Chapter III

CHAPTER III

MATERIAL AND METHODS

1. Pathogens:
Pathogenic isolates of Aspergillus fumigatus [ITCC4517 (IARI, Indian
Agricultural Research Institute, Delhi), ITCC1634 (IARI, Delhi), 190/96 (VPCI,
Vallabhbhai Patel Chest Institute, Delhi)], Aspergillus flavus [ITCC5192 (IARI,
Delhi), ITCC5076 (IARI, Delhi), 223/96 (VPCI, Delhi)] and Aspergillus niger
[ITCC5405 (IARI, Delhi), 56/96 (VPCI, Delhi)] were employed in the current study.
These pathogenic species of Aspergillus were cultured in laboratory on Sabouraud
dextrose agar plates. The plates were inoculated with stock cultures of A. fumigatus,
A. flavus and A. niger incubated at 370C for 96- hrs. These cultures were used as the
source of spores required for performing experiments.

1.1 Aspergillus spore suspension:

The Aspergillus species were grown on Sabouraud dextrose agar plates at
370C for 96-hrs and their homogenous spore suspension was prepared in the
Sabouraud dextrose broth. Spores from the fungal colonies of 96-hrs cultures in the
plates were picked up by an applicator and transferred to sterile physiological saline
in a tube or harvested by adding 1.0 ml of sterile physiological saline directly to the
plates and recovering back the spore suspension into screw cap tubes. The tubes were
vortexed gently for 3 to 4 min at room temperature to prepare a homogeneous
suspension of the spores. The spores were counted by using haemocytometer and the
number was adjusted to 1x108 spores/ml.

2. Bacterial Strains:
Lactococcus lactis (MTCC 440), Escherichia coli DH5 (MTCC 1652),
Escherichia coli (MTCC 1674), Streptococcus thermophilus (MTCC 1938),
Staphylococcus epidermidis (MTCC 3615), Staphylococcus epidermidis (MTCC
6810), Streptococcus subsp.equi (MTCC 3522), Bacillus amyloliquefaciens (MTCC
2248), Bacillus pumilus (MTCC 2299), Bacillus subtilis (MTCC 1133), Bacillus
licheniformis (MTCC 1483), Streptomyces hygroscopicus (MTCC 4003),
Pseudomonas fluorescens (MTCC 1748) were purchased from Institute of Microbial
Technology, Chandigarh, India. These strains were cultured in Luria Bertini (LB)
broth for 3 days at 370C in an incubator shaker.
2.1 Preparation of bacterial supernatant (Secretory Protein) and lysate
(Cytosolic Protein):
The 72-hrs cultures of all above mentioned bacterial strains were centrifuged
at 3256 g for 30 min. The supernatant was collected in a flask for testing the
antifungal activity and pellet was washed with phosphate buffer saline (PBS) thrice
and suspended in sonication buffer (50 mmol l-1 TrisHCl, 50 mmol l-1 EDTA,
5mmol l-1 DTT, 1 mmol l-1 PMSF). The cell suspension was sonicated at 20 s bursts at
200 W and 10 s cool period using a sonicator (Bio-Age, Sonicator Mohali, India). The
sonicate was centrifuged at 16350 g for 30 min using Sigma refrigerated centrifuge
(Osterode, Germany). The supernatant was collected and used as lysate. The lysate
was dialysed against distilled water at 40C for 24-hrs with several changes of water
and lyophilized. Protein concentration of bacterial supernatant and lysate was
determined by Bradford method.

2.2. Protein estimation by Bradford Method:

The Bradford protein estimation assay described by Bradford (1976) was used
to estimate the concentrations of proteins in bacterial supernatant and lysates. Biorad
Bradford reagent (1X) was used as the working solution. The Bradford reagent and
diluted protein samples were mixed in a ratio of 10: 1 and incubated at 37 0C
temperature for 20 min. The optical density (OD) was recorded at 595nm. The BSA
was used as standard protein in the assay. The concentration of protein in different
samples was estimated with the help of standard graph plotted.

3. Evaluation of bacterial products for antifungal activity:

The antifungal activity of bacterial secretory and cytosolic proteins against
various Aspergillus species (A. fumigatus, A. flavus, A. niger) was analysed by
microbroth dilution assay as described by Chhillar et al.2006. Each experiment was
repeated at least three times and performed in duplicates.
3.1 Microbroth dilution assay:
The test was performed in 96-well culture plates (Tarson, Kolkata India).
Autoclaved Sabouraud dextrose broth (90 l) was added to the wells of culture plates.
Various concentrations of bacterial products in the range of 1000.03.9 g/ ml) were
prepared in the wells by twofold dilution method and these wells were inoculated with
10 l of spore suspension containing 1 x 108 spores/ml. The plates were incubated at
370C and examined macroscopically after 48 h for the growth of Aspergillus mycelia.
Appropriate control wells treated with amphotericin B or without any treatment were
included in the experiment. A protein was considered to be active if the wells
appeared clear without any visible growth of Aspergillus and the results were
expressed as minimum inhibitory concentration (MIC).
The results of preliminary experiments with microbroth dilution assay
indicated that bacterial strains showed mild to moderate activity. The cytotoxicity of
the most active antifungal bacterial fractions (Bacillus amyloliquefaciens,
Pseudomonas fluorescens, Streptomyces hygroscopicus, Bacillus licheniformis, E.coli
DH5) was determined using a haemolytic assay.

4. Haemolytic Assay:
The basic method of Latoud et al. (1986) with slight modifications was
employed to determine the haemolytic effect of bacterial proteins with antifungal
activity. Human blood samples, collected from apparently healthy individuals, were
washed three times with PBS by centrifugation at 1500 r.p.m. for 10 min. A 2%
erythrocyte suspension was incubated at 370C for 1-hr with different concentrations of
lysate ranging from 1000.00g/ml to 15.6g protein/ ml. After incubation, cells were
pelleted at 5000 r.p.m. for 10 min.The supernatant was collected and the absorbance
at 450 nm was determined using a spectrophotometer (UV Vis Spect 2450, Shimadzu,
Japan). In negative control sets, only buffer was used for background lysis, whereas in
positive controls, lysis buffer was used for completely lysing the erythrocytes. For
each sample the percentage of maximum haemolytic activity was determined.
Initial experiments demonstrated that the lysate of E. coli DH5 was better than all
other bacterial fractions with respect to anti-Aspergillus properties as well as
cytotoxicity. Therefore we chose to focus only on E. coli DH5 lysate with an aim to
isolate biologically active molecule(s) having possible antimycotic potential.
5. Anti Aspergillus activity of E.coli DH5 lysate:
The antifungal activity of lysate of E. coli DH5 was determined against
various species of Aspergillus (A. fumigatus, A. flavus and A. niger) using various
antifungal susceptibility assays.

5.1 Microbroth Dilution Assay:

The anti-Aspergillus activity of E. coli lysate was determined by
microbroth dilution assay. The antifungal assay was performed as described under
section 3.1.
5.2 Disc diffusion assay:
The disc diffusion test was performed in radiation sterilized Petri plates of
10.0 cm diameter (Tarsons, Kolkata, India). Different concentrations ranging from
50.00 to 3.20 g of proteins/disc of the lysate of E. coli DH5 were impregnated on
the sterilized discs (5.0 mm in diameter) of Whatman filter paper No. 1. These discs
were placed on the surface of the agar plates, which were already inoculated with
Aspergillus spores (1x108 spores/ml). The plates were incubated at 370C and
examined after 48- hrs for zone of inhibition, if any, around the discs. The diameter of
zone of inhibition was recorded. The concentration, which developed the zone of
inhibition of at least 6.0 mm diameter, was considered as MIC. Ampho.B was used in
assay as a standard control drug. An additional control disc without any sample but
impregnated with equivalent amount of solvent was also used in the assay.
5.3 Percentage Spore germination inhibition assay:
The Aspergillus species were grown on Sabouraud dextrose agar plates and
their homogenous spore suspension was prepared in the Sabouraud dextrose broth.
Various concentrations (10003.90 g/ ml) of the lysates of E. coli DH5 in 90 l of
culture medium were prepared in 96-well flat bottom microculture plates (Tarsons,
Kolkata, India) by double dilution method. The wells were prepared in duplicates for
each concentration.The wells were inoculated with 10l of spore suspension
containing 100 5 spores. Appropriate control wells treated with Ampho.B or
without any treatment were included in the study. The plates were incubated at 37 0C
for 16-hrs and then examined for spore germination under compound microscope
(Labomed, USA). The number of germinated and nongerminated spores was counted
and the percent spore germination inhibition (PSGI) was calculated using following
formula:
PSGI = (100- No. of spores germinated in drug treated well
/ No. of spores germinated in control well) X100
The activity of the preparations was represented as the MIC90 which inhibited the
germination of spores in the range of 90100%.
As the E.coli DH5 lysate show good antifungal potential as well as ground level
cytotoxicity, further work on purification and characterization for identification of
antifungal molecule was carried out with E.coli DH5 lysate only.

6. Purification of active molecule from E. coli DH5 Lysate:

The lysate of E coli DH5 which showed activity against various Aspergillus
spp. was fractionated to identify and isolate the active protein molecule. Various
chromatographic methods i.e. anion exchange, cation exchange and ultra performance
liquid chromatography were employed for this purpose. The tests for antifungal
susceptibility were performed after every step of purification as described in section 5.
6.1 Anion exchange chromatography:
Akta Purifier 10 (GE Healthcare Life Sciences) equipped with a triple
wavelength UV monitor that allows detection of a wide range of proteins along with
the optional Monitor pH/C-900 that allows temperature, pH and conductivity
measurement in the flow path was used. UNICORN control software was used to
generate high-quality purification results based on its flexible and intuitive method
concept supported by advanced tools for result assessment.
6.1.1 Column:
HiPrep 16/10 Q XL prepacked, ready to use columns for anion exchange
chromatography was used. The column matrix was made up of 6% highly cross-
linked spherical agarose having a mean particle size of 90 m, bed volume of 20 ml,
bed height of 100 mm,i.d.of 16 mm and composed of medical grade polypropylene
with M6 Connectors, having a flow rate range of 110 ml/min (60300 cm/h) with
maximum flow rate 10 ml/min (300 cm/h). The charged group which acted as a strong
anion exchanger was N+(CH3)3. The column was equilibrated with 10 mM Tris-HCl,
pH 7.4 previously filtered through a membrane of 0.22 m pore size (Millipore).
6.1.2 Sample loading:
An amount of 20.00 mg protein of E. coli DH 5 lysate was dissolved in 10
mM TrisHCl, pH 7.4 and injected into the HiPrep 16/10 Q XL column of Akta
purifier (GE Healthcare), pre-equilibrated with the same buffer. The protein solution
was filtered through 0.22 m filter (Millipore) before injecting. Precautions were
taken to avoid entrapment of air bubbles in the protein solution.

6.1.3 Elution:
After the sample had been injected onto the column of Akta purifier (GE
Healthcare), it was left for 2-hrs to bind the protein to the column. The bound proteins
were eluted using salt gradient 0.0 to 1 M NaCl as elution buffer. The column was run
at a flow rate of 1.2 ml/min & fraction volume was set as 5ml. Total 52 fractions of
5.0 ml each were collected in glass tubes which were transferred to an ice bucket. The
OD of the fractions was measured using Spectrophotometer (UV Vis 2450,
Shimadzu). The fractions were pooled and concentration of protein recovered in
different peaks was quantitated by the Bradford method. The contents of different
peaks were named as F I, II, III, etc. All the ion exchange chromatographic fractions
were analyzed for antifungal efficacy and the active fraction was used to study the
protein profile by SDS-PAGE and further sub fractionated to purify the active
component.
6.2 Cation Exchange Chromatography:
6.2.1 Column:
HiPrep 16/10 SP XL prepacked, ready to use columns for cation exchange
chromatography was used. The column matrix was made up of 6% highly cross-
linked spherical agarose having a mean particle size of 90 m, bed volume of 20 ml,
bed height of 100 mm, i.d.of 16 mm and composed of medical grade polypropylene
with M6 Connectors, having a flow rate range of 110 ml/min (60300 cm/h) with
maximum flow rate 10 ml/min (300 cm/h).The charged group which acted as a strong
cation exchanger was SO3-. The column was equilibrated with 10 mM Tris-HCl, pH
7.4 previously filtered through a membrane of 0.45 m pore size (Millipore).

6.2.2 Sample Loading:

The concentrated anion exchange chromatographic active fraction (3.0 mg of
protein) dissolved in 10 mM Tris-HCl, pH 8.4 was applied onto the pre-equilibrated
column. The protein solution was filtered through 0.22 m filter (Millipore) before
injecting. Precautions were taken to avoid entrapment of air bubbles in the protein
solution.
6.2.3 Elution:
After the sample had been injected into the loop of the column of Akta purifier
(GE Healthcare), it was left for 2 hours to bind the protein to the column. The bound
proteins were eluted using salt gradient 0.0 to 1 M NaCl as elution buffer. The column
was run at a flow rate of 1.2 ml/min & fraction volume was set as 5ml. Total 46
fractions of 5.0 ml each were collected in glass tubes which were transferred to an ice
bucket. The OD of the fractions was measured using Spectrophotometer (UV Vis
2450, Shimadzu). The concentration of protein recovered in different peaks was
quantitated by the Bradford method. The contents of different peaks were named as
SF I, II, III, etc. All the cation exchange chromatographic fractions were analyzed for
antifungal efficacy and the active fraction was used to study the protein profile by
SDS-PAGE and further sub fractionated to purify the active component. The active
sub fraction was further subjected to ultra performance liquid chromatography for
identifying and separating out pure active molecule.

6.3 Ultra performance liquid chromatography (UPLC):

UPLC was performed using ACQUITY UPLC system (Central Facility, Delhi
University) fitted with two LC 10AD pumps and a highly sensitive SPD 10A detector
having variable wavelengths options. The system was connected to a monitor
(Empower 2 software) which could analyze the data to give all the necessary
information such as retention time, relative concentrations of components, peak height
and their area. The reverse phase column C4 (BEH C4100mm Waters) was used to
check the purity and resolve the components of active cation exchange
chromatographic sub fractions. All the chromatographic solvents were of UPLC
grades and purchased from WATERS Technology. These solvents were filtered
through 0.22 m filters (Millipore) and degassed. The mobile phase used for
separation of sample components was acetonotrile: 0.1% formic acid in a ratio of
70:30.
6.3.1. Sample loading and elution:
The column was washed with acetonitrile and water sequentially. After proper
washing with acetonitrile, the column was equilibrated with acetonitrile: 0.1% formic
acid (70:30). The equilibration was continued until the baseline of the column was
stabilized properly. After proper equilibration, 5.0 l of sample which was dissolved
in the mobile phase solvent and filtered through 0.22 m filter, was injected into the
loop of column with the help of UPLC loading syringe. The detector was set to 220
nm to read the absorbance. The flow rate of the column was adjusted to 0.5 ml/min
and eluted protein component(s) were collected. The UPLC chromatogram was
obtained by printing the data using EMPOWER 2 software. The UPLC purified active
sub fractions containing purified protein of E. coli DH5 was stored at 700 C.

7. Characterization of purified active protein molecule:

The antifungal molecule isolated from E.coli DH5 was named as AAP (Anti
Aspergillus Protein) and was subjected to biochemical, biophysical and biological
characterization.
7.1.1 SDS-PAGE:
SDS-PAGE was carried out by using the basic method of Laemmli and Favre
(1973). A 12.5 % polyacrylamide gel containing 0.1 % SDS was prepared using
Biorad gel casting apparatus. The proteins for analysis were taken into a reducing dye,
1M Tris HCI buffer (pH 6.8) containing 2 % SDS, 10 % glycerol, 4 % -
mercaptoethanol and 0.01 % bromophenol blue. The samples were boiled for 10 min
before loading in the wells. An amount of 10.0 g of protein in 30 l of samples was
loaded into the wells using Hamilton syringe. Molecular weight markers in the range
of 17 kDa to 130 kDa were applied in one of the wells for simultaneous
electrophoresis. The proteins were electrophoresed under an electric current at 100 V.
The gels were removed and the separated protein band(s) were visualized by staining
the gels with silver stain.
7.1.2 Silver Staining:
The silver staining was carried out as described by Blum et al. (1987). The gel
was removed from the chamber and transferred to the fixative solution (50 %
methanol and 7 % acetic acid in distilled water) for 2 h. The gel was soaked in Hypo
solution (20 mg of sodium thiosulfate in 100 ml of distilled water) for 1 min and
rinsed with distilled water three times for 1 min each. It was then transferred to
staining solution (100 mg of Ag2NO3 in 100 ml of distilled water and 75 l of
formaldehyde) and stained for 20 min. After proper washing with distilled water, the
gels were developed in a solution of 100.0 ml of water containing 2.0 gm of Na2CO3
and 50 l of formaldehyde. After the development, the reaction was stopped by 0.1 %
citric acid.
7.2 Peptide mass fingerprinting:
The peptide mass fingerprinting of purified protein was carried out by
MALDI-TOF MS.

7.2.1 MALDI-TOF MS:

In-gel protein digests were prepared as described previously (Gao et al.1994).
The silver-stained purified band from SDS-PAGE was excised from the gel and sliced
into about 1.0 mm in thickness. After washing with distilled water, the gel slices were
incubated for 20 min in wash solution (50 % acetonitrile in 50 mM NH4HCO3) to
remove the staining dye. Gel pieces were treated with 150 l of 10 mM DTT in 100
mM NH4HCO3 for 10 min and incubated in 5 % acetonitrile for 1 h at 550C. The gel
pieces were dehydrated in 100 l of 100 mM NH4HCO3 and 100 % of acetonitrile for
10 and 20 min respectively. For alkylation, 100 l of 50 mM iodoacetamide in 100
mM NH4HCO3 was added to the gel pieces and incubated in dark at room temperature
for 30 min. The gel pieces were washed with 100 l of 100 mM NH4HCO3 for 10 min
and then dried in a vacuum centrifuge for 15 min. Subsequently, 20 l of trypsin
solution (10ng/ml in 50 mM NH4HCO3) was added to tubes having gel pieces and
incubated at 370C for 16-hrs. After digestion, peptides were extracted in buffer (5%
trifluoroacetic acid in 60% acetonitrile) and concentrated by Speed-Vac centrifugation
for 3-hrs. The lyophoilized peptides were re-solubilized in a re-suspension solution
(1% trifluoroacetic acid in 50% acetonitrile) and then centrifuged for 2 min to obtain
the supernatant. The peptide solution was mixed with matrix in the ratio of 1:1 and
subjected to MALDI TOF MS analysis.
7.2.2 Mascot search:
The peptide sequences along with their masses of purified protein acquired
from MALDI-TOF were analyzed by the MASCOT search of Matrix Science to find
out the peptide matches present in MSDB protein database.
8. Biochemical Analysis of Purified Protein AAP:
8.1. Stability of AAP:
The stability of protein at temperatures ranging from 4 to 1000C was
determined by incubating AAP in 10 mM Tris-HCl buffer (pH 7.5) for 20 min at
different temperatures. The solutions of protein was cooled to 40C and examined for
anti-Aspergillus activities. The stability of AAP under acidic and alkaline conditions
was tested by using citrate phosphate buffer (pH 2.5 to 8.0) and Tris-HCl buffer (pH
7.5 to 10.5). AAP was incubated in each buffer at various pHs at 40C for 20 min. The
anti-Aspergillus activity was assayed after the pH of each AAP solution was
readjusted to 7.5.

8.2 Cytotoxicity of AAP:

The cytotoxicity of AAP was evaluated by haemolytic assay as described
earlier under section 4.
8.3 In vitro time kill kinetics:
Sabourd dextrose agar plates were inoculated with stock cultures of A.
fumigatus and incubated at 370C for 96-hrs. The freshly revived fungal plates were
used for preparing spore suspension.The homogenous spore suspension was made &
diluted with 0.9% NaCl to get 1x 108 spores/ml. Aliquots with 5 ml of RPMI 1640 as
culture medium were inoculated with spore suspension of 1x 108 spores/ml. Out of
these one third were treated with protein concentration equal to 1/2 xMIC, one third
with MIC and remaining one third with 2x MIC. For each concentration
corresponding controls without any drug treatment were preared simultaneously. The
aliquots were incubated at 370C with shaking for up to 48-hrs. At defined time
intervals (6, 12, 24, 48-hrs) aliquots were removed and the number of viable colonies
per millilitre were determined on Sabouraud dextrose agar plates after serial dilution
in 0.9% NaCl. The results were expressed as log10 cfu/ml against time of incubation at
drug concentration tested. The protein concentration which causes an average
reduction of 3 log10 in colony forming units per ml as compared to control at 0-hrs
was considered as fungicidal (Manavanthu et al.2000).

8.4 Scanning Electron Microscopy:

Aspergillus fumigatus was cultured in 100ml L-asparagine broth using a spore
suspension of 1x108 spores/ml for 24 hrs in a BOD incubator shaker at 370C at 100
r.p.m. Half of the flasks were treated with MIC50 of E .coli DH5 purified protein
(AAP) to examine the effect on fungus morphology and spore germination while
remaining half were observed as control. Fungal mass recovered after culture was
used for SEM studies. It was fixed with 2.5% glutaraldehyde in 0.1 mol/l phosphate
buffer (pH 7.2) at 480C for 2- hrs. All specimens were washed four times with
phosphate buffer and postfixed with phosphate- buffered 2% osmium tetroxide at
480C for 4-hrs. After washing overnight with the same buffer; the specimens were
dehydrated in graded ethanol and finally freeze-dried in t-butyl alcohol. The
specimens were coated with gold film (approximately 10 nm). Samples were
examined using a Carl Zeiss EV040 Gemini field emission scanning electron
microscope with an acceleration voltage of 20 kV using the Everhart Thornley SE
detector and the in-lens SE detector in a 50:50 ratio at AIRF (JNU, New Delhi).