Titration and biochemical assay of leukotoxin of Fusobacterium

necrophorum isolated from bovine liver abscesses

Suha A. Hussein1 and Essam F. Al-Jumaily2

1
Dept. of microbiology, College of Veterinary Medicine, University of Sulaimani, Sulaimaniyah,
Kurdistan Region- Iraq (suhaali68@yahoo.com).
2
Biotechnology Dept., Genetic Engineering and Biotechnology Institute for post graduate
studies, Baghdad University, Baghdad- Iraq.

Abstract
This study was conducted with the aim of extraction, titration and studying the biochemical
properties of the leukotoxin of a Fusobacterium necrophorum subsp. necrophorum isolate
recovered from an abscessed liver obtained from slaughtered cattle in Sulaimaniyah region.
Leukotoxin-containing culture supernatant of F. necrophorum subspecies necrophorum was
extracted and then concentrated using an ultrafiltration cell and purified by gel filtration
chromatography. The 5-fold concentration raised the titer of the leukotoxin by a factor 2 and the
purification had created six peaks, however, the tetrazolium dye reduction test revealed
leukotoxin activity only in the third peak. The molecular weight of the purified leukotoxin was
approximately equal to 316 kilo Dalton using the gel filtration chromatography whereas the
sodium dodecyl sulfate polyacrylamide gel electrophoresis had revealed a single band which is
approximately equal to 48 kilo Dalton. The comparative analysis of leukotoxicity titers of the
crude, concentrated crude and purified leukotoxin revealed that the concentrated crude has the
highest leukotoxicity titer.

Key words: Fusobacterium necrophorum; leukotoxin, tetrazolium.

Intoduction
Fusobacterium necrophorum, a gram-negative, anaerobic bacterium and a normal inhabitant of
the rumen, is the primary etiologic agent of bovine liver abscesses [1- 2]. It is classified into two
subspecies, F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme [3- 4].
The pathogenicity of F. necrophorumis attributed mainly to its leukotoxin which seems to be an
important virulence factor in the pathogenesis of hepatic and interdigitalnecrobacillosis and it is
indicated to be cytotoxic to leukocytes, macrophages, ruminal epithelial cells, and possibly
hepatocytes [5- 6].
Although the pathogenicity and virulence factors of F. necrophorum have been studied
widely for many years, attempts to develop an effective vaccine against liver abscess have not
been successful commercially [7]. However, previous studies have indicated that antileukotoxin
immunity reduced the incidence of hepatic abscesses and interdigital necrobacillosis [5].
The aim of the present study was to extract, titrate and study the biochemical
characteristics of the leukotoxin of F. necrophorum subsp. necrophorum isolated locally from
liver abscesses of slaughtered cattle in Sulaimaniyah region.
Materials and Methods
The Fusobacterium necrophorum subspecies necrophorum isolate used in this study was
recovered from an abscessed liver obtained froma slaughtered cattle in the abattoir of

1
Sulaimaniyah governorate [8]. This isolate was used for the production and extraction of
leukotoxin according to [9] and [10] respectively.
The leukotoxin-containing culture supernatant was concentrated 5 folds using an
ultrafiltration cell (Amicon 8200, Australia) containing a PM-50 filter membrane with a cut-off
value of 10000 dalton.
Purification of the concentrated crude leukotoxin containing culture supernatant was done
by gel filtration chromatography according to [11], briefly, a total of 3 ml of the concentrated
crude leukotoxin-containing culture supernatant was purified in a Sepharose CL 6B column (60
x 2.1cm) which was prepared and packed according to the instructions of the manufacturing
company (Pharmacia, Sweden). The column was equilibrated with a 0.2 M phosphate buffer (pH
7.2) at a flow rate of 30 ml / hour. The protein content of the fractions was estimated by
measuring the absorbance value at 280 nm wavelength by a spectrophotometer. The major peaks
were determined by plotting the absorbance value of the protein fractions versus the elution
volumes. The leukotoxin activity was determined for the fractions of each major peak by the
tetrazolium (MTT) dye reduction assay [10].
Protein quantification of the crude, concentrated crude and purified leukotoxin of F.
necrophorum subspecies necrophorum was performed using the absolute method [12] and the
molecular weight of the purified leukotoxin was determined by gel filtration chromatography
[11], in which the void volume (Vo) of the Sepharose CL 6B (60 x 2.1 cm) column was
determined using the blue dextran 2000 solution while the leukotoxin elution volume (Ve) was
determined for the dissolved fraction of the purified leukotoxin by following the absorbance
value at 280 nm wavelength. Different standard proteins were used for standardization of the
leukotoxin molecular weight by gel filtration chromatography including the bovine serum
albumin (67 Kilo Dalton-KD), aldolase (158 KD), catalase (232 KD) and ferritin (440 KD). The
elution volume to the void volume ratio (Ve / Vo ratio) was calculated for each standard protein
and for the dissolved fraction of the purified leukotoxin.
The purity of the sampled leukotoxins (crude, concentrated crude and purified) and the
apparent masses of their variants as well as the molecular weight of the purified leukotoxin were
estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which
was performed according to Laemmli procedure described by [13]. Standard proteins used for
standardization of the leukotoxin molecular weight by SDS-PAGE were alkaline phosphatise (80
KD), bovine serum albumin (67 KD), catalase (60 KD) and ovalbumin (43 KD).
The leukotoxicity determination of the crude, concentrated and purified leukotoxin was
achieved by tetrazolium (MTT) dye reduction assay according to [10], briefly, the formazan
concentration representing PMN leukocyte viability was determined by measuring the
absorbance values in an ELISA reader with dual wavelength (570 nm as test wavelength and 650
nm as reference). The leukotoxicity, expressed in percentage of cell death, and calculated as
follows:
[ 1- (absorbance of toxin treated cells / absorbance of control cells) ] x 100
The titer of leukotoxin was calculated as the reciprocal of the higher culture supernatant
dilution causing 10% loss in viability of leukocytes.

Results
The absolute method revealed that the protein concentration within the crude, concentrated crude
and purified leukotoxin was equal to 9.5, 15.4 and 0.31 respectively (table-1) and the tetrazolium
dye reduction (leukotoxicity) assay displayed that the leukotoxin titer of the crude and

2
concentrated crude was equal to 1024, 2048 respectively. The gel filtration chromatography used
for the purification of the concentrated crude leukotoxin created six peaks (figure 1). However,
the tetrazolium dye reduction assay that was used to determine the leukotoxin activity of the
major peaks revealed that the leukotoxin activity was found only in the third peak which
represents the purified leukotoxin collected from five fractions (fractions 14 18). The
leukotoxin titer of these fractions was 128 for fractions 14 and 18, 256 for fractions 15 and 17,
and 1024 for fraction 16. These fractions were mixed together and their final leukotoxin titer was
recalculated using the tetrazolium dye reduction assay and was equal to 256.

Table 1: Protein concentration in the crude, concentrated crude and purified culture supernatant of F.
necrophorum subspecies necrophorum.

Protein concentration (mg / ml) * Culture supernatant

9.5 Crude culture supernatant
15.4 Concentrated crude culture supernatant
0.31 Purified leukotoxin
* The protein concentration was determined using the absolute method.

0.08 1200

0.07
1000
0.06
800
0.05

0.04 600
Absorbance at 280nm
Absorbance (280 nm) Titre of leukotoxin
Titre of Leukotoxin
0.03
400
0.02
200
0.01

0 0

Figure 1: Leukotoxin purification by gel-filtration chromatography (Sepharose CL 6B

column, 60 x 2.1 cm, 0.2 M phosphate buffer eluent, pH 7.2) at a flow rate of 30 ml / hour.

The results of the molecular weight of the purified leukotoxin (illustrated in figure 2 and
table 2) showed that the Ve / Vo ratio of the purified leukotoxin was equal to 1.313. Accordingly,
the molecular weight of the purified leukotoxin was estimated to be equal to 316 Kilo Dalton.

3
Bovine serum albumin 67 KD

Aldolase 158 KD

Catalase 232 KD

4
Leukotoxin 316 KD
 1.8
Figure 2: Standardization of the leukotoxin molecular weight based on the elution volume to the
1.7ratio (Ve / Vo ratio) by gel-filtration chromatography.
void volume

1.6
Table 2: Standardization of the leukotoxin molecular weight based on the elution volume / void
volume * ratio (Ve / Vo ratio).
1.5 *

(VVe : Vo ratio
e / Vo) ratio Molecular weight (Kilo Dalton) Standard protein
1.4
1.690 67 Bovine serum albumin

1.3
1.569 158 Aldolase

1.2
1.465 232 Catalase

1.1
1.224 440 Ferritin

1
1.313 316 Leukotoxin
4.6 4.8 5 5.2 5.4 5.6 5.8
The void volume of the column used in gel filtration chromatography.
Log Molecular Weight
The SDS-PAGE results (illustrated in figure 3) clearly showed a protein band representing
the purified leukotoxin, and by the aid of the standard curve (figure 4) it was possible to estimate
the molecular weight of the purified leukotoxin which was approximately equal to 48 KD.

1 2 3 4 SP

Crude leukotoxin 1:
Concentrated crude leukotoxin 2:
Purified leukotoxin 3 and 4:
Standard proteins SP:

5
 Alkaline phosphatase (80 KD)
Bovine serum albumin (67 KD)

Catalase (60 KD)

Ovalbumin (43 KD)

Figure 3: SDS-polyacrylamide gel electrophoresis of the leukotoxin obtained from F. necrophorum subsp.

necrophorum.

6
 Alkaline phosphotase 80 KD albumin 67 KD
Bovine serum

Ovalbumin
43 KD
4.95

Catalase 60 KD
4.9
Log Molecular weight

4.85

Leukotoxin 48 KD
4.8

4.75

4.7

4.65
0 0.1 0.2 0.3 0.4 0.5 0.6
Relative mobility
Figure 4: Determination of the molecular weight of the leukotoxin of Fusobacterium necrophorum subsp.
necrophorum by SDS polyacrylamide gel electrophoresis using standard proteins of different
molecular weights.

DISCUSSION
Two subspecies of F. necrophorum can be recovered from bovine liver abscesses, F.
necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, but the former one
produces more leukotoxin [6 - 10 - 14 - 15 16] than the latter and thats why it is encountered
most frequently in bovine hepatic abscesses [1- 7- 8 - 17 -18 - 19] . Therefore, in this study, the
subsp. necrophorum was used for leukotoxin extraction.

7
 The concentration of the crude leukotoxin-containing culture supernatant of F.
necrophorum subsp. necrophorum did not increase the leukotoxin titer quantitatively because a
fivefold reduction in the volume of the culture supernatant had produced only 2-fold increase in
the titer of the leukotoxin. This result is approximately in agreement with that reported by [20]
who reported that a 20-fold reduction in the volume of the culture supernatants of toxigenic
strains of Fusobacterium necrophorum had raised the titer of the leukotoxin by a factor of
between 8 and 12.
Filtration of the concentrated crude culture supernatant using gel filtration chromatography
resulted in deposition of the majority of the leukotoxin activity in the third peak (A 280) at which
the leukotoxin titer (as determined using the MTT dye reduction assay) ranged from 128 to 1024.
The height of the third peak is positively correlated with the leukotoxin titer in the sample
applied to the column. This finding disagrees with that of [20] who reported that filtration of
the concentrated crude culture supernatant of toxigenic strains of F. necrophorum using gel
filtration chromatography on Fractogel TSK HW55 (F) had resulted in deposition of the majority
of the leukocidal activity in the first peak. This disagreement can be ascribed to the difference in
elution volume / void volume ratio (V e / Vo ratio), since the Ve / Vo ratio obtained by these
authors was equal to 1.1 whereas the Ve / Vo ratio obtained in this study was 1.313 indicating a
better purification performed in the current study.
The comparative analysis of leukotoxicity titers of the crude, concentrated and purified
leukotoxin (as determined using MTT dye reduction assay) revealed that the latter has the lowest
leukotoxicity titer (256). This marked reduction in the leukotoxicity titer of the purified
leukotoxin can be attributed to the fact that the level of leukocidal activity found within the
freshly concentrated crude culture supernatant of the F. necrophorum subsp. necrophorum cannot
be maintained as it is after gel filtration because the filtration process perhaps allowed
degradation of the leukotoxin molecule due to salt content of the eluent (0.2 M phosphate buffer)
used in washing of the filtration column, thus reducing the biological activity of the leukotoxin
(9 - 11 -21]. The lack of calcium and magnesium ions in the filtration buffer may also affect the
leukocidal activity [20]. In addition, the filtration process causes removing of a substantial
proportion of cell membrane components found within the concentrated crude culture
supernatant besides the leukotoxin [20]. These cell membrane components may play a
synergistic role for the leukocidal activity of the leukotoxin. Moreover, the dilution of the
purified leukotoxin during the purification process by the elution buffer and the additional
dilution caused by mixing of the five fractions that showed different ranges of leukotoxin activity
may also play a part in reducing the biological activity of the leukotoxin.
The estimated molecular weight of the purified leukotoxin obtained in the current study
using gel filtration chromatography was 316 KD. This result is generally in agreement with those
of [6 22] who reported that the estimated molecular weight of the purified leukotoxin as
determined by gel filtration was more than 300 KD. The SDS-PAGE which was used to
determine the molecular weight of the purified leukotoxin obtained in the present study had
revealed a single band which was approximately equal to 48 KD. This finding showed that the
SDS-PAGE resulted in dissociation of the leukotoxin into several subunits each of which was
approximately equal to 48 KD indicating that this leukotoxin may be a polyvalent toxin. This
result is generally in agreement with findings of [6 -20] who pointed out that upon reduction and
denaturation during SDS-PAGE, the leukotoxin of Fusobacterium necrophorum was dissociated
into several subunits.

8
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