Protective Properties of Nontoxic Recombinant Exotoxin A (Domain I-II) Against Pseudomonas aeruginosa Infection

Document Type: Research Paper

Authors

1 Department of Bacteriology, Faculty of Medical sciences, Tarbiat Modares University, Tehran, IR Iran

2 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran

3 Department of Immunology and Immunology Research Center, Faculty of Medical Sciences, Tabriz University of Medical Sciences, Tabriz, IR Iran

Abstract

Background: Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the development of a vaccine against Pseudomonas aeruginosa infection. Objectives: In this study, recombinant exotoxin A (domains I and II), (ExoA I-II) protein was expressed, purifid and its immunological characteristics were evaluated in a mouse model. Materials and Methods: The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II of exotoxin A was amplifid by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 and the protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affity chromatography was used for recombinant protein purifiation. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A (Domains I, II). Antibody production was evaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 × LD50 (7.5 × 107 CFU) of clinical strain of mucoid P. aeruginosa. Results: Sequencing of the cloned gene showed that the sequence of ExoA I-II gene was in accordance with ExoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-II produced a signifiant amount of specifi IgG antibodies in mice. Also immunization of mice with ExoA I-II increased survival times against intra-peritoneal challenge with an approximate 7.5 × 107 CFU (2 × LD50) of clinical strain of P. aeruginosa. Conclusions: Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a new vaccine candidate against P. aeruginosa.

Keywords


1. Background
Pseudomonas aeuginosa (P. aeruginosa) is an important
pathogen in immunocompromised patients, such as patients suffring from acute immune defiiency (AIDS),
cancer, burn wounds and cystic firosis (CF). Infections
caused by P. aeruginosa are often diffilt to eradicate
because it requires minimal nutrition and can tolerate
a wide range of temperatures (1). This bacterium is commonly isolated from nosocomial infections, especially
from ventilator-associated pneumonia. P. aeruginosa is
also a major cause of pulmonary infection in cystic firosis patients and bacteremia due to this bacterium has
high mortality (2, 3).
On the other hand, P. aeruginosa has the ability to easily acquire antimicrobial resistance. Emergence of multiple drug resistant P. aeruginosa has become asignifiant
problem in clinical settings due to limited therapeutic
options (4, 5). Therefore, alternative ways to treat and prevent Pseudomonas infections are necessary. Diffrent approaches have been used for the development of vaccines
against P. aeruginosa infections (6). Various antigenic and
virulence factors, including lipopolysaccharide, flgellin,
Pili, polysaccharides with high molecular weight, alginate and outer membrane proteins have been investigated as vaccine candidates (7-10), but early trials produced
disappointing results.
Exotoxin A is a major virulence factor that is produced
by most clinical strains of Pseudomonas aeruginosa. Exotoxin A is a potent cytotoxic agent that is lethal for a variety of animals, including subhuman primates. It has
been shown that exotoxin A defiient mutants exhibita
virulence 20 times less than the wild type strain in the
mouse models (11). The mechanism of exotoxin A activity
in mammalian cells has been studied in detail. The toxin binds and enters into the cells by receptor-mediated


endocytosis and then translocates to the cell cytoplasm,
where it inactivates elongation factor 2 (EF-2) and inhibits
protein synthesis. Exotoxin A also has the ability to inhibit host response to infection. In adult mice, the 50% mean
lethal dose (LD50) of intravenously injected exotoxin A
was approximately 1 µg. mouse -1. The role of exotoxin A in
P. aeruginosa infection of burn wound has been examined
in the burned mouse model, which closely resembles P.
aeruginosa infection in human burns. It was shown that
the survival of burned mice infected with 2xLD50 doses of
a toxigenic strain of P. aeruginosa (PA103) was enhanced
upon the intravenous injection of anti-exotoxin A serum.
In addition, anti-exotoxin A serum reduced the number
of viable P. aeruginosa microorganisms within the blood
and livers of burned/infected mice (12). These fidings
suggest that exotoxin A is an important antigenic protein
that has been considered as a promising vaccine candidate for pseudomonas infections. Exotoxin A consists of
three domains: domain I (1-252aa, 365-404aa), domain II
(253-364aa), and domain III (405-613aa), which is a toxic
moiety (11). Domain I is responsible for attachment of Exo
A to the cells and domain II is a transporter that enters
exotoxin in to the cells.
2. Objectives
In this study, we reported on the expression and purifiation of ExoA I-II as a nontoxic antigen. We also evaluated the protective activity of anti-ExoA I-II antibodies
against P. aeruginosa infection.
3. Materials and Methods
3.1. Cloning, Expression and Purifiation of Nontoxic ExoA I-II
Genomic DNA of P. aeruginosa strain PAO1 was extracted
by phenol-chloroform extraction and ethanol precipitation method as described elsewhere (13). The ExoA I-II
gene was amplifid by polymerase chain reaction (PCR)
using specifi forward, 5’-GGATCCCCGAGGAAGCCTTCGAC-3’ and reverse 5’-GCCGTCGCCGAGGAACTCCTCGAG-3’
primers containingBamhI and XhoI restriction sites, respectively. The amplifiation conditions were as follows;
initial denaturation at 94°C (4min) and 31 cycles consisting of 94 ° C (1 min), 65 ° C (1 min), 72 ° C (1 min) and a fial
extension at 72 ° C (5 min). PCR product of ExoA-I,II was
gel purifid by a purifiation kit (Macherey Nagel) and
analyzed by electrophoresis.
Cloning of the ExoA-I, II gene was carried out by ligation
of the PCR product into the PTZ57R vector using T-A cloning
method according to the manufacturer’s instructions (Fermentas). The ligate was then transformed into the E. coli
DH5α and screening was performed by PCR and restriction
analysis. Positive clones of PTZ-ExoA I-II were sequenced
(MWG-Germany) for analysis of the sequence integrity.
For recombinant expression of ExoA I-II in E. coli, the insert was removed from pTZ-ExoA vector by digestion with
BamHI and XhoI enzymes and sub-cloned into the pET22b
expression vector. Then, the pET22b-ExoA construct was
transformed into E. coli BL21, and the protein expression
was assessed using 12% (w/v) sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE). An unstained protein molecular weight marker (14.4 kDa to 116 kDa) was
used for monitoring the progress of SDS-PAGE. Ni-NTA
affity chromatography method (Qiagen, Chatsworth,
CA, USA) was used for His tagged fusion protein purifcation. The purifid recombinant protein was dialyzed
against PBS, pH 7.4 for removal of imidazole. SDS-PAGE
was used to analyze the purity of the protein and product concentration was assessed by Bradford method with
bovine serum albumin as the standard (Sigma, Product
Number B6916).
Western blotting was used to evaluate the immunological properties of recombinant ExoA I-II protein. Briefl,
the recombinant protein was separated on SDS-PAGE gel
using pre-stained protein ladder molecular weight marker (10 KDa to 170 KDa) for monitoring the progress of
SDS-PAGE and was then transferred to PVDF membranes
by semi-dry blotting. Next, the reactivity of recombinant
protein with antibody to P. aeruginosa native exoA (Sigma
Product Number P2318) was analyzed. HRP-conjugated
anti-rabbit IgG and DAB substrate were used for reaction
detection. Finally, lipopolysaccharide (LPS) contamination of purifid recombinant protein was assessed by
Limulus amebocyte lysate (LAL) assay method.
3.2. Active Immunization of Mice
6-8 weeks old female BALB/c mice were purchased from
the Pasture Institute (Tehran-Iran). All animals were
housed under pathogen-free conditions at the Tabriz University of Medical Sciences animal house (Tabriz-Iran).
Two groups of 6-8 week old female BALB/c mice (6 mice
per group) were subcutaneously immunized with 20µg
of recombinant ExoA I-II with complete Freund’s adjuvant (test group) or PBS (control group) on days 0, 21 and
42 with incomplete Freund’s adjuvant and day 72 without
adjuvant. This research was approved by the Research
Ethics Committee ofthe Faculty of Medical sciences, Tarbiat Modares University.
3.3. Bleeding From Animals
One week after the last injection, sera were collected
from mice in test and control groups, and stored at -20 °C
until analysis. Housing of one mouse from the test group
and one mouse from the control group was continued for
a further four months. Four months after the last injection, these mice were bled to check decreasing or stability
of anti-exotoxin A level. After bleeding, 20 µg of recombinant ExoA I-II (without adjuvant), as a booster, was injected


to the test group and equal volumes of PBS to the control
group mice, and 1 week after this injection, both mice were
bled again. The sera were stored at -20 °C until analysis.
3.4. Enzyme-Linked Immunosorbent Assay (ELISA)
ExoA I-II specifi IgG antibodies in sera were determined
using an indirect ELISA. The 96 well microtiter plate were
coated with ExoA I-II at a concentration of 5 µg.ml -1 in 0.1
M carbonate/bicarbonate, pH 9.6 and incubated for 45
min at 37 °C. Plates were washed two times with 250 µL
of wash buffr (50 mM Tris, 0.14 M NaCl, o.o5% Tween 20,
pH 8.0) and blocked with PBS-T ( 2% bovine serum albumin in PBS containing 0.05% Tween 20) for 45 min at 37
°C. Plates were washed two times with 250 µL of wash buffer. Sera samples diluted to 1:10000 were added (100 µL
per well) and incubated for 45 min at 37 °C. Plates were
washed fie times with 250 µL of wash buffr; followed
by incubation with peroxidase conjugated anti-mouse
antibody diluted to 1:5000 as a secondary antibody, for
45 min at 37 °C. Plates were washed fie times with 250
µL of wash buffr. Peroxidase activity was detected with
3, 3´, 5, 5´-tetramethyl-benzidine (TMB), after 15 min of
incubation in the dark, stopped with 1 M H2SO4 and the
absorbance was measured at 450 nm.
3.5. Protection Assay
At fist, 24 female BALB/c mice were divided into fie
groups (6 mice per group) for determination of bacterial infection LD 50. Mice were intraperitoneally injected
with diffrent dilutions of the clinical strain of mucoid
P. aeruginosa in PBS (5 × 107, 7.5 × 107, 1 × 108, and 12.5 × 108
CFU). The mice were followed for 10 days, mortality was
recorded, and LD50 was determined according to the
Reed and Muench method (14). Two weeks after the fial
immunization, the immunized and control groups’ mice
were challenged with intraperitoneal injection of 7.5 ×
107 CFU clinical strain of mucoid P. aeruginosa. The survival rate was recorded for 10 days.
3.6. Statistical Analysis
Statistical analysis was performed using the Stata software. The diffrences between the anti-ExoA I-II specifi
IgG-test group and control group were analyzed by student’s t-test. Diffrences were considered to be statistically signifiant with a P-value of P ≤ 0.05.
4. Results
4.1. Cloning, Expression, Purifiation and Characterization of ExoA I-II
Amplifiation of ExoA I-II from P. aeruginosa PAo1 resulted in a PCR product of 1212 bp that was in accordance with
the expected product size (Figure 1).

Sub-cloning of theinsert (ExoA I-II) into the expression vector, pET-22b wasverifid by digestion with BamhI and XhoI
restriction enzymes (Figure 2) and the integrityof ExoA I-II
in the construct was confimed by DNA sequencing.

Analysis for expression of recombinant ExoA I-II-(His)
6-tag protein revealed that the protein was highly expressed in E. coli, which appeared as a 45 KDa protein
in SDS-PAGE. Both supernatant and the pellet of cell lysates were tested for the presence of recombinant protein. The majority of the expressed protein was detected in inclusion bodies. Figure 3 shows the commassie
blue-stained SDS-PAGE gels of E. coli lysates before and
after induction, and the purified recombinant ExoA I-II
protein.

Western blot analysis of the purifid protein showed
that ExoA I-II could be recognized by an antibody against
native Exotoxin A antibody (Figure 4).
Analysis for reactivity of recombinant protein with
sera from patients with P. aeruginosa infections showed
that all sera contained antibody against exotoxin A and
highly reacted with purifid recombinant ExoA I-II protein (Figure 5).
4.2. Antibody Responses
Monitoring of antibody production in immunized and
control groupmice after four doses of immunization
with recombinant ExoA I-II protein showed signifiant
(P < 0.0001) amounts of specifi antibody production
(Table 1). No specifi antibodies were detected in mice in
the negative control group that were injected with PBS


4.3. Protection Assay Results
Analysis of survival rate from bacterial challenge test
of immunized and control group mice showed a partial
protection, and compared to control group, 40% of mice
immunized with recombinant ExoA I-II survived after a
challenge with 7.5 X 10 7 CFU (2XLD50) of clinical strain
of mucoid P. aeruginosa (survival rate 40%) whereas 80%
mice in control groups died one day after the challenge
(Figure 6).

5. Discussion
P.aeruginosa is an aerobic, gram-negative opportunistic pathogen, responsible for nosocomial infections especially in Intensive Care Units (ICU) and burn wards.
Infections caused by P. aeruginosa are usually diffilt to
eradicate because of its diverse virulence factors, inherent
resistance to many antibiotics and ability to acquire resistance to diffrent antibiotics. Thus, an effctive immune
prophylactic/therapy method is desirable to supplement
conventional antibiotic therapy for treatment of P. aeruginosa infections (15). Exotoxin A secreted by P. aeruginosa is
a toxic pathogenic factor, which inhibits protein synthesis
by host cells. Exotoxin A is extremely lethal, causes histopathological changes within the liver, induces apoptosis of
the hepatocytes and enhances the expression of diffrent
pro-inflmmatory cytokines (12). Research has shown that
these damaging effcts are neutralized in the presence of
anti-exotoxin A antibodies that are essential for protection
against P. aeruginosa infections (12, 16, 17). Native exotoxin
A is toxic and its modifiation into toxoid form by diffrent methods and its immunologic properties have been
reported in several in vitro and in vivo studies (7, 12, 16, 17).
In this research, we reported a new recombinant nontoxic
form of exotoxin A. We constructed a nontoxic exotoxin A
(ExoA I-II) by deletion of the enzymatic domain from the
carboxyl terminus of toxin. This nontoxic exotoxin A retains most of its antigenicity and can induce protective immunity. Some studies have used the native Exotoxin A purifid from bacterial culture for investigation but it has been
shown that purifiation of native exotoxin A from culture
medium is very diffilt and proteases secreted into the
culture medium decreases the yield of toxin (18). Therefore,
production of recombinant ExoA I-II is preferred for preparation of ExoA I-II with high quality and quantity. In this
study ExoA I-II gene was isolated from P. aeruginosa PAO1,
then cloned in to the pET22b vector and overexpressed in
E. coli as inclusion bodies aggregates. pET22b vector carries
six histidine residues in the c-terminal of the protein. The
His-tag facilitates purifiation of recombinant protein by
Ni2+-sepharos resin. In our study, highly purifid recombinant protein was obtained after purifiation. Purifid
recombinant ExoA I-II effctively refolded using dialysis
against urea gradients and PBS without protein aggregation or precipitation.
Reaction of sera from patients infected with P. aeruginosa
with recombinant ExoA I-II demonstrated the presence
of a high titer of anti-exotoxin A antibody in all of these
sera. This fiding is consistent with the results reported by
cross et al. (19). They reported, that, there are distinct differences in the titers of anti-exotoxin A antibody in sera
obtained from dead and surviving patients after P. aeruginosa infection (19). These observations thus indicate that
anti-exotoxin A antibody might be one of the most effctive factors in protecting humans from life-threatening
P. aeruginosa infection. Thus, to avoid the pathogenesis of
Pseudomonas infection, development of a vaccine against
exotoxin A might be an appropriate approach. These results also indicate that this non-toxic exotoxin A protein
may be used as a serodiagnostic antigen for rapid diagnosis of P. aeruginosa infections. Analysis of sera from
mice immunized with recombinant non-toxic exotoxin
A revealed that the protein induced production of a signifiant level of IgG antibodies. We also found stability
in the level of antibody in the immunized group for four


months, which was doubled by a booster injection (Table
1). This fiding verifid the highly immunologic property
of non-toxic exotoxin A proteins. Several in vitro and in vivo
studies showed that the antibodies induced by non-toxic
exotoxin A can neutralize the exotoxin A cytotoxicity (7, 12).
Therefore non-toxic exotoxin A can be used in active and
passive immunization for neutralization of pathological
effcts of exotoxin A. The protective efficy of P. aeruginosa exotoxin A is a subject of controversy. Manaf A et al.
reported a 93.8% protection in burned mice following active immunization with a toxoid of exotoxin A (7). DenisMize and colleges also reported an increase in survival rate
and protection properties in mice immunized by toxoid
of exotoxin A (19). However, Palovskiset al. found that the
survival rate did not increase signifiantly following active
immunization with a toxoid of exotoxin A and infection
with P. aeruginosa in burned mice (20), while Chen et al.
reported less protection for burned mice immunized with
nontoxic exotoxin A (15). In our results, recombinant ExoA
I-II showed a protective efficy of 40% against challenge
with 7.5 × 10 7 CFU (2XLD50) of clinical strain of mucoid P.
aeruginos . Immunization with recombinant ExoA I-II also
increased the survival time in immunized mice compared
to mice in the control group (Figure 6). The variation in
protection effiency may be related to diffrences in the
challenge strain and method, immunization doses and
routes, infection site and etc, but the increase in survival
time is a common fiding that has been reported by most
studies. Therefore, it is suggested that use of exotoxin A in
combination with other antigens in vaccine preparations
would be benefiial. In conclusion, the recombinant nontoxic exotoxin A protein described in the present study is a
useful antigen to include in vaccine preparation for induction of an effient immune response.
Acknowledgements
We thank Dr. Ashraf Mobarez (Dept of Bacteriology,
Faculty of Medical Sciences, Tarbiat Modares University)
and Hamid Mostafazadeh (Biotechnology Research Center, Tabriz University of Medical Sciences) for their useful
suggestions.
Authors’ Contributions
All author had participated equally in the present study.
Financial Disclosure
The authors declare that they have no competing interests to disclose.
Funding/Support
The study is self funded.








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