Vaccine 27 (2009) 5855 5864
Contents lists available at ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Purification and characterization of an immunogenic outer membrane
protein of Shigella flexneri 2a
Debasis Porea, Pinki Chowdhurya,1, Nibedita Mahataa, Amit Pala, Shinji Yamasakib,
Dilip Mahalanabisc, Manoj K. Chakrabartia,"
a
Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Kolkata 700010, West Bengal, India
b
Laboratory of International Prevention of Epidemics, Department of Veterinary Science, Graduate School of Life and Environmental Sciences,
Osaka Prefecture University, Osaka, Japan
c
Society for Applied Studies, CF 198, Sector-I Salt Lake, Kolkata 700064, West Bengal, India
a r t i c l e i n f o a b s t r a c t
Article history:
In the present study we purified 34 kDa major outer membrane protein (MOMP) of Shigella flexneri 2a
Received 11 May 2009
for the first time, which was cross-reactive and antigenically conserved among Shigella spp. and the
Received in revised form 13 July 2009
epitope was surface exposed on the intact bacterium. The purified antigen was found to be glycosylated,
Accepted 18 July 2009
which aids in binding to macrophages and up-regulated the production of nitric oxide, granulocyte-
Available online 4 August 2009
colony stimulating factor and IL-12p70, indicating that the MOMP is immunogenic and has the ability to
commence protective immune responses against intracellular pathogens, thereby it may be considered
Keywords:
as a potential vaccine candidate.
Vaccine
© 2009 Elsevier Ltd. All rights reserved.
Major outer membrane proteins
Shigellosis
Immunogen
1. Introduction teobacteria [14], but the detail characterization of these proteins
has not yet been done.
Shigellosis is a gastrointestinal disease caused by Shigella spp. Vaccines that consist of native antigenic structures are more
[1]. Still it is a major public health concern among infants and likely to stimulate an immune response that mimics the response
young children who make up 69% of all cases, particularly in the induced after natural infection, which is most readily achieved
developing countries [2,3]. Globally in each year millions of cases with live-attenuated vaccines as well as with subunit vaccines. Pre-
of shigellosis occur with over 600,000 cases resulting in death [4]. viously we have shown that oral immunization with heat-killed
The clinical management of the disease has become difficult due whole-cell S. flexneri 2a gives protection against challenge with
to rapid escalation in antibiotic resistance of Shigella spp., even to homologous strain in rabbits [17]. It has been observed that among
the newest antibiotics [5 6]. Consequently, high priority has been different outer membrane proteins, the gel cut band of 34 kDa
given by the World Health Organization for the development of a (according to SDS-PAGE) protein is capable of providing signifi-
safe and effective vaccine to help in the control of Shigellosis [1]. cant protection in rabbits against the challenge with virulent S.
Vaccine strategies must consider the need for protection against flexneri 2a [18]. Moreover it has also been found that electroeluted
four species of Shigella (Shigella flexneri, Shigella dysenteria, Shigella gel cut band of 34 kDa OMP induces the release of IL-12, tumor
boydii, Shigella sonnei). Despite many years of extensive research necrosis factor alpha (TNF- ) and production of nitric oxide (NO)
to develop an effective Shigella vaccine [7 13], a practical vaccine by murine peritoneal macrophages in a dose dependent manner
is still not available. Recently, several immunogenic proteins of which showed itself as a potent antigen [18].
S. flexneri 2a have been identified by immunoproteomics analysis In continuation to our previous findings [17,18], an attempt has
[14 16] and the peptide masses of few of those proteins have been been made to more precisely identify the active immunological
found to match with the theoretical peptide masses of other pro- and protective component(s) which may play a vital role towards
the development of a Shigella vaccine. The present study has been
directed towards purification and characterization of the 34 kDa
protein of S. flexneri 2a and analyzing its potential as protective
"
Corresponding author. Tel.: [phone]; fax: [phone].
antigen after the removal of LPS. It has been shown that the puri-
E-mail address: mkc [e-mail] (M.K. Chakrabarti).
1 fied protein is glycosylated, which plays role in the conservation of
Present address: Institute of Molecular Medicine, University of Texas Health
Science Center, Houston, TX, USA. the epitope as revealed by the macrophage binding study. It has also
0264-410X/$  see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2009.07.054
5856 D. Pore et al. / Vaccine 27 (2009) 5855 5864
been found that the purified protein is much more immunogenic 2.4. Determination of protein concentration
than that of the previously obtained electroeluted SDS-PAGE gel cut
protein. Immunogenicity and antigenic relatedness of the purified The protein concentration was routinely estimated by the
MOMP among Shigella spp. have also been studied here. method of Bradford [23], with BSA as standard. Protein estimation
was done using Bio-Rad microassay with manufacturer s protocol.
2. Materials and methods
2.5. Removal of lipopolysaccharide
2.1. Bacteria
To remove endotoxin, the purified protein was passed through
Detoxi-Gel endotoxin-removing resin (Pierce product) using man-
S. flexneri 2a (N.Y-962/92), Shigella dysentery type1 (AO-14971),
ufacturer s protocol.
S. boydii (BCH-621) and S. sonnei (D-80) were obtained from
the Pathophysiology Division of National Institute of Cholera and
2.6. SDS-PAGE
Enteric Diseases, Kolkata, India. The virulence of the Shigella spp.
was routinely examined by the Sereny test [19].
Samples for electrophoresis were solubilised in SDS sample
ć%
buffer [24] at 100 C for 5 7 min. SDS-PAGE was carried out
2.2. Preparation of outer membrane protein
on a 12.5% separating gel with the discontinuous buffer system
described by Laemmli [24] and the bands were visualized by
Outer membrane protein was prepared according to the method
Coomassie brilliant blue staining. Fermentus molecular weight
of Filip et al. [20]. Briefly, the Shigella spp. (N.Y-962/92) was grown
marker was run in parallel for the determination of molecular
ć%
in tryptic soy broth (Difco Laboratories) at 37 C for 18 h under
weights.
shaking conditions. The cells were harvested by centrifugation at
10,000 × g (Beckman) for 10 min and washed twice in 100 mM
2.7. Amino-terminal sequencing
HEPES (N-2 hydroxyethyl piperazine-N -ethanesulfonic acid, pH
7.0) buffer (Sigma). The harvested cells were suspended in 50 ml
The purified 34 kDa protein was electrophoresed by SDS-PAGE.
of HEPES buffer (pH 7) and the cells were then disrupted with
After SDS-PAGE, the protein was electrophoretically transferred
ć%
an ultrasonic disintegrator (MICROSON) at 4 C with intermittent
onto a polyvinylidene difluoride membrane (Bio-Rad) according
bursts of 1 min. The unbroken cells were removed by centrifu-
to the method of Matsudaria [25], with modifications. The section
ć%
gation at (10,000 × g) for 10 min at 4 C. The supernatant was
of the membrane containing the 34 kDa protein was excised and
then centrifuged at 105,000 × g (Sorvall) for 1 h to obtain the
applied to an automated protein sequencer (model 491; Applied
envelope fraction. The envelope fraction was treated with 0.5%
Biosystem, Tokyo, Japan).
(weight/volume) N-laurylsarcosine sodium salt (Sigma Chemical
Co) for 20 min at room temperature to selectively solubilise the
2.8. Development of antisera
inner membrane part. The insoluble outer membrane protein frac-
ć%
tion was recovered by centrifugation at 100,000 × g for 1 hat 4 C.
BALB/c mice, originally obtained from the Jackson laboratory
(Bar Harbor, Maine), were reared and maintained in the animal
2.3. Purification of major outer membrane protein
facility at the National Institute of Cholera and Enteric Diseases,
Kolkata, India. The mice were immunized intraperitoneally with
The major outer membrane protein (MOMP) fraction was iso- purified 34 kDa protein emulsified with two volumes of Freund s
lated by the methods of Munson and Granoff [21] and Murphy et
complete adjuvant (Gibco). Two booster immunizations were fol-
al. [22], with modifications. The sarcosyl-insoluble OMP fraction
lowed at an interval of 10 days and the sera were collected by
was suspended in buffer A (1% [w/v] 3-[(3-cholamidopropyl)- puncturing of the supraorbital plexus.
dimethyl-ammonio]-1-propanesulfonate [CHAPS], 20 mM Tris-HCl
and 10 mM EDTA [pH 8.0]) and incubated at room temperature
2.9. Immunoblotting
for 30 min. The insoluble residue was harvested by centrifu-
gation at 100,000 × g for 30 min, suspended and incubated in
Proteins recognized by antisera were identified by immunoblot-
buffer A, and recovered. The insoluble residue was extracted in
ting [26]. The whole-cell antigens of Shigella spp., the purified
20 mM Tris HCl (pH 8.0) 0.1 M NaCl 10 mM EDTA 0.4% CHAPS
MOMP were separated by SDS-PAGE and transferred electrophoret-
(Sigma) [extraction buffer] for 30 min. The suspension obtained
ically to nitrocellulose membrane (Transblot; Bio-Rad Laboratories,
was centrifuged at 100,000 × g for 1 h. The soluble fraction was
USA) under wet transfer condition at 100 V for 1 h in 50 mM
concentrated and applied (5.8 mg of protein) to a Sephacryl S- Tris HCl, 40 mM glycine, and 0.04% SDS, 25% (v/v) methanol (pH
200 HR (Pharmacia) column (1.8 cm × 48 cm) equilibrated with
8.3). Free sites on nitrocellulose strips were then blocked with TBS-
ć%
the extraction buffer. Proteins were eluted at a flow rate of
5% (w/v) defatted milk (Bio-Rad) powder for overnight at 4 C. They
0.5 ml/min in a Biologic Due Flow (Bio-Rad) system with the
were then incubated sequentially with mouse antiserum (1:1000,
extraction buffer and the elution profiles were monitored by
v/v), raised against the purified 34 kDa OMP of S. flexneri 2a [for
absorbance at 280 nm. The fraction containing the 34 kDa MOMP
cell-lysate] and rabbit antisera (1:1000, v/v) produced by challenge
was identified by SDS-PAGE and pooled and dialysed in buffer
(according to the method of Rabbani et al. [27]) with live whole S.
B (0.4% CHAPS, 20 mM Tris HCl and 10 mM EDTA [pH 8.0]).
flexneri 2a [For MOMP] respectively in TTBS (TBS containing 0.1%
The dialysed fraction was then concentrated and applied (4.2 mg
Tween 20 and 0.5% non-fat dry milk) at room temperature for 1 h
of protein) to a DE-52 (Whatman) column equilibrated with
and alkaline phosphatase-conjugated goat anti-mouse IgG (1:2000,
buffer B. The column was washed with two column volume of
v/v [Jackson]) in TBS (containing 0.5% non-fat dry milk) for 1 h at
buffer B, and the MOMP was eluted in 1.5 ml fraction with a
room temperature. Non-specifically bound proteins were removed
linear gradient of 0 to 0.5 M NaCl in buffer B. The fraction con- by washing nitrocellulose strips with TTBS thrice between two
taining the purified 34 kDa MOMP was identified by SDS-PAGE,
incubation steps. Immunoreactive components were visualized by
pooled, dialysed in 0.4% CHAPS-10 mM EDTA and finally stored at
color development with alkaline phosphatase using 5-bromo-4-
ć%
-20 C.
chloro-3 indolyl phosphate (BCIP, 3.75 mg in 250 l of 100% DMF
D. Pore et al. / Vaccine 27 (2009) 5855 5864 5857
[Sigma]) and nitroblue tetrazolium (NBT, 7.5 mg in 250 l of 70% 2.13. Identification of glycoproteins
DMF [Sigma]) in carbonate buffer, pH 9.8, containing 0.1 M NaHCO3
and 1 mM MgCl2. Protein glycosylation was analyzed by blot of the purified protein
according to the method of Haselbeck and Hosel [31] with modifica-
tion. Briefly, the purified protein was electrophoresed by SDS-PAGE.
2.10. Immunofluorescence
After SDS-PAGE, the protein was electrophoretically transferred to
a nitrocellulose membrane. The membrane was equilibrated with
The technique is a modification of a method by Klauser et
0.1 M acetic acid for 10 min and carbohydrates were oxidized with
al. [28]. Briefly, S. flexneri 2a was harvested from mid-log-phase
10 mM sodium metaperiodate in 0.1 M acetic acid by incubation
cultures, washed with PBS (Bio-Rad), applied to a microscope cov-
for 20 min at room temperature in the dark. The membrane was
erslip, and fixed with 2% (v/v) paraformaldehyde. The coverslip was
washed twice with 0.1 M acetic acid and once with 0.05% Tween 20
then incubated for 1 h with mouse anti MOMP antibody (1:100
and 0.1 M acetic acid. Biotin-hydrazide (Bio-Rad) in 0.05% Tween 20
ć%
in PBS with 1% (w/v) BSA [Sigma]) at 4 C, washed twice with
and 0.1 M acetic acid was then added and allowed to react for 60 min
ć%
PBS, and finally incubated for 1 h at 4 C with fluorescein isothio-
at room temperature to label the aldehydes that resulted from car-
cyanate (FITC)-conjugated goat anti-mouse IgG antiserum (dilution
bohydrates oxidation. After three washes with 0.05% Tween 20 in
1:500 in PBS containing 1% BSA; Jackson Immuno Research Lab-
TBS (TBST), the membrane was blocked for 30 min and incubated
oratories Inc., West Grove, Pa.). After two washes with PBS, the
with a 1:2000 solution of streptavidin-alkaline phosphatase con-
coverslip was gently aspirated dry and mounted upside down
jugate (Bio-Rad). The membrane was washed again with TBST and
on a clean glass microscope slide and sealed with acrylic nail
stained with BCIP/NBT (Sigma) as substrate.
polish. Bacteria were photographed by using an Olympus AX70
microscope equipped with phase-contrast and epifluorescence
2.14. Enzymatic deglycosylation
illumination, standard FITC filters and a 100× achromatic oil
immersion lens.
The deglycosylation of the purified protein was assessed
using the Enzymatic Deglycosylation kit (Calbiochem) according
2.11. Antibody absorption to the indications of the manufacturer s. The enzymes used in
this study included N-glycosidase F, 2-3,6,8,9-neuraminidase,
Antiserum was absorbed by the method of Loeb [29]. Mid- endo- -N-acetylgalactosaminidase, 1,4-galactosidase and -N-
log-phase cells of S. flexneri 2a (1.5 × 1011 CFU) were suspended in acetylglucosaminidase.
0.8 ml of PCM (PBS [pH 7.2] containing 0.5 mM MgCl2 and 0.15 mM
ć%
CaCl2) at 4 C. An equal volume of the 1:100 dilution of protein 2.15. Preparation of murine peritoneal macrophages
A-purified antiserum in PCM containing 1% non-fat dry milk (Bio-
ć%
Rad) was added to the cell suspension and incubated at 4 Cfor 1h Macrophages were isolated according to the method of Ref. [32].
with gentle agitation. No change in the live bacterial cell numbers Briefly, the peritoneal washing containing the macrophages was
ć%
was confirmed after incubation. Absorbed antiserum was obtained collected on sterile Petri dishes and incubated at 37 C in 5% CO2
by centrifugation and then examined for antibody reactivity in for 2 h. The cells of the monocyte macrophage lineage adhered on
immunoblot analysis. the surface of the Petri dishes to form a confluent cell monolayer.
The non-adherent peritoneal cells were removed by repeated wash-
ing of the plates with cold PBS. The adhered peritoneal cells were
2.12. Analytical gel filtration chromatography
removed from the surface with a rubber scraper. The cells were
washed thoroughly by suspending in PBS and subsequent centrifu-
Analytical gel filtration chromatography was performed to
gation at 400 × g for 5 min. The cell pellet obtained was suspended
determine the molecular weight (M.W.) and stokes radius (Rs)ofthe
in RPMI 1640 medium (Gibco). Cells (200 l) were seeded per well
purified MOMP. Gel filtration chromatography was carried out with
at a concentration of 1 × 106 cells/ml in 96-well flat-bottomed tis-
a Superdex 200 (Pharmacia) column (1 cm × 40 cm) using BioLogic
ć%
sue culture plates (BD Falcon). Macrophages were cultured at 37 C
Duo-Flow (Bio-Rad) pre-equilibrated with two column volumes
in a humidified 5% CO2 atmosphere, with the absence and presence
of 20 mM Tris HCl (pH 8.0) and 100 mM NaCl, at a flow rate of
of purified 34 kDa MOMP in RPMI 1640 supplemented with 5 U/ml
1 ml/min, at room temperature. The purified MOMP and a mixture
penicillin G, 5 g/ml streptomycin, 0.1% gentamycin, 2% fetal bovine
of standard proteins (Sigma) alcohol dehydrogenase (molecular
serum and 0.1% insulin-transferrin-selenium. The integrity of the
mass 150 kDa; radius 46 Å), bovine serum albumin (molecular mass
monolayer was monitored by inverted light microscopy (Olympus,
66.5 kDa; radius 35.5 Å), ovalbumin (molecular mass 43 kDa; radius
USA). Macrophages were 85 90% viable as determined by trypan
30.5 Å), and carbonic anhydrase (molecular mass 29 kDa; radius
blue exclusion.
23.9 Å) were loaded onto the column and their elution profiles
were monitored by absorbance at 280 nm. The molecular weight
2.16. Macrophage binding assay
was estimated [30] by a linear fit of the logarithm of M.W. of the
standard proteins against the relative elution volumes (Ve/V0), and
To assess the nature of binding of the purified native and
calibration standard curve of the Stokes radii of the standard pro-
deglycosylated antigen to macrophages (saturation and qualita-
teins versus the partition coefficient Kav [30] was used to interpolate
tive features), an ELISA was developed. For this assay, peritoneal
the Rs of the MOMP:
ć%
macrophages (106/well) were cultured (37 C) in 96-well microtiter
plates overnight. The plate was washed three times with PBS and
Ve - V0
Kav = ć%
blocked with 1% BSA in PBS for 1 h at 4 C. The macrophages
Vt - V0
were then treated with serial 1:2 dilution of native and deg-
ć%
where Ve = elution volume of the protein, V0 = void volume and lycosylated pure protein starting at 10 g/well for 1 h at 4 C
Vt = total volume of the column. and macrophages without protein were used as control. After
Blue dextran and tryptophan-methyl-ester were used as mark- the plate was washed with PBS and the reaction was developed
ers of the void volume (V0) and total excluded volume (Vt), by using anti MOMP IgG antibody followed by goat anti-mouse
respectively. horseradish peroxidase-conjugated secondary antibody [Jackson]
5858 D. Pore et al. / Vaccine 27 (2009) 5855 5864
and then incubating with the TMB substrate (ImmunoPure TMB 3. Results
[3,3 ,5,5 -tetramethylbenzidine] substrate kit; Pierce). The reaction
was stopped with 2N H2SO4 and the optical density was read at 3.1. Purification of 34 kDa MOMP
450 nm.
The CHAPS soluble outer membrane fraction was applied to
a molecular-sieve chromatography column. The 34 kDa protein
2.17. Nitrite assay
eluted from the Sephacryl S200 HR column in the second peak
(Fig. 1A). Then the collected fraction was dialysed against buffer
To detect the production of NO by murine macrophages, the
B, which allowed complete removal of NaCl. The next step of the
amount of its stable end product, nitrite accumulated was analyzed
purification was the application of the dialysed fraction to a DE-52
by Griess reaction [33]. Untreated cell was taken as control. Briefly,
chromatography column with a linear 0 to 0.5 M NaCl gradient.
cell free supernatant was collected after centrifugation (400 × g)for
The 34 kDa protein eluted in one peak between 0.15 and 0.20 M
5 min. A 100 l of the supernatant was mixed with equal volume
and thus could be separated from the major contaminant, which
of Griess reagents (modified, Sigma). The reaction was allowed to
eluted earlier in the gradient (Fig. 1B). The SDS-PAGE analysis of
proceed for 10 15 min at room temperature and the absorbance
the protein profile revealed that this peak was essentially pure
was measured at 540 nm. The amount of nitrite in the samples was
34 kDa protein (Fig. 1C).
calculated by extrapolation from a sodium nitrite standard curve.
3.2. Lipopolysaccharide removal
2.18. Cytokine ELISA
The recalcitrant problematic trace levels of lipopolysaccha-
IL-12p70 and G-CSF in 24 h-old macrophage culture supernatant
ride remaining with the purified 34 kDa MOMP was removed
were measured in sandwich ELISA according to the manufacturer s
with Detoxi-Gel endotoxin-removing resin resulting in virtually
instruction (R&DSystem).
pyrogen-free 34 kDa protein containing <0.001 EU of lipopolysac-
charide per ml.
2.19. Statistical analysis
3.3. N-terminal amino acid sequence
The statistical significance of difference between the test groups
was analyzed by Student s t-test (two-tailed) using SPSS 7.5 soft- The amino-terminal analysis revealed that the first 15 amino
ware. Results were expressed as the mean Ä… standard error of the acid residues of the purified MOMP of S. flexneri 2a (Fig. 1D) showed
mean (S.E.M.) where applicable, of three independent experiments. significant homology with N-terminal sequences of the ABC-type
Statistical significance was assumed at p < 0.05. oligopeptide transporter system of Vibrio cholerae RC385 (acces-
Fig. 1. (A) Elution profile (A280) of the S. flexneri 2a MOMP on a Sephacryl S200 HR Column. The sample was applied to a column (1.8 cm × 48 cm) packed with Sephacryl
S200 HR which had been equilibrated with a buffer (20 mm Tris HCl [pH 8.0], 100 mM NaCl, 10 mM EDTA and 0.4% CHAPS). The flow rate was 0.5 ml/min. SDS-PAGE analysis
revealed that the MOMP was in the second peak. (B) Elution profile (A280) of the S. flexneri 2a MOMP on a DE-52 Column. The sample was applied to a column packed with
DE-52 in a buffer (20 mm Tris HCl [pH 8.0], 10 mM EDTA and 0.4% CHAPS). The column was washed with the same buffer and bound proteins were eluted in 1.0 ml fraction
with a linear gradient of 0 to 0.5 M NaCl. SDS-PAGE analysis revealed that the 34 kDa OMP is in the second peak. (C) SDS-PAGE showing the purification of the 34 kDa OMP
of S. flexneri 2a. Lane 1: Fermentus broad range molecular weight marker. Lane 2: N-laurylsarcosyl-insoluble OMP (5.8 g). Lane 3: Sephacryl S-200 fraction (3.5 g) Lane 4:
DE-52 fraction (2.5 g). (D) The N-terminal amino acid sequence of the purified 34 kDa major outer membrane protein (MOMP).
D. Pore et al. / Vaccine 27 (2009) 5855 5864 5859
Fig. 2. (A) SDS-PAGE analysis of the whole-cell lysate of S. flexneri 2a (N.Y-962/92). Lane 1: Fermentus broad range molecular marker; Lane 2: whole-cell lysate preparation.
(B) Western blot analysis of the whole-cell antigen of S. flexneri 2a (N.Y-962/92) with anti-MOMP antibody (Lane 1), Western blot analysis of the purified antigen with antisera
from virulent S. flexneri 2a challenged rabbits (Lane 2) and Western blot analysis of the whole-cell lysate of S. flexneri 2a (N.Y-962/92) with antisera raised from virulent S.
flexneri 2a challenged rabbits (Lane 3). Molecular mass standards are noted on the left. (C) Immunoblot analysis of whole-cell preparations from representative S. flexneri
strains with murine antibody. Four reactive patterns were observed (Lanes 1 4). Lane 1: S. flexneri 2a B 290, Lane 2: S. flexneri 1b NK 2683, Lane 3: S. flexneri 3a NK 3327 and
Lane 4: S. flexneri 4b NK 2258. Approximately 3 g protein per lane was resolved. Molecular mass standards are noted on the left. (D) Western blot analysis of whole-cell
antigens of Shigella spp. The 34 kDa antisera raised in mice was incubated at RT with blotted whole-cell lysate of S. flexneri 2a (Lane 1), S. boydii (Lane 2), S. sonnei (Lane 3), S.
dysenteriae type 1 (Lane 4). Molecular mass standards are noted on the left.
sion number: ZP 00753763) and Aeromonas salmonicida (accession immunofluorescence microscopy was performed. The antiserum
number: YP 001141527.1), the extracellular solute-binding protein showed positive staining on the bacterial cell surfaces (Fig. 3A).
of Enterobacter sp. (accession number: YP 001176214.1) and the To confirm the question of surface exposure, the 34 kDa antiserum
putative periplasmic murein peptide-binding protein of Yersinia was absorbed with log-phase whole cells of S. flexneri 2a. Absorp-
enterocolitica (accession number: YP 001006350.1). tion of antiserum reduced the antibody reactivity with 34 kDa OMP
(Fig. 3B). These results indicated that the epitope recognized by the
34 kDa antiserum was exposed on the surface of the intact bac-
3.4. Antigenicity and antigenic conservation of 34 kDa MOMP of
terium.
S. flexneri 2a
Murine antibody raised against the purified 34 kDa MOMP of S.
flexneri 2a recognized the MOMP in the whole-cell preparation of
S. flexneri 2a in an immunoblot analysis (Fig. 2B, lane 1) and the
purified protein showed reactivity (Fig. 2B, lane 2) with rabbit anti-
sera produced by challenged with whole-cell virulent S. flexneri 2a
(method not described here), suggesting the strong immunogenic-
ity of the purified MOMP. To assess the degree of conservation of the
antigen recognized by MOMP antiserum among S. flexneri 2a strains,
obtained from diseased persons were tested in immunoblot anal-
ysis. Whole-cell preparations were used in this study. The murine
antibody reacted with S. flexneri 2a B 290, S. flexneri 1b NK 2683, S.
flexneri 3a NK 3327 and S. flexneri 4b NK 2258 tested (Fig. 2C).
3.5. Cross-reactivity of MOMP murine antiserum with other
Shigella spp.
To evaluate the cross-reactivity of the antigen among Shigella
spp., the MOMP antiserum was used to probe the whole-cell anti-
gens of S. dysentery type 1, S. boydii, and S. sonnei (Fig. 2D). The
antiserum reacted with all Shigella spp. tested. The apparent molec-
ular mass of the antigen varied among strains from 34 to 38 kDa.
Fig. 3. (A) Immunofluorescence microscopy of S. flexneri 2a. Bacteria harvested from
mid-log cultures were treated with anti MOMP antibody (diluted 1:100 in PBS) and
FITC-conjugated secondary antibody. The phase-contrast image is on the left-hand
3.6. Surface exposure of the 34 kDa protein
Side (A I) and the IF image is on the right-hand side (A II). (B) Immunoblot analysis
of the reactivity of the 34 kDa antiserum absorbed with log-phase whole cells of S.
To determine whether MOMP antibody recognized an epitope
flexneri 2a. Lane 1: unabsorbed 34 kDa antisera; Lane 2: absorbed 34 kDa antisera.
that was exposed on the surface of an intact S. flexneri 2a bacterium,
Molecular mass standards are noted on the left.
5860 D. Pore et al. / Vaccine 27 (2009) 5855 5864
Fig. 4. (A) Analytical gel filtration chromatography. Conditions are described in the experimental section. Typical elution profiles (A280) are shown with the positions of
calibration markers: (A) alcohol dehydrogenase; (B) bovine serum albumin; (O) ovalbumin; (C) carbonic anhydrase; (PI) purified 34 kDa OMP. The void volume is indicated
by V and AU, absorbance units. (B) Linear fit of the relative elution volume versus the logarithm of molecular weight of standard proteins. The arrow indicates the molecular
weight of the purified MOMP is 30.5 Ä… 0.07. (C) Linear fit of known Stokes radii as a function of the measured partition coefficient (Kav). The arrow shows the partition
coefficient of purified MOMP, corresponding to 24.6 Ä… 0.06.
3.7. Analytical gel filtration chromatography
Analytical gel filtration chromatographic analysis showed that
the purified MOMP was a monomeric protein (Fig. 4A). Standard
proteins with known molecular weights and stokes radii were
loaded onto a gel filtration column and their elution volumes were
plotted against their molecular weights and stokes radii (Fig. 4B and
C) to predict the molecular weight and stoke radius of the MOMP.
The molecular weight and stokes radius of MOMP were determined
to be about 30.5 Ä… 0.07 kDa and 24.6 Ä… 0.06 Å, respectively.
3.8. Fluorescent spectroscopic characterization
Fig. 5. Emission fluorescence spectra of purified MOMP with excitation at 295 nm
and emission from 310 to 400 nm in 20 mM Tris HCl (pH 8.0) and 100 mM NaCl.
The intrinsic tryptophan fluorescence was measured using an
excitation wavelength of 295 nm to avoid tyrosine emission. Fluo-
rescence spectroscopy of tryptophan (Trp) is a powerful technique
to access information about the environment where this residue
is located as Trp is very sensitive to the polarity of the environ-
Fig. 6. (A) Glycosylation analysis of the purified MOMP of S. flexneri 2a. The MOMP was separated by SDS-PAGE, transferred to a nitrocellulose membrane and stained with
carbohydrate-specific periodate oxidation and biotin-hydrazide conjugation. Molecular mass standards are noted on the left. (B) Enzymatic deglycosylation of the purified
MOMP. Native (Lane 1), N-linked specific deglycosylated (Lane 2) and O-linked specific deglycosylated (Lane 3) MOMP was transferred to a nitrocellulose membrane and
reacted with anti-MOMP antibody. Molecular mass standards are noted on the left.
D. Pore et al. / Vaccine 27 (2009) 5855 5864 5861
ment. Moreover, the emission fluorescence spectroscopy of Trp duction of IL-12p70 (maximum of 10.23 pg, p < 0.05) on treatment
can be used to access the folded state of a protein [34]. The puri- with deglycosylated antigen.
fied MOMP showed maximum fluorescent intensity at 335 nm
(Fig. 5).
3.14. Dose kinetics of purified MOMP induced macrophage G-CSF
release
3.9. Detection of glycosylation
Macrophages on treatment with purified MOMP for 24 h pro-
Glycosylation assay of the purified protein was performed as it
duced granulocyte-colony stimulating factor (G-CSF) in a dose
plays numerous cellular functions of protein including: (i) aiding
dependent manner (Fig. 7D). The optimum dose was 2 g/0.2 ml,
in protein folding, (ii) maintenance of protein stability and sol-
which released approximately 38.48 pg G-CSF, p < 0.05.
ubility, (iii) modulation of biological activity (immune response),
and (iv) protection from proteolytic degradation [35]. The puri-
4. Discussion
fied protein was labelled with biotin-hydrazide after oxidation with
sodium periodate. Glycosylation was observed in the purified pro-
Shigella spp. is the etiological agent of Shigellosis and an impor-
tein (Fig. 6A).
tant cause of childhood mortality and morbidity [1 3]. It has been
reported earlier that among different outer membrane proteins,
3.10. Enzymatic deglycosylation analysis
34 kDa gel cut protein can be considered as a potent antigen,
which can elicit significant protection against challenge with vir-
The nature of the glycans attached to the purified protein
ulent S. flexneri 2a in rabbit model of shigellosis [17]. Our initial
was characterized by treating the protein with the N-linked and
observations also showed that the dose dependent increase in the
O-linked specific glycosidases. Increase in the electrophoretic
production of IL-12, NO and TNF- by gel cut electroeluted 34 kDa
mobility was found in the protein treated with N-linked glycosi-
OMP-treated M of BALB/c mice [18]. Protein obtained from the
dases but not in O-linked glycosidases (Fig. 6B). Therefore, these
previously described gel cut electroelution techniques was not pure
enzymes, which were specific for carbohydrate moieties commonly
enough for structural and functional analysis because denaturation,
present in N-glycoproteins, were able to hydrolyze the glycans
lipopolysaccharide and ionic detergent might interfere functional
present in the purified protein.
integrity of the protein. We, therefore, optimized a purification
protocol by using 0.4% CHAPS (a non-ionic detergent and can be
3.11. Macrophage binding
ć%
dialyzed completely even at 4 C), which improves the purity of the
protein so that we could get native well-folded active conformation.
To assess the impact of glycosylation on immunogenicity of the
In this study, we have purified and characterized the 34 kDa major
purified MOMP, macrophage binding assay was performed by com-
outer membrane protein (MOMP) of S. flexneri 2a for the first time.
paring the interactive properties of the native and deglycosylated
Purification of the S. flexneri 2a MOMP to apparent homogeneity
antigens. The native glycoprotein bound to macrophage (Fig. 7A).
has been achieved by molecular-sieve and ion exchange chromato-
Although the deglycosylated protein also adhered to macrophage,
graphic techniques with a yield of 100 g per liter culture. In the
its binding capacity was significantly reduced (p < 0.01) with
present study we almost completely removed LPS, which excluded
respect to native glycoprotein.
the possibility of immunogenicity of the purified protein owing to
LPS contamination because LPS can induce protective immunity in
3.12. Dose kinetics of native and deglycosylated MOMP induced
shigellosis [37] and also can modulate cytokine release by M [38].
release of nitric oxide
The purified preparation contained <0.001 EU of LPS per ml.
Hydrophobic surfaces usually contribute to the interaction
The production of highly reactive free radical nitric oxide (NO)
between proteins including cell surface receptors [39] and tryp-
generated by the inducible isoform of NOS (iNOS) in response to a
tophan ranks as one of the most hydrophobic amino acids. Purified
bacterial antigen has been demonstrated to have a beneficial effect
protein gives a fluorescence emission maximum at 335 nm, indi-
in host defence mechanisms against various pathogenic bacteria
cating that most of the tryptophans are partially buried, which
and protozoa [36]. In this study we thus monitored the ability of
reveals that the protein retains high degree of tertiary structure
the purified native and deglycosylated antigen to elicit NO produc-
after purification. This result suggests that the conditions used for
tion by murine peritoneal macrophages. Macrophages on treatment
the purification of MOMP does not significantly disturb the folding
with native antigen for 40 h released NO in a dose dependent man-
of the protein.
ner with an optimum dose of 2 g/0.2 ml (Fig. 7B), corresponding
The analytical gel filtration chromatography demonstrates that
to 18.789 MNO(p < 0.05).
the purified protein is a monomer with molecular weight and stokes
Macrophage on treatment with deglycosylated antigen for 40 h
radius of 30.5 kDa and 24.6 Å, respectively.
produced significantly decreased level (Fig. 7B) of NO, (maximal
The N-terminal protein sequence (15 amino acids) analysis indi-
NO level of 8.345 M, p < 0.05) suggesting the importance of glycan
cates that the purified MOMP of S. flexneri 2a exhibits considerable
residues in the purified native antigen.
sequence homology with the ABC-type oligopeptide transporter
protein of V. cholerae and A. salmonicida. It also overlaps with the
3.13. Time kinetics of purified native and deglycosylated antigen extracellular solute-binding protein of Enterobacter sp. and with the
induced IL-12p70 production in macrophage putative periplasmic murein peptide-binding protein of Y. entero-
colitica. This corroborates with the earlier findings that the first 12
Production of proinflammatory cytokines by macrophages in amino acid residues of 37 kDa heat-modifiable OMP of H. somnus
response to the bacterial antigen was also implicated in the ability showed a 67% overlap with the E. coli ompA and a 100% overlap
of the antigen to modulate the protective immune response. The with the A. actinomycetemcomitans 29 kDa sequence [40]. Moreover
production of IL-12p70 by macrophages in response to purified this observation also correlates with the findings of Peng et al. that
native (2 g/0.2 ml) or deglycosylated (2 g/0.2 ml) antigen was several immunogenic proteins of S. flexneri 2a showed molecular
observed to peak at 12 h (Fig. 7C). Native antigen induced IL-12p70 mass homology with other proteobacteria [14]. Further studies are
(maximum of 22.123 pg, p < 0.05) was significantly higher to pro- needed to identify the exact nucleotide sequence of the purified
5862 D. Pore et al. / Vaccine 27 (2009) 5855 5864
MOMP, which will help to predict the amino acid sequence of the It is known that glycosylation is required for proper antigen
full-length protein and to determine conservation of the gene and conformation and it also plays important role in the functional
respective protein amongst Shigella and other Enterobacteriaceae. immunogenicity of protein vaccine [41]. The present study indicates
The outer membranes of gram-negative bacteria are immuno- that the purified MOMP is N-glycosylated. Binding of the purified
logically important structures. It has been found in this study that native MOMP with murine macrophages, production of NO and
antibody to the purified major outer membrane protein was present IL-12p70 have noticeably been reduced when MOMP is enzymat-
in sera of mice immunized with purified MOMP. The purified anti- ically deglycosylated. These results provide evidence that glycan
gen also reacted strongly with antisera produced by challenge in residues present in purified antigen play crucial role in interaction
rabbits with live whole-cell bacterium. These results suggest that with macrophage and the production of macrophage cell signalling
the antigen is accessible to host defence mechanisms, i.e. immuno- molecules, which may probably due to conservation of the epitope
genic. Whole-cell antibody absorption study reveals that the MOMP with the glycoside residues.
antiserum recognized an epitope that is expressed on the sur- Immunogenicity of the purified MOMP has been shown
face of an intact bacterium. The surface exposure of MOMP was by demonstrating the release of NO, IL-12p70 and G-CSF by
also confirmed by immunofluorescence microscopy. Accessibility macrophages. Macrophages have been treated with different con-
of epitopes on the bacterial surface is crucial for antibodies to be centrations of the purified antigen to find out the optimum dose.
protective and is likely to play a vital role in specific binding to cel- The activity or immunogenicity of the purified antigen in the
lular receptors. From the point of view of vaccine development, present study has been found to be maximum at a concentration
identifying common surface antigens among strains is essential of 2 g/0.2 million cells (macrophages) as compared to our earlier
because immunization with a single common antigen might induce findings with the electroeluted gel cut antigen, which showed a
protection from disease due to many strains. Characterization of maximum activity at 10 g/0.2 million cells (macrophages) [18].
the conservation of the antigen reactive with MOMP antiserum So the purified antigen in the present study shows excellent
among different S. flexneri strains isolated from infected patients immunogenicity in terms of release of NO, IL-12p70 and G-CSF
shows that this antigen is present in all S. flexneri strains tested. by macrophages compared to what we have reported previously
The cross-reactivity of the murine antibody with other Shigella spp. with the electroeluted gel cut antigen suggesting the presence of
was also examined. This result suggests that although the molecular properly folded active antigen or in other word we can say that the
weight of the cross-reactive antigens slightly varied among Shigella purified antigen has the ability to induce immune response even at
spp. but they share common epitope, which may play important a much more lower concentration.
role in cross-protection study. The presence of a conserved, sur- The purified protein has been found to induce dose dependent
face exposed epitope on the MOMP suggests that it may have to be increase in the production of NO, which may be important in the
considered as a potential antigen for the development of a novel development of the protective immune response leading to killing
candidate vaccine against Shigellosis. of phagocytosed S. flexneri 2a.
Fig. 7. (A) Binding of glycosylated and deglycosylated purified MOMP to murine macrophage. Native and deglycosylated MOMPs were assayed in vitro for their ability to
bind to macrophage surface proteins. Binding was expressed as the optical density at 450 nm. Data represent mean Ä… S.E.M. of three independent experiments and p < 0.05
denotes statistically significant difference between native and deglycosylated protein macrophage binding. (B) Effect of native and deglycosylated purified 34 kDa antigens
with increasing doses on nitric oxide production by murine M . Data represent mean Ä… S.E.M. of three independent experiments. (C) Time kinetics of IL-12p70 production
in macrophages treated with native (2 g/0.2 ml) and deglycosylated (2 g/0.2 ml) purified MOMP antigen. Data represent mean Ä… S.E.M. of three independent experiments.
(D) Quantification of M released G-CSF by ELISA for increasing doses of purified 34-kDa antigen. Data represent mean Ä… S.E.M. of three independent experiments.
D. Pore et al. / Vaccine 27 (2009) 5855 5864 5863
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