Journal of Medical Microbiology Papers in Press. Published April 10, 2014 as doi:10.1099/jmm.0.069559-0
1
A novel Omp25-binding peptide screened by phage display can 2
inhibit Brucella abortus 2308 infection in vitro and in vivo 3
Authors 4 5
Junbo Zhang 1, Fei Guo 2, Xiaoqiang Huang3, Chuangfu Chen1*, Ruitian Liu4*, Hui Zhang1, Yuanzhi Wang2, Shuanghong Yin2, Zhiqiang Li1
6
1
7
China
8
2
College of Medicine, Shihezi University, Shihezi, 832003, China
9
3
College of Life Sciences, Shihezi University, Xinjiang 832000, China
10
4
College of Life Sciences, Tsinghua University, Beijing 100083, China
*
Corresponding author:
College of Animal Science and Technology, Shihezi University, Shihezi 832003,
11 12 13 14
E-mail address: [email protected] (CC); [email protected] (RL)
15
Fax: +86-0993-2058612
16
Tel: +86-0993-2058002
17 18 19 20 21 22
23
Abstract
24
Brucellosis is a globally distributed zoonotic disease that causes animal and
25
human diseases, while current antibiotic and vaccine strategies are not optimal. The
26
surface-exposed protein OMP25 is involved in Brucella virulence and play an
27
important role in Brucella pathogenesis during infection, suggesting that Omp25 is
28
an useful target for selecting potential therapeutic molecules to inhibit Brucella
29
pathogenesis. In this study, we identified, for the first time, the specific peptides
30
bind specifically to Omp25 protein of pathogen using a phage panning technique,
31
After four rounds of panning, 42 plaques of eluted phages were subjected to
32
pyrosequencing. Four phage clones bound better than the other clones were
33
selected through the confirmation of ELISA and affinity constants. Selected
34
peptides could inhibit significantly Brucella abortus 2308 (S2308) internalization
35
and intracellular growth in RAW264.7 macrophages, and induce significantly
36
secretion of tumor necrosis factor alpha (TNF-α) and interleukin-12 in peptide- and
37
S2308-treated cells. Any observed peptide (OP11, OP27, OP35, or OP40) could
38
inhibit significantly S2308 infection in BALB/c mice. Moreover, the peptide 0P11
39
was the best candidate peptide in inhibiting S2308 infection in vitro and in vivo.
40
These results suggest that peptide OP11 has potential for exploitation as a peptide
41
drug in resisting S2308 infection.
42
Keywords: Brucella abortus 2308, Phage-display, Peptides, Inhibition infection
43 44
1. Introduction
45
Bacteria of the genus Brucella are gram-negative and facultative intracellular
46
pathogens that pose threats to animal and human health throughout the world. The
47
main pathogenic species worldwide are B.abortus and B. melitensis, which can cause
48
abortions and infertility in cattle and sheep, resulting in heavy economic losses. The
49
human disease is also caused by Brucella melitensis, Brucella abortus or Brucella
50
suis. Infection in humans can cause fever, arthritis, spondylitis, dementia, and
51
meningitis or endocarditis on rare circumstances (Elzer et al., 2002; Godfroid et al.,
52
2005; Hamdy et al., 2002).
53
Brucella spp. contain three groups of major outer membrane proteins (Omps)
54
(Dubray & Bezard, 1980; Verstreate et al., 1982). The group 3 Omps consist of two
55
related proteins of 25 (Omp25) and 31 (Omp31) kDa (Cloeckaert et al., 1996;
56
Vizcaino et al., 1996). Analysis of the omp25 gene indicates that it is highly
57
conserved in Brucella species, biovars, and strains (Cloeckaert et al., 1995). OMP25
58
are surface-exposed, a protein known to be involved in Brucella virulence, which play
59
an important role in the attachment or invasion to the host cell. Omp25 mutant of B.
60
melitensis, B. abortus, and B. ovis strains have been found to be attenuated in mice,
61
goats (B. melitensis), and cattle (B. abortus) (Edmonds et al., 2001; 2002a; 2002b).
62
Phage display technique was set up in 1985 by Smith (Smith 1985), which has
63
been proven to be a powerful tool for studying protein–protein interactions, including
64
host–pathogen interactions, bacterial adhesins, and receptor–ligand (Bair et al., 2008;
65
Pande et al., 2010). Phage random peptide library contains a pool of billions of
66
peptides that can be produced by the fusion of random nucleic acid sequences to the N
67
terminus of one of the capsid protein genes (pVIII or pIII) of a filamentous
68
bacteriophage (Cwirla et al., 1990; Devlin et al., 1990; Scott & Smith, 1990). The
69
most important feature of this technique is the direct link between the experimental
70
phenotype and its encapsulated genotype (Azzazy & Highsmith, 2002). Phage display
71
is now playing an important role in discovering peptides and antibodies that may
72
serve as novel therapeutics (Nelson et al., 2010; Fjell et al., 2011).
73
In this study, we identified, for the first time, the specific peptides bind
74
specifically to Omp25 protein of pathogenic using a phage panning technique. Our
75
results showed that the peptide 0P11 was the most effective in inhibiting S2308
76
infection compared with other three selected peptides (OP27, OP35, and OP40) in
77
vitro and in vivo, which may be useful for developing new drugs against Brucella
78
infections.
79 80
2. Materials and methods
81
Recombinant Omp25 and 32A expression and purification
82
The Omp25 open reading frames were amplified by PCR from S2308 genome.
83
The designations of the primers were based on S2308. The primer sequences
84
omp25 gene (642 bp) were as follows: sense primer, 5'-GAATTCATGCGCACTCTT-
85
AAGTCTCTCGTA-3'
86
5'-GTCGACTTAGAACTTGTAGCCGATGC 3' (SalⅠsite underlined). The amplified
87
OMP25 gene was then cloned into the pET32a vector (Novagen) to generate the
88
recombinant plasmid pET32a-Omp25. The recombinant plasmid pET32a-Omp25 was
(EcoR
Ⅰ
site
)
underlined
and
antisense
of
primer,
89
then transformed into E. coli BL21 (DE3). Expression of recombinant protein was
90
induced with 1 mM IPTG generating N-terminally His-tagged fusion proteins. The
91
recombinant proteins (45.4 kDa) expressed from pET32a-Omp25 purified by affinity
92
chromatography with Ni2-conjugated Sepharose, and concentrated by Centricon 3
93
concentrators (Amicon). The pET32a empty vector was also transformed separately
94
into E. coli BL21 (DE3), and the protein (32A, 20.4kDa) contained the thioredoxin
95
and histidine fusion tag alone expressed and purified using the same method as above,
96
preparing for the control. The two proteins were confirmed by SDS-PAGE. Total
97
protein assays were carried out using BCA protein assay kit (Pierce, Rockford, IL,
98
USA).
99 100
Biopanning procedure
101
A cyclic 12-mer random peptide library (from New England BioLabs) was used in
102
this report. Biopanning was performed by coating a micro-plate well with 200µl of
103
purified Omp25 at a concentration of 40 µg/ml in 0.1 M NaHCO3 (pH 8.6) at 4°C
104
overnight in a humidified container. The wells were then blocked with 200 µl of
105
blocking solution (0.5% BSA + 0.1 M NaHCO3, w/v, pH 8.6) at 37°C for 1 h. The
106
phage library Ph.D. C7C were input at 2×1011 pfu/well in 100 µl of wash buffer
107
[1×TBS + 0.1% (v/v) Tween-20] and 1 h at 37°C with gentle agitation. The wells
108
were washed ten times with wash buffer to remove unbound phages. Bound phages
109
were
110
temperature and neutralized with 20 µl of 1 M Tris (pH 9.1). The eluted phage were
eluted with 200 µl of 0.2 M glycine-HCl (pH 2.2) for 10 min at room
111
titered and used to infect E. coli 2738 cells for amplification for the next round of
112
panning. Four rounds of panning were performed.
113 114
Phage tittering
115
ER2738 was inoculated and grown at 37°C to mid-log phase (OD600 ~0.5).
116
Ten-fold serial dilutions of phage containing suspension were prepared in LB-medium
117
(101 to 104 for unamplified panning eluates, 108 to 1011 for amplified cell culture
118
supernatants). Ten microliters of each phage dilution was added to 200 µl of mid-log
119
phase E. coli ER2738 and incubated with 4.0 ml of molten top agar (45° C) for 5
120
minutes. Infected cells were mixed with preheated agarose top, poured onto
121
prewarmed (37°C) LB/IPTG/X-Gal plates, and cool at room temperature for 10
122
minutes, then incubated overnight at 37°C. Phage titers were calculated by
123
multiplying the number of blue plaques by the dilution factor.
124 125
Plaque amplification
126
An overnight culture of E. coli ER 2738 was diluted 1:100 in LB and 200 µl was
127
dispensed into each microtiter well. Individual blue plaques were transferred to each
128
well and incubated at 37°C for 5 h with shaking. The microtiter plates were
129
centrifuged at 6000×g for 15 min at 4°C and the upper 80% of phage-containing
130
supernatant was collected and stored in 4°C.
131 132
Phage ssDNA extraction
133
Selected amplified phage (1 ml) from individual clones was transferred to a fresh
134
microcentrifuge tube. PEG-NaCl (400 µl) was added and incubated at room
135
temperature for 10 min. After centrifugation (15,000×g at 4°C for 15 min), the
136
supernatant was discarded, and the pellet was suspended thoroughly in 100 µl of
137
iodide buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 4 M NaI). Then 250 µl of
138
ethanol was added. The mixtures were incubated for 10 min at room temperature. The
139
mixture was centrifuged at 15,000×g for 15 min and and the supernatant was
140
discarded. The pellet was washed with 70 % ethanol, dried, and suspended in 40 µl of
141
sterile double-distilled water.
142 143
Nucleotide sequencing
144
DNA content was verified by electrophoresis on agarose gels. Single-stranded
145
DNA of individual isolated phage clones was purified according to the manufacture’s
146
protocol using Qiagen miniprep kit (Valencia, CA), then sequenced with the -96gIII
147
sequencing primer (New England Biolabs) performed by the Sangon Biotech Co. Ltd.
148
(Shanghai, China).
149 150
ELISA with isolated phage clones
151
The purified fusion proteins Omp25 and 32A were applied to 96-microtiter-well
152
plates (Nunc) (10 µg/well) and incubated overnight at 4°C. Unbound proteins were
153
removed, and the wells were blocked with blocked with blocking buffer (TBST
154
contained 3% BSA) at 37°C for 1 h. Selected phage clones (1×1010/well) were added
155
and incubated for 1.5 h at 37°C. Unbound phage was removed by washing buffer
156
(PBS–0.05% Tween 20) five times for 3 min. Peroxidase-labeled anti-M13-phage
157
antibodywas added for another hour. After washing four times with washing buffer,
158
the reaction was visualized with ABTS (Sigma, Munich, Germany) and optical
159
density read at 450 nm.
160 161
Affinity constant determination
162
The affinity (Kaff) constant of peptides (OP11, OP27, OP35, and OP40) was
163
determined by the previous method using a solid-phase non-competitive enzyme
164
immunoassay (Beatty et al., 1987).
165 166
MTT assay
167
RAW264.7 macrophages were seeded at 1×104 cells/ml in 96-well plates and
168
cells were washed thrice with PBS at 0, 12, 24 and 48 hours after incubation with
169
OP11, OP27, OP35, and OP40 peptides (20 µM), and were assessed for cell viability
170
using 3-(4,5-dimethy- lthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma).
171
Fifty µl of MTT was added to each well and incubated at 37°C for 4 h. Then the
172
supernatants was removed and 100 µl of Me2SO (Sigma, USA) was added to each
173
well. Optical density was determined by eluting the dye with dimethyl sulfoxide
174
(Sigma), and the absorbance was measured at 570 nm. Three independent
175
experiments were performed.
176
177
Cell test
178
Murine macrophage RAW264.7 cells were obtained from Type Culture
179
Collection of the Chinese Academy of Sciences (Shanghai, China) and were cultured
180
in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal
181
bovine serum (FBS). The cells were routinely cultured at 37°C in a 5% CO2
182
atmosphere. To analyze the effect of peptides on S2308 internalization, RAW264.7
183
cells in 6-well plates were incubated with peptides (10 µM) and S2308 (100 MOI) at
184
37°C for 30 and 45min. Cells were washed once with medium and then incubated
185
with in DMEM with gentamicin (30µg/ml) for 30 min to kill any remaining
186
extracellular and adherent bacteria. The cells were washed three times with PBS and
187
lysed and the live bacteria were enumerated by plating on TSA plates. To determine
188
the effect of peptides on S2308 intracellular growth, the infected cells were incubated
189
at 37°C for 40 min, and the media were replaced with DMEM, peptides, and
190
gentamicin, then incubated for 4, 24, and 48 h. Cell washing, lysis, and plating
191
procedures were the same as for the analysis of S2308 internalization. At 12 and 24 h
192
postinfection, the levels of TNF-α and IL-12 in the supernatants were measured using
193
an ELISA Quantikine Mouse kit (R&D Systems). Three independent experiments
194
were performed.
195 196
Mice experiments
197
All 6-week-old female BALB/c mice were obtained from the Experimental
198
Animal Center of Academy of Military Medical Science (Beijing, PRC). The mice
199
were divided into six groups (10 mice/group). Experimental groups of mice were
200
separately injected subcutaneously with S2308 (1 × 106 CFU/mouse) and any peptide
201
OP11, OP27, OP35, and OP40 (200 µg/mouse), and the control group was injected
202
with S2308 (1 × 106 CFU/mouse) and PBS (200 μl/mouse). At weeks 2, 5, and 7,
203
experimental groups of mice were given any peptide (200 µg/mouse) and control
204
groups were given PBS (200 μl/mouse) by subcutaneously injection. At 3, 6, and 9
205
weeks, the mice were euthanised, and their spleens were removed aseptically. The
206
spleens were collected aseptically for quantitation of bacterial burden, and spleens and
207
bodies were weighed. The ratio of spleen weight to body weight was then calculated.
208
The suspensions were diluted in sterile saline and plated on TSA. The plates were
209
incubated at 37°C, and the number of CFU was counted after three days. The
210
experiments were repeated thrice with similar results.
211 212
Statistical analysis
213
The data were analyzed using Student's t-test, and were expressed as mean value
214
± standard error of the mean (S.E.M.). The P values of < 0.05 were considered
215
statistically significant.
216 217
Results
218 219 220
Recombinant Omp25 expression and purification After induction with IPTG (1.0 mM) at 37°C for 4h, E. coli BL21 (DE3)
221
harboring pET32a-Omp25 or pET32a exhibited a high level of expression (data not
222
shown). The recombinant Omp25 protein were purified using Ni-NTA affinity column.
223
The 32A protein (20.4 kDa) expressed by pET32a empty vector itself were also
224
purified as described above. The results showed that two clear target bands appeared
225
approximately at the 46 kDa and 20 kDa positions (Fig. 1).
226 227
Phage display screening
228
In order to identify peptides that specifically bind to Omp25, four rounds of
229
bio-panning were performed using a random cyclic 7-mer peptide library. A gradual
230
increase in enriched phage from 5.5 × 104 to 2 × 108 pfu was monitored after each
231
round of panning for the antigen (Table 1). Forty-two phage clones were randomly
232
picked from the fourth round output phage and were subjected to sequencing and
233
more analysis.
234 235
In vitro confirmation of binding
236
In order to confirm the binding ability of selected phage clones to Omp25,
237
forty-two independent phage clones which were randomly picked after the fourth in
238
vitro panning were tested by ELISA, and 32A protein and PBS were used as the
239
control. Phage clones OP11, OP27, OP35, and OP40 appeared to bind better than the
240
other clones (Fig. 2). When the sequences of these phage clones were analyzed by
241
DNA sequencing, it was shown that OP11, OP27, OP35, and OP40 contained the
242
peptide sequences TTSLKTF, STPSSQT, SLTTSSN, and MSPSSNT (Table 2),
243
respectively, and they have a similarity of 46.43 % analyzed using the software
244
DNAMAN with default parameters and the ClustalW algorithm for multiple sequence
245
alignment. Moreover, the affinity constants (Kaff) of the different peptides was
246
measured (Table 3), which was consistent with the results of ELISA.
247 248
Effects of peptides on the growth of RAW264.7 macrophages
249
We next investigated whether TTSLKTF, STPSSQT, SLTTSSN, and MSPSSNT
250
peptides affect the growth of RAW264.7 macrophages. The cell viability of
251
RAW264.7 macrophages incubated with peptides (20 µM) was analyzed by MTT
252
assay at 0h, 12h, 24h, and 48h. There was no loss of viability of the peptide-treated
253
macrophages compared with that of control cells (P>0.05; Fig. 3). These results
254
suggest that these peptides do not affect the survival of RAW264.7 macrophages.
255 256
Inhibition of S2308 infection by the peptides in RAW264.7 macrophages
257
The mixture of S2308 and peptides were incubated with macrophages to test
258
whether the selected peptides inhibit S2308 internalization and intracellular growth.
259
At 30 min post-infection, a significant ( P
1
A novel Omp25-binding peptide screened by phage display can 2
inhibit Brucella abortus 2308 infection in vitro and in vivo 3
Authors 4 5
Junbo Zhang 1, Fei Guo 2, Xiaoqiang Huang3, Chuangfu Chen1*, Ruitian Liu4*, Hui Zhang1, Yuanzhi Wang2, Shuanghong Yin2, Zhiqiang Li1
6
1
7
China
8
2
College of Medicine, Shihezi University, Shihezi, 832003, China
9
3
College of Life Sciences, Shihezi University, Xinjiang 832000, China
10
4
College of Life Sciences, Tsinghua University, Beijing 100083, China
*
Corresponding author:
College of Animal Science and Technology, Shihezi University, Shihezi 832003,
11 12 13 14
E-mail address: [email protected] (CC); [email protected] (RL)
15
Fax: +86-0993-2058612
16
Tel: +86-0993-2058002
17 18 19 20 21 22
23
Abstract
24
Brucellosis is a globally distributed zoonotic disease that causes animal and
25
human diseases, while current antibiotic and vaccine strategies are not optimal. The
26
surface-exposed protein OMP25 is involved in Brucella virulence and play an
27
important role in Brucella pathogenesis during infection, suggesting that Omp25 is
28
an useful target for selecting potential therapeutic molecules to inhibit Brucella
29
pathogenesis. In this study, we identified, for the first time, the specific peptides
30
bind specifically to Omp25 protein of pathogen using a phage panning technique,
31
After four rounds of panning, 42 plaques of eluted phages were subjected to
32
pyrosequencing. Four phage clones bound better than the other clones were
33
selected through the confirmation of ELISA and affinity constants. Selected
34
peptides could inhibit significantly Brucella abortus 2308 (S2308) internalization
35
and intracellular growth in RAW264.7 macrophages, and induce significantly
36
secretion of tumor necrosis factor alpha (TNF-α) and interleukin-12 in peptide- and
37
S2308-treated cells. Any observed peptide (OP11, OP27, OP35, or OP40) could
38
inhibit significantly S2308 infection in BALB/c mice. Moreover, the peptide 0P11
39
was the best candidate peptide in inhibiting S2308 infection in vitro and in vivo.
40
These results suggest that peptide OP11 has potential for exploitation as a peptide
41
drug in resisting S2308 infection.
42
Keywords: Brucella abortus 2308, Phage-display, Peptides, Inhibition infection
43 44
1. Introduction
45
Bacteria of the genus Brucella are gram-negative and facultative intracellular
46
pathogens that pose threats to animal and human health throughout the world. The
47
main pathogenic species worldwide are B.abortus and B. melitensis, which can cause
48
abortions and infertility in cattle and sheep, resulting in heavy economic losses. The
49
human disease is also caused by Brucella melitensis, Brucella abortus or Brucella
50
suis. Infection in humans can cause fever, arthritis, spondylitis, dementia, and
51
meningitis or endocarditis on rare circumstances (Elzer et al., 2002; Godfroid et al.,
52
2005; Hamdy et al., 2002).
53
Brucella spp. contain three groups of major outer membrane proteins (Omps)
54
(Dubray & Bezard, 1980; Verstreate et al., 1982). The group 3 Omps consist of two
55
related proteins of 25 (Omp25) and 31 (Omp31) kDa (Cloeckaert et al., 1996;
56
Vizcaino et al., 1996). Analysis of the omp25 gene indicates that it is highly
57
conserved in Brucella species, biovars, and strains (Cloeckaert et al., 1995). OMP25
58
are surface-exposed, a protein known to be involved in Brucella virulence, which play
59
an important role in the attachment or invasion to the host cell. Omp25 mutant of B.
60
melitensis, B. abortus, and B. ovis strains have been found to be attenuated in mice,
61
goats (B. melitensis), and cattle (B. abortus) (Edmonds et al., 2001; 2002a; 2002b).
62
Phage display technique was set up in 1985 by Smith (Smith 1985), which has
63
been proven to be a powerful tool for studying protein–protein interactions, including
64
host–pathogen interactions, bacterial adhesins, and receptor–ligand (Bair et al., 2008;
65
Pande et al., 2010). Phage random peptide library contains a pool of billions of
66
peptides that can be produced by the fusion of random nucleic acid sequences to the N
67
terminus of one of the capsid protein genes (pVIII or pIII) of a filamentous
68
bacteriophage (Cwirla et al., 1990; Devlin et al., 1990; Scott & Smith, 1990). The
69
most important feature of this technique is the direct link between the experimental
70
phenotype and its encapsulated genotype (Azzazy & Highsmith, 2002). Phage display
71
is now playing an important role in discovering peptides and antibodies that may
72
serve as novel therapeutics (Nelson et al., 2010; Fjell et al., 2011).
73
In this study, we identified, for the first time, the specific peptides bind
74
specifically to Omp25 protein of pathogenic using a phage panning technique. Our
75
results showed that the peptide 0P11 was the most effective in inhibiting S2308
76
infection compared with other three selected peptides (OP27, OP35, and OP40) in
77
vitro and in vivo, which may be useful for developing new drugs against Brucella
78
infections.
79 80
2. Materials and methods
81
Recombinant Omp25 and 32A expression and purification
82
The Omp25 open reading frames were amplified by PCR from S2308 genome.
83
The designations of the primers were based on S2308. The primer sequences
84
omp25 gene (642 bp) were as follows: sense primer, 5'-GAATTCATGCGCACTCTT-
85
AAGTCTCTCGTA-3'
86
5'-GTCGACTTAGAACTTGTAGCCGATGC 3' (SalⅠsite underlined). The amplified
87
OMP25 gene was then cloned into the pET32a vector (Novagen) to generate the
88
recombinant plasmid pET32a-Omp25. The recombinant plasmid pET32a-Omp25 was
(EcoR
Ⅰ
site
)
underlined
and
antisense
of
primer,
89
then transformed into E. coli BL21 (DE3). Expression of recombinant protein was
90
induced with 1 mM IPTG generating N-terminally His-tagged fusion proteins. The
91
recombinant proteins (45.4 kDa) expressed from pET32a-Omp25 purified by affinity
92
chromatography with Ni2-conjugated Sepharose, and concentrated by Centricon 3
93
concentrators (Amicon). The pET32a empty vector was also transformed separately
94
into E. coli BL21 (DE3), and the protein (32A, 20.4kDa) contained the thioredoxin
95
and histidine fusion tag alone expressed and purified using the same method as above,
96
preparing for the control. The two proteins were confirmed by SDS-PAGE. Total
97
protein assays were carried out using BCA protein assay kit (Pierce, Rockford, IL,
98
USA).
99 100
Biopanning procedure
101
A cyclic 12-mer random peptide library (from New England BioLabs) was used in
102
this report. Biopanning was performed by coating a micro-plate well with 200µl of
103
purified Omp25 at a concentration of 40 µg/ml in 0.1 M NaHCO3 (pH 8.6) at 4°C
104
overnight in a humidified container. The wells were then blocked with 200 µl of
105
blocking solution (0.5% BSA + 0.1 M NaHCO3, w/v, pH 8.6) at 37°C for 1 h. The
106
phage library Ph.D. C7C were input at 2×1011 pfu/well in 100 µl of wash buffer
107
[1×TBS + 0.1% (v/v) Tween-20] and 1 h at 37°C with gentle agitation. The wells
108
were washed ten times with wash buffer to remove unbound phages. Bound phages
109
were
110
temperature and neutralized with 20 µl of 1 M Tris (pH 9.1). The eluted phage were
eluted with 200 µl of 0.2 M glycine-HCl (pH 2.2) for 10 min at room
111
titered and used to infect E. coli 2738 cells for amplification for the next round of
112
panning. Four rounds of panning were performed.
113 114
Phage tittering
115
ER2738 was inoculated and grown at 37°C to mid-log phase (OD600 ~0.5).
116
Ten-fold serial dilutions of phage containing suspension were prepared in LB-medium
117
(101 to 104 for unamplified panning eluates, 108 to 1011 for amplified cell culture
118
supernatants). Ten microliters of each phage dilution was added to 200 µl of mid-log
119
phase E. coli ER2738 and incubated with 4.0 ml of molten top agar (45° C) for 5
120
minutes. Infected cells were mixed with preheated agarose top, poured onto
121
prewarmed (37°C) LB/IPTG/X-Gal plates, and cool at room temperature for 10
122
minutes, then incubated overnight at 37°C. Phage titers were calculated by
123
multiplying the number of blue plaques by the dilution factor.
124 125
Plaque amplification
126
An overnight culture of E. coli ER 2738 was diluted 1:100 in LB and 200 µl was
127
dispensed into each microtiter well. Individual blue plaques were transferred to each
128
well and incubated at 37°C for 5 h with shaking. The microtiter plates were
129
centrifuged at 6000×g for 15 min at 4°C and the upper 80% of phage-containing
130
supernatant was collected and stored in 4°C.
131 132
Phage ssDNA extraction
133
Selected amplified phage (1 ml) from individual clones was transferred to a fresh
134
microcentrifuge tube. PEG-NaCl (400 µl) was added and incubated at room
135
temperature for 10 min. After centrifugation (15,000×g at 4°C for 15 min), the
136
supernatant was discarded, and the pellet was suspended thoroughly in 100 µl of
137
iodide buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 4 M NaI). Then 250 µl of
138
ethanol was added. The mixtures were incubated for 10 min at room temperature. The
139
mixture was centrifuged at 15,000×g for 15 min and and the supernatant was
140
discarded. The pellet was washed with 70 % ethanol, dried, and suspended in 40 µl of
141
sterile double-distilled water.
142 143
Nucleotide sequencing
144
DNA content was verified by electrophoresis on agarose gels. Single-stranded
145
DNA of individual isolated phage clones was purified according to the manufacture’s
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protocol using Qiagen miniprep kit (Valencia, CA), then sequenced with the -96gIII
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sequencing primer (New England Biolabs) performed by the Sangon Biotech Co. Ltd.
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(Shanghai, China).
149 150
ELISA with isolated phage clones
151
The purified fusion proteins Omp25 and 32A were applied to 96-microtiter-well
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plates (Nunc) (10 µg/well) and incubated overnight at 4°C. Unbound proteins were
153
removed, and the wells were blocked with blocked with blocking buffer (TBST
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contained 3% BSA) at 37°C for 1 h. Selected phage clones (1×1010/well) were added
155
and incubated for 1.5 h at 37°C. Unbound phage was removed by washing buffer
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(PBS–0.05% Tween 20) five times for 3 min. Peroxidase-labeled anti-M13-phage
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antibodywas added for another hour. After washing four times with washing buffer,
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the reaction was visualized with ABTS (Sigma, Munich, Germany) and optical
159
density read at 450 nm.
160 161
Affinity constant determination
162
The affinity (Kaff) constant of peptides (OP11, OP27, OP35, and OP40) was
163
determined by the previous method using a solid-phase non-competitive enzyme
164
immunoassay (Beatty et al., 1987).
165 166
MTT assay
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RAW264.7 macrophages were seeded at 1×104 cells/ml in 96-well plates and
168
cells were washed thrice with PBS at 0, 12, 24 and 48 hours after incubation with
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OP11, OP27, OP35, and OP40 peptides (20 µM), and were assessed for cell viability
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using 3-(4,5-dimethy- lthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma).
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Fifty µl of MTT was added to each well and incubated at 37°C for 4 h. Then the
172
supernatants was removed and 100 µl of Me2SO (Sigma, USA) was added to each
173
well. Optical density was determined by eluting the dye with dimethyl sulfoxide
174
(Sigma), and the absorbance was measured at 570 nm. Three independent
175
experiments were performed.
176
177
Cell test
178
Murine macrophage RAW264.7 cells were obtained from Type Culture
179
Collection of the Chinese Academy of Sciences (Shanghai, China) and were cultured
180
in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal
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bovine serum (FBS). The cells were routinely cultured at 37°C in a 5% CO2
182
atmosphere. To analyze the effect of peptides on S2308 internalization, RAW264.7
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cells in 6-well plates were incubated with peptides (10 µM) and S2308 (100 MOI) at
184
37°C for 30 and 45min. Cells were washed once with medium and then incubated
185
with in DMEM with gentamicin (30µg/ml) for 30 min to kill any remaining
186
extracellular and adherent bacteria. The cells were washed three times with PBS and
187
lysed and the live bacteria were enumerated by plating on TSA plates. To determine
188
the effect of peptides on S2308 intracellular growth, the infected cells were incubated
189
at 37°C for 40 min, and the media were replaced with DMEM, peptides, and
190
gentamicin, then incubated for 4, 24, and 48 h. Cell washing, lysis, and plating
191
procedures were the same as for the analysis of S2308 internalization. At 12 and 24 h
192
postinfection, the levels of TNF-α and IL-12 in the supernatants were measured using
193
an ELISA Quantikine Mouse kit (R&D Systems). Three independent experiments
194
were performed.
195 196
Mice experiments
197
All 6-week-old female BALB/c mice were obtained from the Experimental
198
Animal Center of Academy of Military Medical Science (Beijing, PRC). The mice
199
were divided into six groups (10 mice/group). Experimental groups of mice were
200
separately injected subcutaneously with S2308 (1 × 106 CFU/mouse) and any peptide
201
OP11, OP27, OP35, and OP40 (200 µg/mouse), and the control group was injected
202
with S2308 (1 × 106 CFU/mouse) and PBS (200 μl/mouse). At weeks 2, 5, and 7,
203
experimental groups of mice were given any peptide (200 µg/mouse) and control
204
groups were given PBS (200 μl/mouse) by subcutaneously injection. At 3, 6, and 9
205
weeks, the mice were euthanised, and their spleens were removed aseptically. The
206
spleens were collected aseptically for quantitation of bacterial burden, and spleens and
207
bodies were weighed. The ratio of spleen weight to body weight was then calculated.
208
The suspensions were diluted in sterile saline and plated on TSA. The plates were
209
incubated at 37°C, and the number of CFU was counted after three days. The
210
experiments were repeated thrice with similar results.
211 212
Statistical analysis
213
The data were analyzed using Student's t-test, and were expressed as mean value
214
± standard error of the mean (S.E.M.). The P values of < 0.05 were considered
215
statistically significant.
216 217
Results
218 219 220
Recombinant Omp25 expression and purification After induction with IPTG (1.0 mM) at 37°C for 4h, E. coli BL21 (DE3)
221
harboring pET32a-Omp25 or pET32a exhibited a high level of expression (data not
222
shown). The recombinant Omp25 protein were purified using Ni-NTA affinity column.
223
The 32A protein (20.4 kDa) expressed by pET32a empty vector itself were also
224
purified as described above. The results showed that two clear target bands appeared
225
approximately at the 46 kDa and 20 kDa positions (Fig. 1).
226 227
Phage display screening
228
In order to identify peptides that specifically bind to Omp25, four rounds of
229
bio-panning were performed using a random cyclic 7-mer peptide library. A gradual
230
increase in enriched phage from 5.5 × 104 to 2 × 108 pfu was monitored after each
231
round of panning for the antigen (Table 1). Forty-two phage clones were randomly
232
picked from the fourth round output phage and were subjected to sequencing and
233
more analysis.
234 235
In vitro confirmation of binding
236
In order to confirm the binding ability of selected phage clones to Omp25,
237
forty-two independent phage clones which were randomly picked after the fourth in
238
vitro panning were tested by ELISA, and 32A protein and PBS were used as the
239
control. Phage clones OP11, OP27, OP35, and OP40 appeared to bind better than the
240
other clones (Fig. 2). When the sequences of these phage clones were analyzed by
241
DNA sequencing, it was shown that OP11, OP27, OP35, and OP40 contained the
242
peptide sequences TTSLKTF, STPSSQT, SLTTSSN, and MSPSSNT (Table 2),
243
respectively, and they have a similarity of 46.43 % analyzed using the software
244
DNAMAN with default parameters and the ClustalW algorithm for multiple sequence
245
alignment. Moreover, the affinity constants (Kaff) of the different peptides was
246
measured (Table 3), which was consistent with the results of ELISA.
247 248
Effects of peptides on the growth of RAW264.7 macrophages
249
We next investigated whether TTSLKTF, STPSSQT, SLTTSSN, and MSPSSNT
250
peptides affect the growth of RAW264.7 macrophages. The cell viability of
251
RAW264.7 macrophages incubated with peptides (20 µM) was analyzed by MTT
252
assay at 0h, 12h, 24h, and 48h. There was no loss of viability of the peptide-treated
253
macrophages compared with that of control cells (P>0.05; Fig. 3). These results
254
suggest that these peptides do not affect the survival of RAW264.7 macrophages.
255 256
Inhibition of S2308 infection by the peptides in RAW264.7 macrophages
257
The mixture of S2308 and peptides were incubated with macrophages to test
258
whether the selected peptides inhibit S2308 internalization and intracellular growth.
259
At 30 min post-infection, a significant ( P
