pellet in the

rehydration buffer. Then the mixture was centrifuged at 18,000g for 15 minutes at 4°C”

(Sharma et al., 2010). “The supernatant was transferred to a new tube and its protein

concentration was estimated using the Bradford method” (Bradford, 1976).

Rehydration buffer

Urea 8 M

CHAPS (non-ionic or zwitterionic detergent) 2%

DTT (dithiothreitol) 50 mM

Bio-Lyte 3/10 ampholyte 0.2%

Bromo phenol blue 0.001%

2.1.2.8.5 Bradford method for protein estimation

Principle

59

“This method is based on the interaction of the dye, Coomassie brilliant blue,

with the protein. Free dye has absorption at 470 nm and 650 nm but when bound to

protein it has an absorption maximum at 595 nm. The amount of dye binding appears

to vary with the content of the basic amino acid arginine and lysine in the protein”

(Sambrook et al., 1989).

Standard protein solution: 1 mg/ ml BSA, Bradford reagent.

Procedure

The spectrophotometer was warmed up before use

1. In a series of clean and dry test tubes 5, 10, 15, 20, 25 µl of standard BSA

solutions were pipetted. Volume in each test tube was then made up to 50µl

with distilled water.

2. Spectrophotometer was adjusted to a wavelength of 595nm, and the blank to

zero using the tube which contains no protein.

3. To estimate the protein in the cell extract, 5µl of protein extract was taken and

volume made up to 50µl with distilled water.

4. “1.5 ml Bradford reagent was then added to each tube, incubated at room

temperature for a minimum of 15 minutes and then the absorbance was taken at

595nm” (Bradford, 1976).

5. Concentration versus absorbance curve was plotted.

2.1.2.8.6 Rehydration of the IPG strip

Procedure

“2D PAGE was carried out using the method of in gel rehydration” (Gorg et al.,

2000). Cell extract (150 µg protein) mixed with rehydration buffer (final volume 124

µl) was applied to immobilized strip (17 cm length, pH 4-7). 124µl of the rehydration

buffer (containing the protein) was spread in a rehydration tray along the wall of one

well on a tray. Plastic coating from the IPG- strip was removed carefully by using

forceps. The gel is present on the back side of the strip. The strip was spread on the

rehydration buffer to cover the whole buffer evenly. Thus the gel remains in contact

with the buffer. Care was taken to prevent the formation of bubbles. The side

containing the gel was kept downwards. Mineral oil was applied on the IPG-strip to

60

prevent it from drying up. The rehydration tray was placed in the IEF unit to maintain a

constant temperature of 20°C for passive rehydration. The program was fed and run for

passive rehydration in IEF cell.

First dimension separation: Isoelectofocussing (IEF) - IEF was performed for the

rehydrated strip. This was performed at 20°C in an IEF unit using the following

procedure:

0-250 V Linear mode for 1 hour

250 V Rapid mode- constant for 1hour

250-3000V Linear mode for 4 hour

3000V Rapid mode constant until 15 kVh, slow mode

The current limit was set at 50mA/strip.

Procedure

In the IEF tray the electrode wire was covered by electrode wicks (made by

filter paper), these wicks are moistened with distilled water. IPG strips were taken out

from rehydration tray. After rehydration the strip swelled up (The strip was cleared

from that side on which the gel is not present, to remove the mineral oil). The strip was

layered on the electrode wick in the IEF-tray. The positive sign shown on the strip was

always kept on the lower side, in contact with electrode wick. This tray was then fixed

in IEF unit and the program was run.

Equilibration of IPG strips prior to SDS-PAGE

Procedure

IEF tray containing the IPG strips is transferred to rehydration tray containing

about 2 ml of Equilibration buffer I and shaken for 15 minutes.

Later the IPG strips were again transferred in another well of the rehydration tray

containing about 2 ml of Equilibration buffer II and were shaken for 15 minutes.

Test Reagents

Equilibration buffer I

0.375 M Tris, (pH 8.8) 2.5 ml

6M Urea 3.6 g

61

20% Glycerol 2 ml

2% SDS 0.2 g

20% Glycerol 2 ml

130mM DTT 0.2 g

The volume was made up to 10ml with distilled water and it was stored at room

temperature after filtration.

Equilibration buffer II (reagents for 10 ml)

0.375 M Tris, pH 8.8 2.5 ml

6M Urea 3.6 g

20% Glycerol 2 ml

2% SDS 0.2 g

20% Glycerol 2 ml

Iodoacetamide (135mM) 0.25 g

2.1.2.8.7 Separation in the second dimension: SDS-Page

“The strip was loaded on top of a vertical SDS-polyacrylamide gel (12%) and

sealed in 1% low melting agarose dissolved in electrode buffer. The molecular markers

are loaded in a separate well by the side of the strip. Electrophoresis was performed as

described in Section 2.1.2.7(b) with a constant current of 25 mA until the indicator dye

reached the bottom edge of the gel. Proteins were stained by Coomassie brilliant blue R

250” (Sambrook et al., 1989). The molecular masses of the protein were determined

with the help of the molecular weight marker, in the range of 6.5 kDa-194.2 kDa,

loaded in the second dimension.

Sample buffer

Distilled water 4 ml

0.5ml Tris HCl pH 6.8 1.0 ml

Glycerol 1.8 ml

10% SDS 1.6 ml

0.05% Bromo phenol blue 0.2 ml

DTT 0.4 ml

62

Marker was diluted in the ratio of 1:19 in the sample buffer

Marker 5 µl

Sample buffer 95 µl

DTT 1-2 pinch

Marker, sample buffer and DTT was heated and cooled for 1-2 minutes before using.

Analysis of the image

The 2-D gels were scanned with a gel scanning densitometer. Analysis includes

quantification of spots and matching the gels with the help of PD Quest Advanced

software.

Statistical analysis

Student t-test statistical tool was applied for the statistical study by PDQuest

software. This ensures that only significant changes in the value of protein spots were

taken for analysis. The program picks up the spots with a differential intensity of

significant levels built in the system. Same amount of protein (150µg) was loaded onto

all the gels.

2.1.2.9 Protein identification with MALDI-TOF MS

Differenentially-expressed WCP spots in each of the forms, that is, planktonic,

AS and biofilm forms of MTCC-2958 were chosen for MALDI-TOF.

2.1.2.9.1 “In gel digestion of selected protein spots (Coomassie stained) with

trypsin” (Shevchenko et al., 1996)

Chemicals

NH4HCO3 M= 79 g/mol; 50 mM = 4 mg/ml

Dithiothreitol (DTT) M=154 g/mol; 10 mM = 10.2 mg/ml

Iodacetamide (IAA) M=185 g/mol; 55 mM =10.2 mg/ml

Trypsin C= 20-25 ng/µl

Excision of protein bands from polyacrylamide gels:

1. “Gels were washed with water (2 times, 10 minutes each)” Shevchenko et al.,

1996).

2. “Protein spots of interest were excised manually from the 2-D gels. Excised

pieces were cut into 1 mm-cube”.

63

3. Gel pieces were transferred to a 0.5 µl micro centrifuge tube.

“Washing the gel pieces” (Shevchenko et al., 1996)

1. “For destaining the gel pieces it was cut into bits and washed with water and 50

mM NH4HCO3 -50% acetonitrile [1:1 (v/v)] for 15 minutes”.

2. “The remaining liquid was removed and enough acetonitrile was added to cover

the gel pieces. The gel bits shrink and stick together”.

3. “Acetonitrile was removed”.

4. “The gel pieces were rehydrated in 50 mM NH4HCO3” (Shevchenko et al.,

1996).

5. “After 5 minutes, an equal volume of acetonitrile was added” (Shevchenko et

al., 1996)

6. “All the liquid was removed after 15 minutes of incubation” .

7. Again enough acetonitrile was added to cover the gel pieces.

8. When the gel pieces had shrunk, the acetonitrile was removed.

9. The gel pieces were dried in air or in a vacuum centrifuge.

“Reduction and alkylation” (Shevchenko et al., 1996)

1. “The gel pieces were rehydrated in 10 mM DTT in 50 mM NH4HCO3 (freshly

prepared)”.

2. “They were then incubated for 45 minutes at 56°C”.

3. “The tubes were brought to room temperature”.

4. The excess liquid was removed and replaced quickly by roughly the same

volume as above of freshly prepared 55 mM iodoacetamide in 50 mM

NH4HCO3.

5. This was incubated for 30 min at room temperature in the dark.

6. The iodoacetamide solution was removed.

7. The gel pieces were washed with 50 mM and NH4HCO3 in acetonitrile (1+1;

v/v), one or two changes each, 15 min per change.

8. Again enough acetonitrile was added to cover the gel pieces.

9. After the gel pieces had shrunk, it was removed.

10. The gel particles were dried in air or in vacuum centrifuge.

64

“In-gel digestion” (Shevchenko et al., 1996)

Preparation and addition of trypsin solution: Sequencing grade/ Mass

spectrometry grade trypsin (Promega) is recommended. Trypsin cuts C-terminal side of

K (lysine) and R (arginine) unless next residue is P (proline). The solid powder was

dissolved in 1 mM hydrochloric acid giving a concentration of 100 ng/ µl. It was stored

in 10 µl aliquots at -20°C. Freshly prepared enzyme solution was added (in 25 mM

NH4HCO3) to cover the gel.

1. This was incubated at 37°C for 30 min.

2. The excess enzyme solution was removed.

3. About 25 mM NH4HCO3 was added (approx. 2-3 µl) to keep the gel wet

overnight, but excess liquid was avoided.

4. This was incubated overnight at 37°C.

“Extraction of peptides” (Sharma et al., 2010)

“Peptides were extracted twice, from the gel pieces using the extraction buffer

[1:1] mixture of 70% ACN and 0.1% trifluoroacetic acid (TFA)]. Extraction was

supported by ultrasonication for a few minutes; to improve the extraction yields.

Recommended time frame for extraction was 30 minutes at room temperature”

(Sharma et al., 2010).

2.1.2.9.2 Mass spectrometry

Digested samples were desalted and concentrated on C-18 ZipTips (Millipore,

Billerica, MA, USA) employing manufacturer’s protocol”. “ZipTips were extracted on

MTP 384 target plate using 2 µl saturated solution of α-cyano-4-hydroxycinnamic acid

dissolved in 50 % ACN (v/v), 0.2 per cent trifuoroacetic acid. Autofex II TOF/TOF 50

(Bruker Daltonik GmbH, Leipzig, Germany) in positive refectron mode, in the

detection range of 500-3000 m/z was used to acquire mass spectra of digested protein”

(Sharma et al., 2010). “Mascot tool was used to evaluate the proteolytic masses.

Mascot wizard (Matrix Science, UK) was used for peak detection in MALDI spectra

and submission of the peak lists to the database. The parameters used for peptide mass

tolerance was fixed to 50 ppm with carbamidomethyl-cystein, oxidation of methionine

as variable modifcation and 1 missed cleavage site permitted” (Sharma et al., 2010).

65

2.1.3 RESULTS

2.1.3.1 Whole cell protein (WCP) analysis

“WCP profiles of planktonic, biofilm and agar-surface associated cells of the C.

sakazakii isolates displayed variability in their molecular weights” (Sharma and

Prakash, 2013a). (Figure 2.3 a, b, c). WCP profiles of the three forms (planktonic,

biofilm and agar-surface associated) of the C. sakazakii isolates with their molecular

weights are listed in Tables 2.1 a and b. “Though WCP profile of the three forms was

more or less similar for each respective isolate, but distinct variations in

presence/absence of protein bands were observed” (Sharma and Prakash, 2013a).

(Figure 2.3 a, b, c; Table 2.1 a, b).

“Prominent proteins (expressed in WCP profiles of more than 30% isolates)

expressed in planktonic phase, but not in agar-surface associated and biofilm included

WCP bands of 104.0 kDa, 93.1 kDa, 61.3 kDa, 39.2 kDa, 34.9 kDa, 22.1 kDa, 17.1

kDa, 15.2 kDa. Prominent proteins (expressed in WCP profiles of more than 30%

isolates) common in planktonic and agar-surface associated cells included WCP bands

of 24.2 kDa, 29.2 kDa, 70.7 kDa, and 82.2 kDa. Prominent proteins (expressed in WCP

profiles of more than 30% isolates) expressed in agar-surface associated form, but not

in planktonic and biofilm included WCP bands of 103.4 kDa, 90.4 kDa, 46.1 kDa, 35.5

kDa, 13.4 kDa” (Sharma and Prakash, 2013a) .

“Prominent proteins (expressed in WCP profiles of more than 30% isolates)

common to agar-surface associated cells and biofilm included WCP bands of 94.4 kDa,

87.2 kDa, and 27.3 kDa. Prominent proteins (expressed in WCP profiles of more than

30% isolates) expressed in, biofilm but not in planktonic and agar-surface associated

form included WCP bands of 118.0 kDa, 107.4 kDa, 98.5 kDa, 75.8 kDa, 65.1 kDa, 57

kDa, 48.2 kDa, 34.4 kDa, and 15.8 kDa. Prominent proteins common (expressed in

WCP profiles of more than 30% isolates) in planktonic form and biofilm included

WCP bands of 20.6 kDa and 55.1 kDa. Bands common to all the three forms

(expressed in WCP profiles of more than 30% isolates) are 91.6 kDa, 31.5 kDa, 28.1

kDa and 19.7 kDa” (Sharma and Prakash, 2013a).

2.1.3.2 Protein estimation

66

Cell lysate of C. sakazakii isolate grown as planktonic, AS and biofilm cells

was prepared as described earlier. By employing Bradford’s method, a standard curve

(Figure 2.4) was obtained for protein estimation “using bovine serum albumin (BSA)

as standard” (Bradford, 1976). The protein concentrations of planktonic, AS and

biofilm cells were found to be 17µg, 20µg and 31 µg respectively (Table 2.2). Table

2.3 shows the calculation for the protein sample (µl) and rehydration buffer which is

loaded on the IEF strip.

2.1.3.3 Analysis of proteome

Proteome maps of C. sakazakii planktonic, biofilm and agar surface associated

cells after 24 hours of growth were analysed and compared.

1. Qualitative analysis includes absence of protein spots among the three

proteome maps.

2. Quantitative analysis includes proteins exhibiting an increase or decrease by a

factor of two or greater which were chosen arbitrarily for further analysis and

are summarized in (Table 2.4 a-g; Figure 2.5 a, b, c).

Proteins exhibiting an increase or decrease by a factor of two or greater were chosen

for analysis. The results from this analysis are summarized in Table 2.4 a-g.

The study focused on the proteomic analysis of planktonic, agar-surface associated

and biofilm grown cells of C. sakazakii (MTCC-2958). The proteome of the three

growth forms showed an overall similarity in WCP spot patterns (Figure 2.5 a, b, c).

However analysis of the 2-D electrophoregrams by PD-Quest software showed up-

regulated and/or down regulated and growth form specific unique proteins present in

each of the three growth forms (planktonic, agar-surface associated and biofilm cells

(Figure 2.5 and 2.6 a, b,c).

A total of 31 proteins were analysed by PD-Quest software, of which 9 (spot 1 to

spot 9) were successfully identified by MALDI-TOF mass spectrometry (Table 2.6;

Figure 2.5 a, b, c). Mass spectrums of these nine proteins (spot 1-spot 9) are shown in

Figure 2.7 a, b, c).

0

Figure 2.3 “Electrophoregrams displaying whole cell protein profiles of (a) planktonic, (b) agar surface-associated and (c) biofilms

forms of C. sakazakii isolates. Lanes (a)1, (b)1 and (c)1 : stained protein ladder (69.0, 43.0 29.0, 20.0, 14.3 kDa) (Sigma, USA);

Lanes (a) 2- (a) 12 : isolates Jal 1, Jal 2, Jal 3, Jal 4, Jal 5, Jal 6, Jal 7, Jal 8, Jal 9, Jal 9 and MTCC-2958, (b) 2-(b) 11,and (c) 2-

(c) 11,: isolates Jal 1, Jal 2, Jal 3, Jal 4, Jal 5, Jal 6, Jal 7, Jal 8, Jal 9 and MTCC-2958” (Sharma and Prakash, 2013a) respectively.

1 2 3 4 5 6 7 8 9 10 11 12

kDa

69

43

29

20

14.3

11 10 9 8 7 6 5 4 3 2 1 11 10 9 8 7 6 5 4 3 2 1

(a) (b) (c)

67

Table 2.1 (a) “Whole cell protein profiles of planktonic (P), biofilm (B), and agar-surface associated (AS) forms of C. sakazakii

isolates (Jal 1, Jal 2, Jal 3, Jal 4) and MTCC-2859” (Sharma and Prakash, 2013a).

Jal 1 Jal 2 Jal 3 Jal 4 MTCC-2858

Isolate

growth

form

P

AS

B

P

AS

B

P

AS

B

P

AS

B

P

AS

B

Molecular

weight

(kDa)

118 118 118

107 107.4

104

103.4 103.4 103.4 103.4

98.5 98.5 98.5 98.5

93.1 93.1 94.4 94.4 94.4 94.4 94.4 94.4 94.4 94.4

91.6 91.6 91.6 91.6 91.6 91.6 91.6 91.6 91.6

90.5 90.4 90.4

87.6

87.2 87.2 87.2 87.2

82.2 82.2 82.2 82.2 82.2

75.8 75.8 75.8

70.7 70.7 70.7 70.7 70.7 70.7 70.7

65.1 65.1 64.6

61.3 61.3 61.9 61.3

57.0 57.0 57.0

55.1 55.1 55.1 55.1

48.2 48.2 48.2

46.1 46.1 46.1

68

39.2 39.2 39.2

35.5 35.5 35.5

34.9 34.9

34.4 34.4

31.5 31.5 31.5 31.5 31.5 31.5 31.5 31.5 31.5

29.2 29.2 29.2 29.2 29.2 29 29

28.1 28.1 28.1 28.1 28.1 28.1

27.3 27.3 27.3 27.3 27.3 27.3

24.2 24.2 24.2 24.2 24.2 24.2

22.1 22.1 22.1

20.6 20.6 20.6 20.6

19.7 19.7 19.7 19.7 19.7 19.7 19.7 19.7

17.1 17.1 17.0 17.1

15.8 15.8 15.8 15.8

15.2 15.2 15.2

13.4 13.4 13.4

Total no.

of bands

12 9 10 11 14 13 10 10 10 10 4 8 7 10 11

69

Table 2.1 (b) “Whole cell protein profiles of planktonic (P), biofilm (B), and agar-surface associated (AS)

forms of C. sakazakii isolates (Jal 5, Jal 6, Jal 7, Jal 8, and Jal 9)” (Sharma and Prakash, 2013a).

Jal 5 Jal 7 Jal 7 Jal 8 Jal 9

Isolate

growth

form

P

AS

B

P

AS

B

P

AS

B

P

AS

B

P

AS

B

Molecular

weight

(kDa)

118 115

107.4 107.4

104.0 104.0 104.0 104.0

103.6 103.4

98.5

96.8

91.9 94.4 94.4 94.3 94.4

93.8

93.1 93.1

91.6 91.6 91.6 91.6 91.6 91.6

91.1 91.1 91.1

90.4 90.4 90.4

87.7 87.0 87.2 87.2

82.4 82.2 82.2 82.2 82.2 82.2 82.2

75.8 75.8

70.7 70.7 70.7 70.9 70.7 70.7

70.4

63.0

65.1 65.1 64.6

62.4

61.3 61.9 61.3

60.1 57.0 57.0

55.1 55.1

48.2 48.2 48.2

70

47.8 46.1 46.3 46.7 46.1

35.5 35.0 35.5 35.5

34.2 34.9

34.9 34.4 34.4

31.5 31.5 31.5

29.2 29.2 29.2 29.2 29.2 29 29

28.1 28.1 28.1 28.1 28.1 28.1

27.3 27.3

24.2 24.2

22.1 22.1 22.5

21.9

20.6 20.6 20.6

19.7 19.7 19.7 19.7 19.7

17.1 17.1 17.2 17.0 17.1

15.8 15.8

15.4 15.4 15.2 15.5 15.9

13.4 13.4

Total no. of

bands

12 8 5 11 13 11 12 10 8 8 4 7 5 7 6

71

Table 2.2 Protein concentrations of planktonic, AS and biofilm cells

Form of growth Obtained O.D of

the protein sample

Protein

concentration

(µg)

Protein

concentration

in µg/ml

Planktonic 0.17 17 17/5 =3.4

AS 0.20 20 20/5= 4.0

Biofilm cells 0.31 31 31/5=6.2

Table 2.3 Calculation for the loading protein (150 µg) sample on the IEF strip

Form of

growth

Protein

concentration

in µg/ml

Protein sample in

(µl) loaded for IEF

(Final amount

desired= 150 µg)

Rehydration

buffer

added

Final

volume

(desired)

Planktonic 3.4 44.11 79.89 µl 124 µl

AS 4.0 37.50 86.50 µl 124 µl

Biofilm cells 6.2 24.19 99.81 µl 124 µl

Figure 2.4 Standard curve of BSA for protein estimation by Bradford method.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 10 20 30 40 50

O.D

595

nm

Protein concentration (µg)

a

194.2

104.2

59.2

41.9

27.8

20.8

15.2

M

(kDa) pH 4 pI pH 7

Figure 2.5 (a) Coomassie stained 2D-gel electrophoretic WCP spot profile of

C.sakazakii (MTCC-2958) grown in planktonic form. Boxes indicate significantly

increased/decreased spots (more than 2-fold) in specific growth form.

b

M

(kDa) pH 4 pI pH 7

194.2

104.2

59.2

41.9

27.8

20.8

15.2

Figure 2.5 (b) Coomassie stained 2D-gel electrophoretic WCP spot profile of

C.sakazakii (MTCC-2958) grown in agar-surface associated form. Boxes indicate

significantly increased/decreased spots (more than 2-fold) in specific growth form.

c

194.2

104.2

59.2

41.9

27.8

20.8

15.2

M

(kDa) pH 4 pI pH 7

Figure 2.5 (c) Coomassie stained 2D-gel electrophoretic WCP spot profile of

C.sakazakii (MTCC-2958) grown in biofilm form. Boxes indicate significantly

increased/decreased spots (more than 2-fold) in specific growth form.

d

(a) (b) (c)

Figure 2.6 Coomassie stained 2D-gel electrophoresis WCP spot profiles of C. sakazakii (MTCC-2958) grown in (a) planktonic, (b)

agar-surface associated and (c) biofilm forms. Numbers 1 to 10, indicate significantly increased/decreased spots (more than 2-fold )

in the specific growth form.

e

Figure 2.7 (a) Mass spectrum of protein spot 1, 2 and 9 generated by MALDI

TOF/TOF MS

2856.326

1928.8551459.760

1605.817804.284

1189.545 2075.0552989.4262283.079 2524.080961.537

D1\0_N1\1\1SRef

0.0

0.5

1.0

1.5

2.0

5x10

Inte

ns

. [a

.u.]

0

1654.884

818.422

1280.6422601.513

1083.502 1937.027

1487.793

2232.228

1134.580

1518.778

1765.802

2716.443

D2\0_N2\1\1SRef

0

2

4

6

5x10

Inte

ns

. [a

.u.]

1000 1500 2000 2500 3000

m/z

1677.289

1486.137

942.716

2263.7061933.4791148.974

1798.393

D9\0_N9\1\1SRef

0

1

2

3

4

5x10

Inte

ns

. [a

.u.]

1000 1500 2000 2500 3000

m/z

Spot 1

Spot 2

Spot 9

f

Figure 2.7(b) Mass spectrums of protein spots 3, 4 and 5 generated by MALDI

TOF/TOF MS.

1654.891

1409.711

818.4722601.390

1937.030 2232.197

1083.5601518.806

1144.633

1179.628

2716.333

D3\0_N3\1\1SRef

0

1

2

3

4

5

5x10

Inte

ns

. [a

.u.]

1500.877

1295.712

1087.605

2028.171

2211.205 2435.3921707.861842.502 3030.6332746.628

D4\0_M4\1\1SRef

0

1

2

3

4

5x10

Inte

ns

. [a

.u.]

1592.983

1757.176

1481.887

1903.202914.632 2211.3351277.848 2676.629

D5\0_N5\1\1SRef

0

1

2

3

4

5

5x10

Inte

ns

. [a

.u.]

1000 1500 2000 2500 3000

m/z

Spot 3

Spot 4

Spot 5

g

Figure 2.7 (c) Mass spectrum of protein spot 6, 7 and 8 generated by MALDI

TOF/TOF MS.

1189.758

1929.170

1460.001

2856.815

806.564 2211.389

1675.099

2515.766 2989.969

1717.0701032.681

980.626

D6\0_N6\1\1SRef

0.00

0.25

0.50

0.75

1.00

1.25

5x10

Inte

ns

. [a

.u.]

1365.895

1473.948

1480.001

1906.2521695.065

809.514 2211.4601165.726 2468.563

2807.753

D7\0_N7\1\1SRef

0

1

2

3

4

5x10

Inte

ns

. [a

.u.]

1473.966

2468.713

1798.247

1285.7631068.725804.473 2149.367 2723.757

D8\0_N8\1\1SRef

0

2

4

6

8

5x10

Inte

ns

. [a

.u.]

1000 1500 2000 2500 3000

m/z

Spot 6

Spot 7

Spot 8

72

Table 2.4 (a) Protein spots common to agar-surface associated and biofilm form but

absent in planktonic form

No. of Spot Planktonic AS Biofilm

23 - ++ ++

20 - ++ ++

19 - ++ ++

Table 2.4 (b) Protein spots upregulated in agar-surface associated and biofilm form

No. of Spot Planktonic Agar-surface

associated

Biofilm

1 + ++ ++

2 + ++ ++

3 + ++ ++

4 + ++ ++

6 + ++ ++

9 + ++ ++

12 + ++ ++

21 + ++ ++

22 + ++ ++

25 + ++ ++

Table 2.4 (c) Protein spots upregulated in agar-surface associated form

No. of Spot Planktonic Agar-surface

associated

Biofilm

11 + ++ +

18 + ++ +

26 + ++ +

27 + ++ +

24 + ++ +

73

Table 2.4 (d) Protein spots present in all three growth forms

No. of Spot Planktonic Agar-surface

associated

Biofilm

13 ++ ++ ++

14 ++ ++ ++

16 ++ ++ ++

28 ++ ++ ++

29 ++ ++ ++

30 ++ ++ ++

31 ++ ++ ++

Table 2.4 (e) Protein spot absent in agar-surface associated form

No. of Spot Planktonic Agar-surface

associated

Biofilm

17 ++ - ++

Table 2.4 (f) Protein spots upregulated in biofilm but not in AS and planktonic form

No. of Spot Planktonic Agar-surface

associated

Biofilm

5 + + ++

7 + + ++

8 + + ++

10 + + ++

Table 2.4 (g) Protein spot upregulated in planktonic and AS form

No. of Spot Planktonic Agar-surface

associated

Biofilm

15 ++ ++ -

74

Table 2.5 Protein expression in C. sakazakii (MTCC-2958) grown as planktonic, AS

and biofilm forms

S.No Presence/absence/upregulation

/downregulation of the protein

spots

Number of

spots

% of total spots

(n=31)

1 Proteins present in all the three

growth forms

7 0.22

2 Proteins present in AS and biofilm

cells absent in planktonic

3 0.09

3 Protein present in planktonic and

biofilm cells absent in AS form

1 0.03

4 Proteins upregulated (in AS and

biofilm) two-fold in a specific growth

form above the levels of the

corresponding spot in other forms

10 0.32

5 Proteins upregulated (in AS) two-fold

in a specific growth form above the

levels of the corresponding spot in

other forms

5 0.16

6 Proteins upregulated in biofilm form 4 0.12

7 Protein upregulated in planktonic and

AS, but absent in biofilm form

1 0.03

75

Table 2.6 Details of proteins identified by mass spectrometry

Protein

spot

no.

Protein

identified

MASCOT

Score

Nominal

mass

(kDa)

pI Sequence

coverage

Accession

number

Function

1 Enolase 233 45.6 5.32 63% ENO_ECO24 It catalyses the conversion of 2-

phosphoglycerate into

phosphoenoylpyruvate. It helps in

carbohydrate catabolism via

glycolysis.

2 Outer membrane

protein A

124 37.1 5.99 38% OMPA_ECO

57

It is required for the action of colicins

K and L and for stabilization of mating

aggregated in conjugation. It serves as

receptor for T-even like phages. Also

acts as a porins with low permeability

3 Outer membrane

protein A

122 37.1 5.99 40% OMPA_ECO

57

It is required for the action of colicins

K and L and for stabilization of mating

aggregated in conjugation. It serves as

receptor for T-even like phages. It also

acts as a porins with low permeability

4 Triosephosphate

isomerase

85 26.8 5.77 17%

TPIS_ENTCL

It catalyses the reversible reaction of

dihydroxyacetone phosphate and D-

glyceraldehyde 3-phosphate.

76

5

Cystine-binding

periplasmic

protein

209 29.0 6.21 64%

FLIY_ECOL6

It is a part of binding-protein-

dependent transport system for

cystine.

6 Enolase 233 45.6 5.32 63% ENO_ECO24 It catalyses the conversion of 2-

phosphoglycerate into

phosphoenoylpyruvate. It helps in

carbohydrate catabolism via

glycolysis.

7 Shikimate kinase 72 20.6 5.85 30% AROK_BAC

FN

It catalyzes the specific

phosphorylation of the 3-hydroxly

group of shikimic acid using ATP as a

cosubstrate

8 Outer membrane

protein X

82 18.5 6.56 42%

OMPX_ECO

5

It belongs to a family of highly

conserved bacterial proteins which are

involved in virulence.

9 Cystine-binding

periplasmic

protein

209 29.0 6.21 64%

FLIY_ECOL6

It is a part of binding-protein-

dependent transport system for cystine

77

2.1.4 DISCUSSION

Conventionally bacteria have been cultured and studied as free living

‘planktonic’ populations. However, bacteria in natural environmental conditions are

commonly found as community of cells organized in biofilms (Davey and O’Toole,

2000). Various strategies have been applied to study the genome and proteome of

planktonic and biofilm cells.

In the present study, the proteomic approach was used to compare the

planktonic, agar-surface associated and biofilm cells of C. sakazakii. To our

knowledge, this is the first study reporting on the differences in the proteins of

planktonic, agar-surface associated and biofilm cells of C. sakazakii. It was

investigated that the three growth forms exhibit differentially and/or uniquely-

expressed proteins.

One-dimensional SDS-PAGE profile of whole cell protein of C. sakazakii

isolates grown as planktonic, agar-surface associated and biofilm revealed specific

phenotypes. Variation in molecular masses of proteins was observed between the WCP

profiles of planktonic, AS and biofilm cells (Tables 2.1 a, b). Several proteins unique

to each of the planktonic, agar-surface associated and biofilm growth forms were

observed for C. sakazakii isolates and MTTCC-2958 (Table 2.1 a, b). In their work on

B. cereus, Vilian and Brozel (2006) have also found that majority of the protein spots

appeared to be uniquely expressed.

Differences between the proteome of free living and sessile bacteria using high

resolving power of two-dimensional electrophoresis have been reported earlier (Sauer

et al., 2002). In the present study this technique has been significantly contributed in

differentiating the WCP expressions between C. sakazakii grown as planktonic, AS and

biofilm cells. Two-dimensional electrophoregrams of WCP of C. sakazakii revealed a

reproducible separation of 31 distinct proteins spots in the pH range of 4 to 7 after

Coomassie brilliant blue staining (Figure 2.5 a, b and c). Ten proteins were selected for

identification, based on the criteria of varied reproducibility in their concentration as a

result of changes in the three growth conditions (planktonic, agar-surface associated

and biofilm cells). Out of a total of 31 spots, nine proteins were characterized by MS

analysis and according to their function (Table 2.6). They are classified as follows:

78

1. Outer membrane proteins (spot no. 2 and 3 and 8)

2. Proteins involved in glycolysis ( spot no. 1, 4 and 6)

3. Protein involved in shikimate pathway (spot no. 7)

4. Periplasmic proteins ( spot no. 5 and 9)

“These categories are related to what has been reported earlier for differentially

expressed proteins in biofilm proteome of other bacteria” (Orme et al., 2006; Hefford

et al., 2005).

WCP spots identified in the 2-D maps of MTCC-2958 were homologous to

several proteins from E. coli (Table 2.6). Spot No 3 (TPIS_ENTCL) was homologous

to protein of E. cloacae.

The whole proteomes of MTCC-2958 were not characterized in the 2-D WCP

electrophoregrams of each growth phase, since proteins falling outside the selected pI

(pH 4-7) and molecular weights (15.2 - 19.4 kDa) will not be detected. Nevertheless,

significant differential protein expression was observed in the selected range of pI and

molecular weights (Figure 2.6 a, b and c).

Spot 1 and spot 6 was recognized as enolase. It is a glycolytic enzyme, which

catalyses the conversion of 2-phosphoglycerate to phosphoenolypyruvate. It is also

known as phosphopyruvate hydratase. Depending on the environmental conditions of

the substrate, it can even catalyze a reverse reaction.

Spot 4 was recognized as “triosephosphate isomerase which catalyses the

reversible reaction of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate”

(Li et al., 2001). The enzyme also plays a role in glycolysis and necessary in energy

production. In E. coli the need for ATP controls the glycolytic flux through the cell (Li

et al., 2001). The increased expressions of glycolytic enzymes (enolase and triose

phosphate isomerase) were observed in the agar-surface associated and biofilm cells

(Figure 2.6 b and 2.6 c). Similar results were observed by Hefford et al., (2005) in

which significant upregulation of enolase and triosephisphate isomerase in biofilm

cells was observed.

The increased expression in agar-surface associated cells and biofilm cells

might be due to the enhanced activity in ATP-consuming biosynthetic reactions. The

decline in the level of intracellular ATP could be expected to increase the production of

79

glycolytic enzymes (enolase and triose phosphate isomerase). It has been well known

that oxygen is deprived to the cells embedded deeper within the biofilms (Xu et al.,

1998). Thus an increased expression of glycolytic enzymes may be in response to

limited amount of oxygen in the cells within the biofilms. Apart from glycolysis,

enolase enzyme when present at the cell surface and binds plasmin, fibronectin and

plasminogen which helps the bacteria in creating an inflammatory response (Pancholi

and Fischetti 1998). The increased expression of this enzyme in agar-surface associated

cells and biofilm cells could be involved in aiding the adhesion process by C. sakazakii

grown as agar-surface associated and biofilm cells.

Spot 2 and spot 3 was identified as Omp A outer membrane protein. It is the

major protein of members belonging to the family of Enterobacteriaceae and this

protein is conserved throughout evolution (Beher et al., 1980). Omp A protein carries

out various functions - helps in structural integrity of the outer membranes (Sonntag et

al., 1978); acts as a target for immune response (Godefroy et al., 2003) and helps in

bacterial conjugation (Ried G and Henning, 1987). It also acts as a receptor for

bacteriophages (Morona et al., 1985). At physiological temperatures Omp A forms a

pore of the size of 1 nm in diameter (Arora et al., 2000). The upregulation of this

protein was observed in AS and biofilm cells (Figure 2.6 b and 2.6 c). “The over-

expression of this protein in biofilm cells is in accordance with the findings of” Orme

et al., (2006) where proteomic study together with immunoassays have shown that

Omp A of E. coli is up-regulated during biofilm formation. Ma and Wook (2009)

reported that E. coli biofilm formation was influenced by OmpA by inhibiting the

cellulose production through the CpxRA stress response system. This protein may

assist in the transfer of polymeric substances which is required for the construction of

EPS formed during biofilm formation. It has also been reported that horizontal gene

transfers occur in biofilms. Transfer of resistance genes is known to occur from

commensals to pathogenic strains (Molin et al., 2003). The up-regulated OmpA protein

in biofilm may assist in DNA exchange. This protein up-regulated in the agar-surface

associated and biofilm may be a related to virulence of C. sakazakii to the host. These

findings suggest that OmpA can be a likely target for biofilm inhibition and may aid in

the designing of biofilm inhibiting strategies.

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Spot 7 was recognized as shikimate kinase. This enzyme catalyses the

conversion of shikimate to shikimate 3 phosphate which is the fifth step of shikimate

pathway. It helps in the biosynthesis of precursors of secondary metabolites and

aromatic amino acids. In E. coli it has been reported (Ely and Pittard, 1979) that the

starvation of aromatic amino acids (tyrosin and tryptophan) results in an enhanced rate

of synthesis of shikimate kinase. In our study, the protein (spot no.7) up regulated in

biofilm cells may be involved in the synthesis of tyrosine and tryptophan (Figure 2.6 a,

b and c). Due to the absence of shikimic pathway in mammalian species, the enzyme

shikimate kinase may be used as a target for generating antimicrobials against C.

sakazakii.

Spot 5 and 9 were recognized as cysteine binding periplasmic proteins (Fli

Y_ECOL6). Fli Y helps in the transport of amino acids and/ or in the regulation of

class III transcription.

In E. coli, the electron transport chain is thought to produce reactive oxygen

species (ROS) such as H2O2 (Imlay, 2003). Two catalases (Kat E and Kat G) and a

peroxidase (AhpCF) help in the elimination of H2O2. However, such enzymes are

present in the cytoplasm and not in the periplasm. FliY, a periplasmic binding protein

which is a component of l-cysteine/l-cystine shuttle system is present in the inner

membrane of E. coli which helps in H2O2 scavenging (Ohtsu et al., 2010). It has been

reported that the transcription of genes which code for FliY is induced by the presence

of hydrogen per oxide (Ohtsu et al., 2010). E. coli cells are exposed to H2O2, which is

produced during phagocytic attack. Therefore, if the cells might detoxify H2O2 in the

periplasmic space before its diffusion into cytoplasm, its toxicity would be diminished.

Upregulation of cysteine binding periplasmic protein (spot 9, Figure 2.6 a, b and c) was

observed in agar-surface associated and biofilm cells of C. sakazakii proves that both

the forms might be more tolerant to phagocytic attack as compared to their planktonic

counterpart. Hall-Stoodley and Paul Stoodley (2009) “reported that bacteria in biofilms

are more resistant to host defense mechanisms”.

Spot 8 was identified as Omp X. “It has been reported that it belongs to a

family of highly conserved bacterial proteins which are involved in virulence” Vogt

and Schulz (1999). “It also plays a role in the defense against attack by the human

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complement system” Vogt and Schulz (1999). Vogt and Schulz (1999) “reported that

the membrane spanning part of this protein is highly conserved in comparison to the

extracellular loops”. “These extracellular loops form a protruding β-sheet, which helps

in binding to external proteins, this binding helps in adhesion and invasion” Vogt and

Schulz (1999). However, Maisnier-Patin et al., (2003) reported that “OmpX protein is

recognized by innate cells but does not activate them, suggesting that OmpX does not

provide a danger signal to APCs” (Antigen-Presenting cells). In our study, we observed

a down regulation of Omp X protein [spot 8, Figure 2.6 (a) and (b)] in planktonic and

AS forms. Upregulation of and/or expression of omp X in biofilm cells (Figure 2.6 c)

may be related to virulence and host invasion of C. sakazakii.

In the present work, a common “proteome with differences in expression levels,

in addition to expression of unique proteins was observed for planktonic, agar surface-

associated and biofilm cells of C. sakazakii" (Sharma and Prakash, 2013a). The

function of putative biofilm proteins was found to be more similar to those of the agar-

surface associated form of MTCC-2959. In the present study, it was shown that

MTCC-2958 biofilm cells proteome differ from proteome of planktonic cells.

Gaining knowledge about the biofilm proteome of C. sakazakii will help to

design strategies that cause its degradation, and will thus help to prevent the

contamination of food by biofilm formation on food contact surfaces by the above

bacteria.