Mansoura University
Faculty of Pharmacy
Department of Microbiology
Role of some proteins and
exotoxin A in protection against
Pseudomonas aeruginosa
infections
A ِِِThesis presented by
Abeer Mohamed Abd EL-Aziz Ibrahim
M. Pharm. Sci., Microbiology
For the Degree of Doctor of Philosophy in Pharmaceutical sciences
(Microbiology)
Supervisors
Prof. Dr.
Prof. Dr.
Wael Abass EL-Naggar
Ramadan H. Ibrahim Hassan
Professor of Microbiology
Faculty of Pharmacy, Mansoura University
Professor and Head of Microbiology
Department
Faculty of Pharmacy, Mansoura University
Dr.
Mohammed Youssif Ibrahim
Lecturer of Microbiology
Faculty of pharmacy, Mansoura University
 2012 
Summary
Pseudomonas aeruginosa is a serious pathogen for specific cohorts of
patients
such
as
those
with
CF,
burn
wounds
or
who
are
immunocompromized. Significant disease burden is associated with a
diverse spectrum of both nosocomial and community acquired infections.
Given the high degree of antibiotic resistance that characterizes P.
aeruginosa and the difficulties inherent in long-term chemotherapy
particularly against inevitable chronic and persistent infections, the
development of a vaccine against P. aeruginosa is an appropriate and
challenging strategy to pursue (Sedlak-Weistein et al., 2005).
Our study aimed to prepare different antigen vaccines against P.
aeruginosa using recombinant DNA technology and to test the efficacy of
these recombinant antigens in protection against P. aeruginosa infection in
murine acute pneumonia model.
To achieve our goal, the genomic DNA of P. aeruginosa PAO1 was
extracted using genomic DNA extraction kit. PCR was used to amplify fliC
(B), OprF, OprI and exo-A using high fidelity DNA polymerase. The
amplicons were separated using agarose gel electrophoresis and their bands
were recovered from the gel using Qiaex II agarose gel extraction kit.
The purified blunt-ended amplicons were A-tailed via an A-tailing
reaction according to Kobs, 1997. Each of the 3'-A tailed amplicon was
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separately ligated to the pGEM-T Easy cloning vector according to
Promega Technical Manual. The ligation product was transformed into
competent E. coli DH5α according to Sambrook and Russell, 2001. The
transformants were selected on LB/amp/IPTG/X-gal plates for blue-white
screening, some white colonies were picked and their plasmids were
isolated by plasmid extraction kit. Cloning was confirmed by EcoRI
enzymatic digestion of the extracted plasmids to screen for clones
containing the correct recombinant vectors.
Recombinant plasmids confirmed to contain the correct amplicons
were selected to subclone the amplified ORFs from pGEM-T Easy vector
into pRSET-B expression vector. The correct recombinant plasmid and
pRSET-B were double digested by the same restriction enzymes to form
complementary sticky ends. pGEM-T/ fliC(B) was double digested with
XhoI and HindIII, both pGEM-T/OprF and pGEM-T/OprI were double
digested with EcoRI and BamHI while pGEM-T/exo-A was digested with
KpnI and EcoRI. Products of digestion were separated by electrophoresis
and gel purification. The purified insert and opened vector were ligated
together using T4 DNA ligase according to Sambrook and Russell, 2001.
Each engineered recombinant expression vector was then transformed into
E. coil DH5α and transformants were selected on LB/amp plates. For each
transformation reaction, some colonies were picked and miniprepped to
screen for the presence of the correct insert by restriction analysis. The
recombinant vectors contained the right size of insert were sequenced using
T7 and T7 reverse primers. The obtained sequences showed 100% identity
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to the corresponding reference sequence from GenBank through multiple
sequence alignment by ClustalW.
For expression of recombinant proteins, the recombinant pRSET-B
vectors containing the inserts with correct sequences were transformed into
E. coli BL21 (DE3) pLysS. The transformants were selected on
LB/amp/chl plates and then, a time course expression experiment was
performed according to Ausubel et al., 1994 through samples collected at
different time points during induction with IPTG. The SDS-PAGE analysis
showed that fliC (B) was highly expressed after 4 hrs of induction and that
it was highly detected in the supernatant. The SDS-PAGE of OprF and
OprI showed that both are best expressed after 5 hrs of induction and they
were also present in the form of soluble proteins. Regarding exotoxin A, it
was not detected in the SDS-PAGE and it was thought that it was not
expressed due to its toxicity to the host bacteria. Large scale production of
each recombinant protein was then performed. The expressed proteins with
6xHis tag were purified using Ni+2 Sepharose 6 Fast Flow packed column.
The SDS-PAGE of the purified proteins demonstrated the presence of a
single band of fliC (B), OprF and OprI at 53, 38 and 7 kDa, respectively.
Western blotting applied to the purified recombinant proteins using antihistidine tag monoclonal antibodies confirmed the identity and purity of
recombinant proteins. The purified proteins were then subjected to
diafiltration using Amicon Centrifugal Filter Devices. The concentrations
of the recombinant proteins were measured using the Bradford protein
assay kit according to the manufacturer’s protocol with BSA as standard.
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Concentrations of recombinant fliC (B), OprF and OprI were 3 mg/ml, 1.5
mg/ml and 2 mg/ml respectively.
Seventy two BALB/c mice were divided into six groups and were
immunized with the prepared recombinant antigen(s) according to
Goudarzi et al., 2009. The first, second and third groups were immunized
with recombinant flagellin (B), OprF and OprI respectively. The fourth
group was immunized with combined antigen vaccine OprF/OprI while the
fifth group was immunized with fliC (B)/OprF/OprI combined antigen
vaccine and the sixth group was used as control. All mice were immunized
with a dose of 50g of recombinant protein(s) administrated
subcutaneously in FCA and were boosted two times with the same dose of
recombinant protein(s) in FIA at days 7 and 14. ELISA was performed on
sera collected from all groups of mice before bacterial challenge where
antigen specific antibodies were detected in the sera of immunized groups
with significant high titers compared to control group.
Two weeks after the second booster dose, all groups were challenged
with a lethal dose (1x107 CFU/mouse) of either P. aeruginosa PAO1 or
PAK strains according to DiGiandomenico et al., 2007 in an acute
pneumonia model. The results of survival analysis and viable bacterial
count in blood demonstrated that immunization with fliC (B) achieved
significant protection (83.3% survival) and significant decrease in
Pseudomonas blood count following challenge with PAO1 strain compared
to control group while non significant difference was observed upon
challenge with PAK strain. Immunization with OprF achieved 66.6 %
protection following challenge with PAO1 and 50% after challenge with
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PAK strains with a corresponding significant decrease in bacterial blood
count. Immunization with OprI afforded the least protection with no
significant difference between the immunized and control groups with
respect to both survival analysis and Pseudomonas blood count. For
OprF/OprI combined vaccine, 66.6% survival was achieved after challenge
with either P. aeruginosa strains with significant decrease in Pseudomonas
blood count. The highest protection against infection was achieved using
fliC (B)/OprI/OprI combined vaccine as all mice (100%) were protected
upon challenge with PAO1 and 66.6 % survived after infection with PAK
with a significant decrease in Pseudomonas blood count.
In conclusion:
 Recombinant DNA technology was successfully applied to
construct prokaryotic expression systems of fliC (B), OprF, OprI and
exo-A.
 Expression of recombinant flagellin B, OprF and OprI were
successfully achieved using BL-21 (DE3) pLysS with high levels of
protein expression. The results showed not only the high efficiency
of protein expression but also the simplicity, the high yield and the
high purity of the expressed proteins following a one step affinity
chromatography protocol using Sepharose columns charged with
nickel.
 The in vivo studies demonstrated that in a mouse acute
pneumonia model, active immunization with fliC (B), OprI and OprF
alone or in combination elicited high level of antigen specific
antibodies. The lowest protection was obtained using OprI alone and
5
the highest protection was obtained using fliC (B)/OprF/OprI
combined antigen vaccine.
 Further studies are needed to evaluate the efficacy of fliC
(B)/OprF/OprI vaccine in different animal models and against
challenge by different clinical isolates of P. aeruginosa.
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