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Page 1 of 21
Protective efficacy of recombinant exotoxin A-flagellin fusion
1
protein against Pseudomonas aeruginosa infection
2 3
Safar Farajnia1, Shahin Najar Peerayeh*2, Asghar Tanomand 3, Jafar Majidi4,
4
Gholamreza Ghoudarzi5, Behrooz Naghili1, Leila Rahbarnia6
5 6
1. Research Center for Infectious and Tropical Disease, Tabriz University of Medical Sciences Tabriz, Iran. Email: [email protected]
8
2. Dept of Bacteriology Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Email: [email protected]
Sciences,
Tarbiat
Modares
University,
9 10
3. Tabriz University of Medical Sciences, Tabriz and Dept of Bacteriology Faculty of Medical
7
Tehran,
Iran.
Email:
[email protected]
11 12 13
4. Dept of Immunology and Immunology research center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. Email: [email protected] 5. Department of Microbiology, Faculty of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran. Email:[email protected] 6. Drug Applied Research Center, Tabriz University of Medical Sciences Tabriz, Iran. Email: [email protected]
14 15 16 17 18 19 20
*Corresponding authors:
21
Shahin Najar Peerayeh, Department of Bacteriology, Faculty of Medical sciences,
22
Tarbiat Modares University, Tehran, I.R. Iran
23
Tel: +98 21 82883870
24
E-mail: [email protected]
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Abstract
25
Pseudomonas (P.) aeruginosa is an opportunistic bacterium causes serious
26
nosocomial infection in immunocompromised patients. The aim of this study was to
27
prepare a fusion protein consisting exotoxin A (ExoA) and flagellin (Fla) from P.
28
aeruginosa and evaluate its potential as a vaccine candidate against P. aeruginosa
29
infection. The genes encoding for ExoA and Fla proteins were cloned in frame and
30
expressed in E. coli. The recombinant ExoA-Fla fusion protein was purified by Ni-
31
NTA affinity chromatography. Mice were immunized subcutaneously with exotoxin
32
A, flagellin, and flagellin - exotoxin A fusion proteins and the humoral immune
33
response was evaluated by ELISA method. The immunized and control group mice
34
were challenged with a 2X LD50 (7.5 x 107 CFU) of P. aeruginosa for protection
35
assay. The results indicated that vaccination with flagellin, exotoxin A and flagellin
36
- exotoxin A fusion proteins produced significant amount of specific IgG antibodies.
37
Immunization of mice with exotoxin A-flagellin fusion protein showed significant
38
protection against intra-peritoneal challenge with 7.5 x 107 CFU (2X LD50) P.
39
aeruginosa. Results of this study suggest that recombinant exotoxin A-flagellin
40
fusion protein is a highly immunogenic protective protein which can be used as a
41
promising vaccine candidate against P. aeruginosa.
42
Keywords: Exotoxin A-flagellin, P. aeruginosa, vaccine candidate, fusion protein
43 44
1. Introduction
45
Pseudomonas (P) aeruginosa is an opportunistic bacterium associated with
46
nosocomial
in
47
immunocompromised patients. The range of diseases caused by this bacterium
48
infections
leading
to
septicemia
and
death
especially
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Page 3 of 21
varies from superficial skin infections to serious systemic infections such as
49
fulminate sepsis. P. aeruginosa is the second most common causative agent of
50
hospital-acquired pneumonia, healthcare-associated pneumonia and ventilator-
51
associated pneumonia. One of the most important features of this bacterium is its
52
resistance to various antibacterial agents (Tavajjohi and Moniri 2011), and even
53
newly developed antibiotics have failed to reduce the mortality rate associated with
54
this organism. This failure in response to antimicrobial treatment has led researches
55
to vaccines and immunotherapy as alternative treatment methods for P. aeruginosa
56
infections. It has been suggested that neutralization of bacterial virulence factors by
57
immunological methods can result in prevention and reduction of mortalities due to
58
P. aeruginosa infections (Doring and Pierb 2008).
59
For this purpose, different antigenic and virulence factors such as outer membrane
60
proteins, toxins, flagella, pilli, and high molecular weight polysaccharides have been
61
evaluated as vaccine candidates (Manafi et al. 2009, Cryz et al. 1987, Eric et al.
62
2009, Doring et al. 2007), but there is no any approved vaccine currently available
63
against P. aeruginosa.
64
Exotoxin A plays an important role in virulence of P. aeruginosa and it has shown
65
that exotoxin A (ExoA) deficient mutants exhibit a virulence 20 times less than the
66
wild type strain in the mouse models (Wolf and Beile 2009). ExoA has 3 structural
67
domains, domain 1 is receptor binding domain, domain 2 is transfer domain and
68
domain 3 is toxic domain. ExoA catalysis inhibition of protein synthesis by ADP-
69
ribosylation of elongation factor 2. Exotoxin A is also an antigenic protein that
70
induces humoral immune responses in animal models. Based on the results of
71
different studies, these antibodies are highly protective. Therefore, exotoxin A has
72
been considered as a promising vaccine candidate for pseudomonas infections
73
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Page 4 of 21
(Hertle et al. 2001, Denis-Mize and Price 2000, Chen and Shang 1999,
74
Campodo´nico et al. 2010).
75
The bacterial flagellum is a strong immunogenic factor and active or passive
76
immunization with flagellar antigens induces antibody production. These antibodies
77
inhibit bacterial distribution, and thus prevent systemic infection in a mouse model
78
of burn and pulmonary infections (Campodo´nico et al. 2010). Studies shown that
79
monoclonal antibodies against P. aeruginosa flagellin are protective against P.
80
aeruginosa infections in various animal models, hence suggested as a vaccine
81
candidate for P. aeruginosa (Matsumoto et al. 1999, Barnea et al. 2009, Nilsson et
82
al. 2007).
83
In this study, we reported preparation and evaluation of recombinant exotoxin A-
84
flagellin fusion protein, as a new vaccine candidate for P. aeruginosa infections.
85
2. Materials and Methods
86
2.1. Preparation of recombinant flagellin - exotoxin A fusion protein
87
Domains I-II of Exotoxin A gene (ExoA) from P. aeruginosa strain PAO1 and N-
88
terminal part of flagellin gene (Fla) from P. aeruginosa strain 8821M were amplified
89
separately by polymerase chain reaction (PCR) using specific primers. PCR products
90
were gel purified separately by purification kit (Macherey Nagel, Germany) and
91
analyzed by electrophoresis. Then, the ExoA-Fla fusion gene was constructed by
92
PCR-mediated overlap extension method using ExoA forward and Fla reverse
93
primers (Heckman and Pease 2007). Cloning of fusion gene was carried out by
94
ligation of PCR product into the PTZ57R vector using T-A cloning method
95
according to the manufacturer instructions (Fermentas, Lithuania).The ligate was
96
transformed into the E. coli DH5α and screening was performed by PCR and
97
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Page 5 of 21
restriction analysis. A positive clone of PTZ-ExoA-Fla was sequenced for analysis
98
of the sequence integrity.
99
For recombinant expression of ExoA-Fla fusion protein in E. coli, the insert was
100
removed from PTZ-ExoA-Fla vector by digestion with BamHI and XhoI enzymes
101
and subcloned into the PET 22b expression vector. Then, the pET22b-ExoA-Fla
102
construct was transformed into E. coli BL21, and the protein expression was
103
assessed using SDS-PAGE. Ni-NTA affinity chromatography method (Qiagen,
104
Chatsworth, CA, USA) was used for His-tagged fusion protein purification
105
according to the manufacturer instructions. The purified recombinant protein was
106
dialyzed against PBS, PH 7.4 for removing imidazole. The purity of protein was
107
analyzed by SDS-PAGE, and product concentration assessed by Bradford method
108
(Sigma, Product Number B6916). Western blotting with antibody to P. aeruginosa
109
native exotoxin A (Sigma Product Number P2318) was used to evaluate the
110
immunological properties of the recombinant ExoA-Fla fusion protein. Finally,
111
lipopolysaccharide (LPS) contamination of purified recombinant protein was
112
assessed by Limulus amebocyte lysate (LAL) assay method.
113
2.2. Preparation of recombinant flagellin (N-terminal) and exotoxin A (Domains I,
114
II) proteins
115
Recombinant flagellin was prepared as described elsewhere (Goudarzi et al 2009).
116
Recombinant exotoxin A (Domains I, II) protein were purified from E. coli BL21
117
carrying the pET22b - exotoxin A (Domains I, II) by Ni-NTA affinity
118
chromatography (Tanomand et al. 2012; Farajnia et al. 2011).
119
2.3. Toxicity test
120
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To test the toxicity of recombinant antigens in mice, various doses (between 100µg
121
and 300 µg) of antigens were subcutaneously injected into 6-8 week old Balb/C
122
mice (6 mice per group). Animal’s mortality was followed for 7 days.
123
2.4. Mice immunization
124
6-8 week old female BALB/c mice were divided into four groups (5 mice per
125
group) and subcutaneously injected with 20 µg recombinant flagellin, exotoxin A
126
(Domains I, II), ExoA-Fla fusion protein, or PBS on days 0 (with complete Freund’s
127
adjuvant), days 21, 42 (with incomplete Freund’s adjuvant), and finally on day 72
128
(without adjuvant). One week after the last injection, the animals were bled from the
129
orbital sinus and sera were prepared and stored at -20 until analysis.
130
2.5. Enzyme-Linkd Immunosorbent Assay (ELISA)
131
ELISAs were performed by standard methods as described previously (11, 18). In
132
brief, microtiter plate was coated with flagellin, exotoxin A or flagellin - exotoxin A
133
fusion protein at a concentration of 5 µg /ml in 0.1 M carbonate/bicarbonate (PH
134
9.6). The plates were incubated for 1 hour at 37°C and then blocked with 2% bovine
135
serum albumin in PBS containing 0.05% Tween 20 (PBS-T), 60 min. between
136
incubation steps, plates were washed three times with PBS-T. Sera samples diluted
137
1:10000 were added and incubated for 60 min, followed by incubation with
138
peroxidase conjugated anti-mouse antibody (abcam) as secondary antibody.
139
Peroxidase activity was detected using 3, 3´, 5,5´-tetramethyl-benzidine (TMB,
140
Sigma), stopped with 1M H2SO4 and the absorbance values was measured at 450
141
nm.
142 143
2.6. Opsonophagocytosis assay
144
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Opsonophagocytosis assay were performed as described previously [11,18] with
145
some modifications. Briefly, a mucoid colony of P. aeruginosa 8821M was grown
146
to mid-log phase on LB broth at 37°C with shaking, washed twice in cold PBS and
147
suspended in PBS at a final concentration of approximately 106 bacteria per ml.
148
Opsonization was performed by mixing 100µl bacteria (106 CFU/ml) for 30 min at
149
37°C in 100µl complement-inactivated sera obtained from various vaccinated and
150
control groups. A complement source were added to these pre-opsonized bacteria
151
and incubated for 90 min at 37°C with gentle shaking. Controls consisted of tubes
152
from which macrophage, complement, or serum was omitted. After 90 min of
153
incubation, 10 µl samples was removed, diluted in PBS and plated for bacterial
154
counts. The plates were incubated overnight at 37°C and bacterial colonies were
155
counted. The kill percentage was calculated as follows:
156
Kill Percentage = [1-(CFU of immune serum: CFU of pre-immune serum] x 100
157
2.7. Bacterial challenge
158
At first, 30 female BALB/c mice were divided into five groups (6 mice per group)
159
for determination of bacterial infection LD50. Mice were intraperitoeally injected
160
with Serial dilutions of P. aeruginosa (2.5 x 107, 5 x 107, 7.5 x 107, 1 x 108, and 12.5
161
x 108 CFU). The mice were followed for 10 days, mortality was recorded, and LD50
162
was determined according to the Reed and Muench method [19]. In challenge
163
experiment, two weeks after the final immunization, the immunized and control
164
group mice were challenged with intraperitoeal injection of 7.5 x 107 CFU of a
165
clinical strain of P. aeruginosa. The survival rate was recorded in all groups for 10
166
days. This strain was a mucoid phenotype, flagellated and Exotoxin A positive strain
167
that was originally isolated from 56 years old male patient with wound and systemic
168
infection, that showed resistance to chloramphenicol , ceftriaxone, piperacillin,
169
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cotrimoxazole and was sensitive imipenem, cotrimoxazole, cefoperazone, amikacin
170
and Ciprofloxacin.
171 172
2.8. Statistical analysis
173
The SPSS version 12 was used for data analysis. Differences in the mean percentage
174
of opsonic kill, and the mean ELISA absorbance were compared by analysis of
175
variance (ANOVA) using a post hoc multiple comparison test. The chi square test
176
was used to analyze the survival data of protection assay. P≤0.05 was considered
177
significant.
178
3. Results
179
3.1. Preparation of recombinant proteins
180
Schematic representation of expression construct that were used for preparation of
181
recombinant proteins has shown in Fig.1. The prepared exotoxin A (Domains I, II) -
182
flagellin(N-terminal) fusion protein composed of 574 amino acids including 404
183
amino acids from exotoxin A (Domains I, II) and 170 amino acids from flagellin(N-
184
terminal).
185
The exotoxin A - flagellin fusion protein, flagellin and exotoxin A were highly
186
expressed in E. coli and purified from inclusion bodies upon solubilization in 8M
187
urea and passage over a Ni-NTA column (Fig 2).
188
Western blotting analysis showed that recombinant flagellin, exotoxin A and
189
exotoxin A– flagellin fusion protein all were reacted with antibody against native
190
Exotoxin A.
191
Evaluation of the Toxicity of recombinant antigens showed that none of mice
192
injected with various doses of antigens (between 100µg and 300 µg) died after 8
193
weeks.
194
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3.2. Immune responses and ELISA results
195
Analysis of antibody production in immunized and control mice showed that
196
vaccination with flagellin, exotoxin A and exotoxin A - flagellin fusion protein
197
produced significant amount of specific antibody. No reacting antibody was detected
198
in mice injected with PBS. As shown in Table 1, the antibody titers in mice
199
vaccinated with exotoxin A - flagellin fusion protein were higher than other
200
vaccinated groups.
201
3.3. Opsonic activity of antisera against P. aeruginosa
202
Opsonophagocytic assay results are shown in Fig 3. In the opsonophagocytic killing
203
assay, antisera from mice immunized with the exotoxin A- flagellin and flagellin
204
alone showed significant phagocytic killing (68%) against the P. aeruginosa 8821M,
205
but antiserum from mice primed with exotoxin A alone was less efficient in
206
mediating phagocytic killing (4%). There were no phagocytic killings in control
207
tubes.
208
3.4. Protection assay results
209
For determining the LD50 of challenge strain, five groups of mice (6 mice per
210
group) were injected with different CFU of bacteria (2.5 x 107, 5 x 107, 7.5 x 107, 1 x
211
108, and 12.5 x 108 CFU). The ratio of killed/total injected animals were 0/6, 4/6,
212
6/6, 6/6 and 6/6, respectively. Therefore, about 3.5 x 107 CFU were selected as
213
LD50 and 7.5 x 107 CFU as 2xLD50 for challenge test in immunized and control
214
group mice.
215
The results of survival rate after bacterial challenge test in immunized and control
216
group mice have shown in Table 2. The mice immunized with exotoxin A– flagellin
217
fusion protein showed significant protection (p≤0.05) against intraperitoneal
218
injection of 7.5 x 107 CFU (2xLD50) clinical strain of P. aeruginosa. However, there
219
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Page 10 of 21
were no significant differences in the rates of survival of mice immunized with
220
exotoxin A (p≤0.49) and flagellin (p≤0.19) in comparing with control group.
221
4. Discussion
222
P. aeruginosa is an important cause of nosocomial infections with high mortality
223
and morbidity rates. One of the most important features of the bacterium is its
224
resistance to various antibacterial agents, and even newly developed antibiotics have
225
failed to reduce the mortality rate associated with this organism. Various new
226
approaches have been investigated to combat pseudomonas infection among them;
227
immunotherapy and vaccine development seems to be promising therapeutic and
228
preventive approaches (Doring and Pierb 2008). Several extracellular products and
229
cell components of P. aeruginosa such as exotoxin A (Shiau et al. 2001), outer
230
membrane proteins (Eric et al. 2009, Chen and Shang 1999), flagellin
231
(Campodo´nico et al. 2010, Neville et al. 2005), pillin (Hertle et al. 2001) and
232
alginate (Kashef et al. 2006) or combination of them have been studied as vaccine
233
candidates(Eric et al. 2009, Chen and Shang 1999, Kashef et al. 2006, Cryz et al.
234
1991, Kamei et al. 2011). In this article, we report development of a chimeric
235
subunit vaccine composed of two important components of P. aeruginosa virulence
236
factor, exotoxin A and flagellin. The exotoxin A is an important pathogenic factor,
237
inhibiting protein synthesis in the host cells through ADP-ribosylation of elongation
238
factor 2 (Cryz et al. 1991). We prepared a deletion mutant from exotoxin A lacking
239
the toxic domain (Chen and Shang 1999). The bacterial flagella are also a strong
240
immunogenic factor and it has shown that active or passive immunization with
241
flagellar antigens induces antibody response. These antibodies inhibit bacterial
242
distribution, and thus prevent disseminated infection in a mouse model of burn and
243
pulmonary infections (Campodo´nico et al. 2010). Analysis of the primary amino
244
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Page 11 of 21
acid sequences of flagellin from different gram-negative bacteria showed that their
245
N terminal part (aa 1-170) are well conserved (Neville 2005). Therefore we used N-
246
terminal 170 amino acids from Fla gene in fusion protein construction.
247
The results of this study showed all three preparations exotoxin A, flagellin and
248
exotoxin A - flagellin fusion were highly immunogenic and induced significant IgG
249
antibodies production in immunized mice (Table 1). We also found that injection of
250
exotoxin A- flagellin fusion protein induced higher antibody titers than two other
251
antigens. Therefore, recombinant exotoxin A-flagellin fusion protein could be
252
considered as an important vaccine candidate for generating anti- exotoxin A and
253
anti-flagellin responses.
254
The results of opsonophagocytic assay showed, sera from mice immunized with
255
either fusion protein or flagellin had moderate opsonic killing activity (52-48%)
256
against P. aeruginosa, but sera from mice immunized with exotoxin A alone did not
257
show any opsonophagocytic activity. These findings could be attributed to the
258
extracellular secretary nature of exotoxin A. The results were consistent with reports
259
of Chen et al (1999) and Campodo´nico et al (2010) and indicated that anti-flagellin
260
antibody is responsible for opsonophagocytic activity of sera from mice immunized
261
with fusion protein.
262
Vaccination with exotoxin A-flagellin fusion protein afforded significant protection
263
against challenge with clinical strain of p. aeruginosa, whereas exotoxin A alone
264
was partially effective in survival rate. These observations were consistent with
265
findings of Chen et al. (1999). They reported a significant protection in burned mice
266
immunized with recombinant fusion protein composed of exotoxin A, outer
267
membrane proteins I and F, but less protection by immunization with exotoxin A
268
alone.
269
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This may not be surprising since the pathogenicity of P. aeruginosa are dependent
270
on both extracellular and cell associated virulence factors. Hence it could be
271
concluded that immunization with cocktail antigens specially those containing both
272
extracellular and cell-associated virulent/antigenic factors can induce more efficient
273
protective response against P. aeruginosa infections than single antigens.
274
Based on our results, the survival rate of immunized mice (as a measure of
275
protection) in different groups was correlated with antibody level. This finding was
276
in line with previous reports (Barnea et al. 2009, Nilsson et al. 2007) and elucidated
277
the role of humoral immune response in immunity against P. aeruginosa infections.
278
In conclusion the results of this study suggested that the exotoxin A (domains I, II) -
279
flagellin (N-terminal) fusion protein is a nontoxic, nonpyrogenic and dual functional
280
immunogenic protein that can be considered as a new protective vaccine candidate
281
for P. aeruginosa infections.
282 283
Acknowledgments:
284
This study was supported by a research grant from Research Center of Infectious
285
and Tropical Diseases, Tabriz University of Medical Sciences, Tabriz, Iran.
286
References
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Neville, L., Barnea, Y., Hammer-Munz, O., Gur, E., Kuzmenko, B., Kahel-Raifer, H., Eren,
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R., et al. 2005. Antibody raised against N-terminal pseudomonas aeruginosa flagellin
337
prevent mortality in lethal murine models of infection. Int J Mol Med. 16:165-171.
338
Nilsson, E., Amini, A., Wretlind, B., Larsson, A. 2007. Pseudomonas aeruginosa infections
339
are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas
340
aeruginosa flagellin. J Chromatogr. 856: 75–80.
341
Reed, L., Muench, H.A. 1938. A simple method for estimating fifty percent end points. Am J Epidemiol. 27: 493-497.
342 343
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Shiau, W.J., Tang, K.T., Shin, L.Y., Tai, C., Sung, Y.Y., Huang, L.J., et al. 2001. Mice
344
immunized with DNA encoding a modified Pseudomonas aeruginosa exotoxin A
345
develop protective immunity against exotoxin intoxication. Vaccine. 19:1106-1112.
346
Tanomand, A., Farajnia, S., Najar Peerayeh, S., Majidi, J. 2012. Cloning, expression and
347
characterization of recombinant exotoxin A-flagellin fusion protein as a new vaccine
348
candidate against Pseudomonas aeruginosa infections. Iran Biomed J; 17:1-7.
349
Tavajjohi, Z., Moniri, R. 2011. Detection of ESBLs and MDR in Pseudomonas aeruginosa in a tertiary-care teaching hospital. IJCID.6:18-23. Wolf, P., Beile, U. 2009. Pseudomonas exotoxin A: From virulence factor to anti-cancer agent. Int J Med Microbiol. 299:161–176.
350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368
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Page 16 of 21
Table1. Analysis of Antibody responses of mice groups immunized with P.
369
aeruginosa flagellin, exotoxin A and exotoxin A - flagellin fusion protein.
370 371
Immunogen*
exoA- fla
Target antigen
OD450
mean difference
P value**
95% Confidence Interval Lower
Upper
exoA -fla
2.965
2.93
Page 1 of 21
Protective efficacy of recombinant exotoxin A-flagellin fusion
1
protein against Pseudomonas aeruginosa infection
2 3
Safar Farajnia1, Shahin Najar Peerayeh*2, Asghar Tanomand 3, Jafar Majidi4,
4
Gholamreza Ghoudarzi5, Behrooz Naghili1, Leila Rahbarnia6
5 6
1. Research Center for Infectious and Tropical Disease, Tabriz University of Medical Sciences Tabriz, Iran. Email: [email protected]
8
2. Dept of Bacteriology Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Email: [email protected]
Sciences,
Tarbiat
Modares
University,
9 10
3. Tabriz University of Medical Sciences, Tabriz and Dept of Bacteriology Faculty of Medical
7
Tehran,
Iran.
Email:
[email protected]
11 12 13
4. Dept of Immunology and Immunology research center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. Email: [email protected] 5. Department of Microbiology, Faculty of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran. Email:[email protected] 6. Drug Applied Research Center, Tabriz University of Medical Sciences Tabriz, Iran. Email: [email protected]
14 15 16 17 18 19 20
*Corresponding authors:
21
Shahin Najar Peerayeh, Department of Bacteriology, Faculty of Medical sciences,
22
Tarbiat Modares University, Tehran, I.R. Iran
23
Tel: +98 21 82883870
24
E-mail: [email protected]
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Page 2 of 21
Abstract
25
Pseudomonas (P.) aeruginosa is an opportunistic bacterium causes serious
26
nosocomial infection in immunocompromised patients. The aim of this study was to
27
prepare a fusion protein consisting exotoxin A (ExoA) and flagellin (Fla) from P.
28
aeruginosa and evaluate its potential as a vaccine candidate against P. aeruginosa
29
infection. The genes encoding for ExoA and Fla proteins were cloned in frame and
30
expressed in E. coli. The recombinant ExoA-Fla fusion protein was purified by Ni-
31
NTA affinity chromatography. Mice were immunized subcutaneously with exotoxin
32
A, flagellin, and flagellin - exotoxin A fusion proteins and the humoral immune
33
response was evaluated by ELISA method. The immunized and control group mice
34
were challenged with a 2X LD50 (7.5 x 107 CFU) of P. aeruginosa for protection
35
assay. The results indicated that vaccination with flagellin, exotoxin A and flagellin
36
- exotoxin A fusion proteins produced significant amount of specific IgG antibodies.
37
Immunization of mice with exotoxin A-flagellin fusion protein showed significant
38
protection against intra-peritoneal challenge with 7.5 x 107 CFU (2X LD50) P.
39
aeruginosa. Results of this study suggest that recombinant exotoxin A-flagellin
40
fusion protein is a highly immunogenic protective protein which can be used as a
41
promising vaccine candidate against P. aeruginosa.
42
Keywords: Exotoxin A-flagellin, P. aeruginosa, vaccine candidate, fusion protein
43 44
1. Introduction
45
Pseudomonas (P) aeruginosa is an opportunistic bacterium associated with
46
nosocomial
in
47
immunocompromised patients. The range of diseases caused by this bacterium
48
infections
leading
to
septicemia
and
death
especially
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Page 3 of 21
varies from superficial skin infections to serious systemic infections such as
49
fulminate sepsis. P. aeruginosa is the second most common causative agent of
50
hospital-acquired pneumonia, healthcare-associated pneumonia and ventilator-
51
associated pneumonia. One of the most important features of this bacterium is its
52
resistance to various antibacterial agents (Tavajjohi and Moniri 2011), and even
53
newly developed antibiotics have failed to reduce the mortality rate associated with
54
this organism. This failure in response to antimicrobial treatment has led researches
55
to vaccines and immunotherapy as alternative treatment methods for P. aeruginosa
56
infections. It has been suggested that neutralization of bacterial virulence factors by
57
immunological methods can result in prevention and reduction of mortalities due to
58
P. aeruginosa infections (Doring and Pierb 2008).
59
For this purpose, different antigenic and virulence factors such as outer membrane
60
proteins, toxins, flagella, pilli, and high molecular weight polysaccharides have been
61
evaluated as vaccine candidates (Manafi et al. 2009, Cryz et al. 1987, Eric et al.
62
2009, Doring et al. 2007), but there is no any approved vaccine currently available
63
against P. aeruginosa.
64
Exotoxin A plays an important role in virulence of P. aeruginosa and it has shown
65
that exotoxin A (ExoA) deficient mutants exhibit a virulence 20 times less than the
66
wild type strain in the mouse models (Wolf and Beile 2009). ExoA has 3 structural
67
domains, domain 1 is receptor binding domain, domain 2 is transfer domain and
68
domain 3 is toxic domain. ExoA catalysis inhibition of protein synthesis by ADP-
69
ribosylation of elongation factor 2. Exotoxin A is also an antigenic protein that
70
induces humoral immune responses in animal models. Based on the results of
71
different studies, these antibodies are highly protective. Therefore, exotoxin A has
72
been considered as a promising vaccine candidate for pseudomonas infections
73
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Page 4 of 21
(Hertle et al. 2001, Denis-Mize and Price 2000, Chen and Shang 1999,
74
Campodo´nico et al. 2010).
75
The bacterial flagellum is a strong immunogenic factor and active or passive
76
immunization with flagellar antigens induces antibody production. These antibodies
77
inhibit bacterial distribution, and thus prevent systemic infection in a mouse model
78
of burn and pulmonary infections (Campodo´nico et al. 2010). Studies shown that
79
monoclonal antibodies against P. aeruginosa flagellin are protective against P.
80
aeruginosa infections in various animal models, hence suggested as a vaccine
81
candidate for P. aeruginosa (Matsumoto et al. 1999, Barnea et al. 2009, Nilsson et
82
al. 2007).
83
In this study, we reported preparation and evaluation of recombinant exotoxin A-
84
flagellin fusion protein, as a new vaccine candidate for P. aeruginosa infections.
85
2. Materials and Methods
86
2.1. Preparation of recombinant flagellin - exotoxin A fusion protein
87
Domains I-II of Exotoxin A gene (ExoA) from P. aeruginosa strain PAO1 and N-
88
terminal part of flagellin gene (Fla) from P. aeruginosa strain 8821M were amplified
89
separately by polymerase chain reaction (PCR) using specific primers. PCR products
90
were gel purified separately by purification kit (Macherey Nagel, Germany) and
91
analyzed by electrophoresis. Then, the ExoA-Fla fusion gene was constructed by
92
PCR-mediated overlap extension method using ExoA forward and Fla reverse
93
primers (Heckman and Pease 2007). Cloning of fusion gene was carried out by
94
ligation of PCR product into the PTZ57R vector using T-A cloning method
95
according to the manufacturer instructions (Fermentas, Lithuania).The ligate was
96
transformed into the E. coli DH5α and screening was performed by PCR and
97
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Page 5 of 21
restriction analysis. A positive clone of PTZ-ExoA-Fla was sequenced for analysis
98
of the sequence integrity.
99
For recombinant expression of ExoA-Fla fusion protein in E. coli, the insert was
100
removed from PTZ-ExoA-Fla vector by digestion with BamHI and XhoI enzymes
101
and subcloned into the PET 22b expression vector. Then, the pET22b-ExoA-Fla
102
construct was transformed into E. coli BL21, and the protein expression was
103
assessed using SDS-PAGE. Ni-NTA affinity chromatography method (Qiagen,
104
Chatsworth, CA, USA) was used for His-tagged fusion protein purification
105
according to the manufacturer instructions. The purified recombinant protein was
106
dialyzed against PBS, PH 7.4 for removing imidazole. The purity of protein was
107
analyzed by SDS-PAGE, and product concentration assessed by Bradford method
108
(Sigma, Product Number B6916). Western blotting with antibody to P. aeruginosa
109
native exotoxin A (Sigma Product Number P2318) was used to evaluate the
110
immunological properties of the recombinant ExoA-Fla fusion protein. Finally,
111
lipopolysaccharide (LPS) contamination of purified recombinant protein was
112
assessed by Limulus amebocyte lysate (LAL) assay method.
113
2.2. Preparation of recombinant flagellin (N-terminal) and exotoxin A (Domains I,
114
II) proteins
115
Recombinant flagellin was prepared as described elsewhere (Goudarzi et al 2009).
116
Recombinant exotoxin A (Domains I, II) protein were purified from E. coli BL21
117
carrying the pET22b - exotoxin A (Domains I, II) by Ni-NTA affinity
118
chromatography (Tanomand et al. 2012; Farajnia et al. 2011).
119
2.3. Toxicity test
120
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To test the toxicity of recombinant antigens in mice, various doses (between 100µg
121
and 300 µg) of antigens were subcutaneously injected into 6-8 week old Balb/C
122
mice (6 mice per group). Animal’s mortality was followed for 7 days.
123
2.4. Mice immunization
124
6-8 week old female BALB/c mice were divided into four groups (5 mice per
125
group) and subcutaneously injected with 20 µg recombinant flagellin, exotoxin A
126
(Domains I, II), ExoA-Fla fusion protein, or PBS on days 0 (with complete Freund’s
127
adjuvant), days 21, 42 (with incomplete Freund’s adjuvant), and finally on day 72
128
(without adjuvant). One week after the last injection, the animals were bled from the
129
orbital sinus and sera were prepared and stored at -20 until analysis.
130
2.5. Enzyme-Linkd Immunosorbent Assay (ELISA)
131
ELISAs were performed by standard methods as described previously (11, 18). In
132
brief, microtiter plate was coated with flagellin, exotoxin A or flagellin - exotoxin A
133
fusion protein at a concentration of 5 µg /ml in 0.1 M carbonate/bicarbonate (PH
134
9.6). The plates were incubated for 1 hour at 37°C and then blocked with 2% bovine
135
serum albumin in PBS containing 0.05% Tween 20 (PBS-T), 60 min. between
136
incubation steps, plates were washed three times with PBS-T. Sera samples diluted
137
1:10000 were added and incubated for 60 min, followed by incubation with
138
peroxidase conjugated anti-mouse antibody (abcam) as secondary antibody.
139
Peroxidase activity was detected using 3, 3´, 5,5´-tetramethyl-benzidine (TMB,
140
Sigma), stopped with 1M H2SO4 and the absorbance values was measured at 450
141
nm.
142 143
2.6. Opsonophagocytosis assay
144
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Page 7 of 21
Opsonophagocytosis assay were performed as described previously [11,18] with
145
some modifications. Briefly, a mucoid colony of P. aeruginosa 8821M was grown
146
to mid-log phase on LB broth at 37°C with shaking, washed twice in cold PBS and
147
suspended in PBS at a final concentration of approximately 106 bacteria per ml.
148
Opsonization was performed by mixing 100µl bacteria (106 CFU/ml) for 30 min at
149
37°C in 100µl complement-inactivated sera obtained from various vaccinated and
150
control groups. A complement source were added to these pre-opsonized bacteria
151
and incubated for 90 min at 37°C with gentle shaking. Controls consisted of tubes
152
from which macrophage, complement, or serum was omitted. After 90 min of
153
incubation, 10 µl samples was removed, diluted in PBS and plated for bacterial
154
counts. The plates were incubated overnight at 37°C and bacterial colonies were
155
counted. The kill percentage was calculated as follows:
156
Kill Percentage = [1-(CFU of immune serum: CFU of pre-immune serum] x 100
157
2.7. Bacterial challenge
158
At first, 30 female BALB/c mice were divided into five groups (6 mice per group)
159
for determination of bacterial infection LD50. Mice were intraperitoeally injected
160
with Serial dilutions of P. aeruginosa (2.5 x 107, 5 x 107, 7.5 x 107, 1 x 108, and 12.5
161
x 108 CFU). The mice were followed for 10 days, mortality was recorded, and LD50
162
was determined according to the Reed and Muench method [19]. In challenge
163
experiment, two weeks after the final immunization, the immunized and control
164
group mice were challenged with intraperitoeal injection of 7.5 x 107 CFU of a
165
clinical strain of P. aeruginosa. The survival rate was recorded in all groups for 10
166
days. This strain was a mucoid phenotype, flagellated and Exotoxin A positive strain
167
that was originally isolated from 56 years old male patient with wound and systemic
168
infection, that showed resistance to chloramphenicol , ceftriaxone, piperacillin,
169
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Page 8 of 21
cotrimoxazole and was sensitive imipenem, cotrimoxazole, cefoperazone, amikacin
170
and Ciprofloxacin.
171 172
2.8. Statistical analysis
173
The SPSS version 12 was used for data analysis. Differences in the mean percentage
174
of opsonic kill, and the mean ELISA absorbance were compared by analysis of
175
variance (ANOVA) using a post hoc multiple comparison test. The chi square test
176
was used to analyze the survival data of protection assay. P≤0.05 was considered
177
significant.
178
3. Results
179
3.1. Preparation of recombinant proteins
180
Schematic representation of expression construct that were used for preparation of
181
recombinant proteins has shown in Fig.1. The prepared exotoxin A (Domains I, II) -
182
flagellin(N-terminal) fusion protein composed of 574 amino acids including 404
183
amino acids from exotoxin A (Domains I, II) and 170 amino acids from flagellin(N-
184
terminal).
185
The exotoxin A - flagellin fusion protein, flagellin and exotoxin A were highly
186
expressed in E. coli and purified from inclusion bodies upon solubilization in 8M
187
urea and passage over a Ni-NTA column (Fig 2).
188
Western blotting analysis showed that recombinant flagellin, exotoxin A and
189
exotoxin A– flagellin fusion protein all were reacted with antibody against native
190
Exotoxin A.
191
Evaluation of the Toxicity of recombinant antigens showed that none of mice
192
injected with various doses of antigens (between 100µg and 300 µg) died after 8
193
weeks.
194
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Page 9 of 21
3.2. Immune responses and ELISA results
195
Analysis of antibody production in immunized and control mice showed that
196
vaccination with flagellin, exotoxin A and exotoxin A - flagellin fusion protein
197
produced significant amount of specific antibody. No reacting antibody was detected
198
in mice injected with PBS. As shown in Table 1, the antibody titers in mice
199
vaccinated with exotoxin A - flagellin fusion protein were higher than other
200
vaccinated groups.
201
3.3. Opsonic activity of antisera against P. aeruginosa
202
Opsonophagocytic assay results are shown in Fig 3. In the opsonophagocytic killing
203
assay, antisera from mice immunized with the exotoxin A- flagellin and flagellin
204
alone showed significant phagocytic killing (68%) against the P. aeruginosa 8821M,
205
but antiserum from mice primed with exotoxin A alone was less efficient in
206
mediating phagocytic killing (4%). There were no phagocytic killings in control
207
tubes.
208
3.4. Protection assay results
209
For determining the LD50 of challenge strain, five groups of mice (6 mice per
210
group) were injected with different CFU of bacteria (2.5 x 107, 5 x 107, 7.5 x 107, 1 x
211
108, and 12.5 x 108 CFU). The ratio of killed/total injected animals were 0/6, 4/6,
212
6/6, 6/6 and 6/6, respectively. Therefore, about 3.5 x 107 CFU were selected as
213
LD50 and 7.5 x 107 CFU as 2xLD50 for challenge test in immunized and control
214
group mice.
215
The results of survival rate after bacterial challenge test in immunized and control
216
group mice have shown in Table 2. The mice immunized with exotoxin A– flagellin
217
fusion protein showed significant protection (p≤0.05) against intraperitoneal
218
injection of 7.5 x 107 CFU (2xLD50) clinical strain of P. aeruginosa. However, there
219
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Page 10 of 21
were no significant differences in the rates of survival of mice immunized with
220
exotoxin A (p≤0.49) and flagellin (p≤0.19) in comparing with control group.
221
4. Discussion
222
P. aeruginosa is an important cause of nosocomial infections with high mortality
223
and morbidity rates. One of the most important features of the bacterium is its
224
resistance to various antibacterial agents, and even newly developed antibiotics have
225
failed to reduce the mortality rate associated with this organism. Various new
226
approaches have been investigated to combat pseudomonas infection among them;
227
immunotherapy and vaccine development seems to be promising therapeutic and
228
preventive approaches (Doring and Pierb 2008). Several extracellular products and
229
cell components of P. aeruginosa such as exotoxin A (Shiau et al. 2001), outer
230
membrane proteins (Eric et al. 2009, Chen and Shang 1999), flagellin
231
(Campodo´nico et al. 2010, Neville et al. 2005), pillin (Hertle et al. 2001) and
232
alginate (Kashef et al. 2006) or combination of them have been studied as vaccine
233
candidates(Eric et al. 2009, Chen and Shang 1999, Kashef et al. 2006, Cryz et al.
234
1991, Kamei et al. 2011). In this article, we report development of a chimeric
235
subunit vaccine composed of two important components of P. aeruginosa virulence
236
factor, exotoxin A and flagellin. The exotoxin A is an important pathogenic factor,
237
inhibiting protein synthesis in the host cells through ADP-ribosylation of elongation
238
factor 2 (Cryz et al. 1991). We prepared a deletion mutant from exotoxin A lacking
239
the toxic domain (Chen and Shang 1999). The bacterial flagella are also a strong
240
immunogenic factor and it has shown that active or passive immunization with
241
flagellar antigens induces antibody response. These antibodies inhibit bacterial
242
distribution, and thus prevent disseminated infection in a mouse model of burn and
243
pulmonary infections (Campodo´nico et al. 2010). Analysis of the primary amino
244
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Page 11 of 21
acid sequences of flagellin from different gram-negative bacteria showed that their
245
N terminal part (aa 1-170) are well conserved (Neville 2005). Therefore we used N-
246
terminal 170 amino acids from Fla gene in fusion protein construction.
247
The results of this study showed all three preparations exotoxin A, flagellin and
248
exotoxin A - flagellin fusion were highly immunogenic and induced significant IgG
249
antibodies production in immunized mice (Table 1). We also found that injection of
250
exotoxin A- flagellin fusion protein induced higher antibody titers than two other
251
antigens. Therefore, recombinant exotoxin A-flagellin fusion protein could be
252
considered as an important vaccine candidate for generating anti- exotoxin A and
253
anti-flagellin responses.
254
The results of opsonophagocytic assay showed, sera from mice immunized with
255
either fusion protein or flagellin had moderate opsonic killing activity (52-48%)
256
against P. aeruginosa, but sera from mice immunized with exotoxin A alone did not
257
show any opsonophagocytic activity. These findings could be attributed to the
258
extracellular secretary nature of exotoxin A. The results were consistent with reports
259
of Chen et al (1999) and Campodo´nico et al (2010) and indicated that anti-flagellin
260
antibody is responsible for opsonophagocytic activity of sera from mice immunized
261
with fusion protein.
262
Vaccination with exotoxin A-flagellin fusion protein afforded significant protection
263
against challenge with clinical strain of p. aeruginosa, whereas exotoxin A alone
264
was partially effective in survival rate. These observations were consistent with
265
findings of Chen et al. (1999). They reported a significant protection in burned mice
266
immunized with recombinant fusion protein composed of exotoxin A, outer
267
membrane proteins I and F, but less protection by immunization with exotoxin A
268
alone.
269
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Page 12 of 21
This may not be surprising since the pathogenicity of P. aeruginosa are dependent
270
on both extracellular and cell associated virulence factors. Hence it could be
271
concluded that immunization with cocktail antigens specially those containing both
272
extracellular and cell-associated virulent/antigenic factors can induce more efficient
273
protective response against P. aeruginosa infections than single antigens.
274
Based on our results, the survival rate of immunized mice (as a measure of
275
protection) in different groups was correlated with antibody level. This finding was
276
in line with previous reports (Barnea et al. 2009, Nilsson et al. 2007) and elucidated
277
the role of humoral immune response in immunity against P. aeruginosa infections.
278
In conclusion the results of this study suggested that the exotoxin A (domains I, II) -
279
flagellin (N-terminal) fusion protein is a nontoxic, nonpyrogenic and dual functional
280
immunogenic protein that can be considered as a new protective vaccine candidate
281
for P. aeruginosa infections.
282 283
Acknowledgments:
284
This study was supported by a research grant from Research Center of Infectious
285
and Tropical Diseases, Tabriz University of Medical Sciences, Tabriz, Iran.
286
References
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Table1. Analysis of Antibody responses of mice groups immunized with P.
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aeruginosa flagellin, exotoxin A and exotoxin A - flagellin fusion protein.
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Immunogen*
exoA- fla
Target antigen
OD450
mean difference
P value**
95% Confidence Interval Lower
Upper
exoA -fla
2.965
2.93
