Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli
Daling Zhua,b, Aihua Lia, Jianguo Wanga , Ming Lia and Taozhen Caia
aThe Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
bGraduate School of the Chinese Academy of Sciences, Beijing 100039, China Received 02 December 2006; revised June 2007
Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli bl21. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His·Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.
Keywords: Aeromonas hydrophila, Cloning, aerA gene, Prokaryotic expression, Recombinant protein AerA-W, Hemolytic activity
Aeromonas hydrophila (genus Aeromonas; family Aeromonadaceae) is a ubiquitous Gram-negative aquatic bacterium which frequently causes harm to the aquaculture and is a causative agent of human diarrhoea and septicaemia1-3. The pathogenesis of A. hydrophila is multifactorial and involves proteases, haemolysin, enterotoxins, acetylcholinsterase and a surface array protein layer4-7. One of the major virulence factors is a toxin aerolysin (a protein), which possesses hemolytic activity against rabbit erythrocytes, cytotoxic activity against Vero cells and enterotoxigenicity in the suckling mouse test8.
Aerolysin is one of the best-characterized bacterial channel-forming toxins9. It is synthesized intracellu- larly as preproaerolysin and secreted extracellularly as an inactive form (proaerolysin) by Aeromonas spp.10. Proaerolysin traverses the inner membrane with aid of a typical N-terminal signal sequence, which is removed before the protein is released into the periplasm, where it folds and dimerizes before it is released from the cell11. Proaerolysin is then converted to aerolysin by proteolytic removal of about 45 amino acids from the C-terminus. Aerolysin binds
to specific surface receptors on target cells and promotes oligomerization, which converts it into an insertion-competent state. Insertion results in the generation of discrete membrane channels causing osmotic swelling and lysis of erythrocytes12.
Aerolysin has been used for detection of glycosyl phosohatidylinositol (GPI)-anchored proteins and to study their function, for example, identification of erythrocyte aerolysin receptor (EAR) and Thy-1 (an GPI-anchored protein that binds aerolysin, found in brain and T-lymphocytes)13,14, diagnosis of paroxysmal noturnal haemoglobinuria or PNH (an acquired hematopoietic stem cell disorder manifested by abnormal hematopoiesis, complement-mediated intra-vascular hemolysis and a propensity towards thrombosis)15, isolation of intracellular parasites from tissues and blood16,17, production of attenuated viral vaccine, purging blood products, cells, or tissues of a population of enveloped viruses, and virus detection18, etc.
Earlier, recombinant protein expressed as inclusion bodies has been purified under denatured condition, but no hemolytic activity has been reported19,20. Aerolysin is harvested from the native organisms, but the expression is very low and is influenced by many culture conditions21. The recombinant aerolysin could, however, overcome these disadvantages. Thus, in this
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Corresponding author E-mail: wangjg@ihb.ac.cnT.
Telefax: 86-02768780720
study, to obtain a significant amount of recombinant aerolysin in the active form, we have expressed the aerolysin in E. coli under the control of T7 RNase promoter. The hemolytic activity in the purified protein is also reported.
Fig. 1—Schematic representation of expression of plasmid pET32a-AerA-W
Materials and Methods
Bacterial strains, vectors, enzymes and chemicals
A. hydrophila XS91-4-1 was isolated from diseased fish22 and cultured in LB broth at 25°C. E. coli DH5α was used as the host for plasmid pGEM-T (Promega) and the recombinant plasmids for cloning and sequencing. E. coli bl21 (DE3) was used to express the gene cloned into plasmid pET32a (Novagen).
E. coli was grown in LB broth or on LB agar plates at 37°C. When required, the medium was supplemented with 100 µg/ml ampicillin and/or kanamycin (15 µg/ml). His·Bind®Resin and His·Bind columns (purchased from Novagen) were used for recombinant protein purification, according to the manufacturer’s instructions. Ex Taq DNA polymerase, restriction endonuclease BamHI and HindIII, T4 DNA ligase and nucleic acid weight marker DL2000 were obtained from TaKaRa Biotechnology Co. (Dalian, China).
Construction of expression vector pET32a-AerA-W
The genomic DNA of A. hydrophila XS91-4-1 was extracted using a bacterial genomic DNA extraction kit (TaKaRa). To obtain the aerolysin in the active form, the aerA gene was cloned omitting only the first 18 bp which encodes the signal peptide. The PCR amplification was performed using primers EAPT-F:
5’-CGCGGATCCATGGGCTTGTCATTGATCATA- TCC-3’ with a BamHI site and EAER-R: 5’- CCCAAGCTTCGTTATTGATTGGCAACTGGC-3’
with a HindIII site. In brief, samples were adjusted to a final volume of 25 µl by adding 0.125 µl TaKaRa Ex Taq polymerase (5 U/µl), 2.5 µl 10×Ex Taq buffer, 2 µl dNTP mixture (2.5 mM respectively), 1 µl of each primer (20 µM) and 1 µl genomic DNA. Reaction mixtures were overlaid with one drop of paraffin oil and incubated for 2 min at 94°C, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 2 min and a final extension at 72°C for 10 min.
The PCR products were purified, digested with BamHI and HindIII, repurified and ligated in-frame into the corresponding site of a pET32a bacterial expression vector to generate the plasmid pET32a- AerA-W (Fig. 1).
Subsequently, the plasmids pET32a-AerA-W were transformed into E. coli bl21 (DE3) cells. Colonies
with correct sizes were further identified by restriction enzyme analysis. The plasmid DNA was purified using a plasmid mini kit (Omega) prior to sequencing. Two clones were sequenced to check that the sequences did not contain PCR-based errors.
Expression and purification of recombinant aerolysin
The expression of aerolysin in E. coli was performed as previously described 23. In brief, cells were grown overnight in LB medium with 100 µg/ml ampicillin, 15 µg/ml kanamycin, 34 µg/ml chloramphenicol and 12.5 µg/ml tetracyline at 37°C and were diluted into 250 ml culture. The culture was incubated (at 220 rpm, 37°C) until an optical density of 0.6 at 600 nm was reached.
Isopropyl-thio-β-D-galactopyranoside (IPTG) was added to a final concentration of 1 mM to induce expression of the T7 RNA polymerase and the culture was incubated for another 4 h at identical conditions.
The cell pellets were harvested by centrifugation (5,000 rpm for 5 min, at 4°C) and resuspended in 50 ml of 1×binding buffer (pH 7.9) containing 0.5 M NaCl, 20 mM Tris-HCl, 0.5 mM imidazole. To obtain soluble or insoluble fusion proteins, the whole cell pellets from bacterial cultures were broken by sonication (13 w, 10 s, interval 10 s) on ice. The supernatants obtained by centrifugation (at 20, 000 g and 4°C for 30 min) were analyzed by SDS-PAGE.
The expressed protein was purified by metal- affinity chromatography using His·Bind resin. The purification of soluble and denatured aerolysin was carried out according to manufacturer’s instructions (Novagen, USA). In brief, for purification the supernatant containing soluble native aerolysin was applied directly to the His·Bind resin chromatography, while the inclusion bodies were first dissolved in 1×binding buffer containing 6 M urea at 4°C overnight, the supernatant containing the solubilized aerolysin was obtained by centrifugation (30 min at 12,000 g and 4°C) and then applied to the His·Bind resin chromatography. After the column was bound, washed and eluted, the native and denatured recombinant proteins were eluted with 1×eluting buffer containing 0.5 M NaCl, 20 mM Tris-HCl, 800 mM imidazole, pH 7.9 or 0.5 M NaCl, 20 mM Tris-HCl, 800 mM imidazole, 6 M urea, pH 7.9, respectively. The collected fractions were analyzed by SDS-PAGE and tested for hemolytic activity on blood agar plates. The 1×eluting buffer and 1×eluting buffer with 6 M urea were used as negative controls.
SDS-PAGE and Western blot analysis
SDS-PAGE was performed using 12%
polyacrylamide gels as described by Laemmli24. The concentration of recombinant protein in crude extract was determined by the Bradford assay using bovine serum albumin as the standard. After SDS-PAGE electrophoresis, proteins were transferred on to nitrocellulose (NC) membrane by electroblotting using a wet blotter (Bio-Rad, Richmond, USA) at 80 v for 90 min. Following the procedure described previously23, antigen bands were probed with the rabbit polyclonal antibodies against the purified recombinant aerolysin AerA-W and sonicated formalin-killed whole cells (WCs) of A. hydrophila.
The NC membranes were then treated with alkaline phosphatase conjugated goat anti-rabbit immunogl- obulin and antigen bands were stained using alkaline phosphatase chromogen kit (BCIP/NBT).
The polyclonal antibody against the recombinant aerolysin (AerA-W) and WCs of A. hydrophila were prepared in an adult New Zealand white rabbit. The former was obtained by injecting 300 µg of recombinant aerolysin in SDS-PAGE gels ground with phosphate-buffered saline (PBS) into soup.
Booster doses were given at 200 µg protein on days 15 and 30 after initial immunization. On day 45, the rabbit was bled and serum was collected and stored in aliquots at -20°C. The polyclonal antibody against the
WCs was obtained by injecting sonicated formalin- killed WCs and stored in aliquots at -20°C.
Hemolytic activity
The hemolytic activity of recombinant aerolysin was determined by the agar diffusive hemolysis test (ADHT)25. After chromatographic purification, hemolytic activity in the elution fractions was detected by ADHT. The medium containing 1% agar, 0.8%
NaCl, 0.02% KCl and 25 mM Tris (pH 7.4) was sterilized at 1.034 × 105 Pa for 15 min. Subsequently, 3% red blood cells of Carrassius auratus ibebio were added, mixed thoroughly and when the medium cooled down to approximately 40°C, then poured immediately into Petri dishes. After the medium was solidified, holes of about 5 mm in diameter were made at the centre of the dishes. The 50 µl of soluble recombinant protein in 1×eluting buffer and 50 µl of denatured recombinant protein in 1×eluting buffer containing 6 M urea were added into the holes. The 50 µl 1×eluting buffer and 1×eluting buffer with 6 M urea were used as negative controls. Finally, the hemolysis of each dish was visualized and scanned for records after incubating at 25°C for 10-12 h.
Results
Expression and purification of recombinant aerolysin AerA-W
To obtain recombinant aerolysin with hemolytic activity, nearly full coding region (aa 7-493) of A. hydrophila aerolysin (preproaerolysin) was isolated
Fig. 2—SDS-PAGE analysis of aerolysin AerA-W expression of at different induction temperatures [Lane M, molecular mass standard; lane C, uninduced E. coli BL21(DE3) cells containing plasmid pET32a-AerA-W; lanes 1, 2, 3: induced cells containing expression plasmid pET32a-AerA-W at 4, 25, and 37°C respectively for 6 h]
Fig. 5—Hemolytic activity of recombinant AerA-W [A, 1×elute buffer (40 mM imidazole; B, recombinant aerolysin AerA-W purified under native condition; C, 1×elute buffer with 6 Murea (40 mM imidazole); and D, recombinant aerolysin AerA-W purified under denaturing condition]
Fig. 3—SDS-PAGE analysis of recombinant aerolysin AerA-W purified at native and denaturing conditions [Lane M, molecular mass standard; lane A, aerolysin AerA-W by denaturing purification; and lane B, aerolysin AerA-W by native purification]
Fig. 4—Fusion proteins detected by western blot using the rabbit polyclonal antibody against purified aerolysin AerA-W (a) and whole cells of A. hydrophila (b) [The arrow indicates the 67.8 kD fusion protein]
b a
by PCR. The segment of aerA gene (acession no.
DQ186611) was inserted into the pET32a vector, generating the expression plasmid pET32a-AerA-W (Fig. 1). The expression of truncated recombinant AerA protein (aa7-493) in E. coli was examined by SDS-PAGE analysis. The results suggested that a 67.8 kDa protein was expressed only in the induced cells (Fig. 2), the same size of the recombinant protein.
The influence of IPTG induction temperatures on expression of recombinant protein was analyzed by SDS-PAGE. The results indicated a little expression of AerA-W at 4°C, partial expression in soluble form and as inclusion bodies at 20 or 25°C and nearly all as inclusion body at 37°C (Fig. 2). Both soluble and denatured recombinant proteins were purified using a nickel column. A single band with a molecular mass about 67.8 kDa was detected in both the elution fractions (Fig. 3).
Western blot analysis
Antigenicity of the recombinant aerolysin AerA-W was demonstrated by Western blot using rabbit anti- sera against the purified AerA-W protein or against the whole cells of A. hydrophila. Two types of sera recognized the recombinant AerA-W protein (Fig. 4).
Hemolytic activity of recombinant aerolysin AerA-W
The soluble recombinant protein produced a visible hemolytic zone and the denatured protein a hemolytic homocentric double-circle on the blood agar plates.
Results indicated that both forms showed hemolytic activity on blood agar plates after diffusion at 25oC overnight (Fig. 5).
Discussion
Although production of A. hydrophila aerolysin in E. coli was reported earlier19,20 , this is the first report when active aerolysin was obtained under the control of the T7 RNase promoter in E. coli. In this study, aerolysin of A. hydrophila was expressed both in soluble and inclusion body form in E. coli. Soluble recombinant aerolysin was produced at 20 or 25oC, but at 37oC, aerolysin was found only in the insoluble form.
Our aim was to obtain a significant amount of recombinant aerolysin in the active form. Thus, a truncated form of the AerA protein, lacking of 6 amino acids at the N terminus (part of the signal peptide) was expressed in E. coli . After optimization of induction 67.8KDa
procedure, the protein was expressed partially in the cytoplasmic compartment in soluble form, but mostly in the form of inclusive bodies in the cytoplasm.
Interestingly, purified recombinant denatured protein demonstrated hemolytic activity on the blood plate.
Possibly, the denatured aerolysin AerA-W refolded during its diffusion outward into the media, suggesting the recombinant aerolysin as inclusion bodies could be renatured.
The active aerolysin is required for various applications such as for diagnosis of the PNH, to isolate intracellular parasites from tissues and blood, for production of attenuated viral vaccine and to purge blood products, cells, or tissues of a population of enveloped viruses13-18. In our laboratory, we have successfully used the recombinant aerolysin in the separation of cellular parasites in fish blood (data not shown). It is likely that the recombinant aerolysin could also be used in the other applications.
In conclusion, the recombinant active aerolysin was expressed in E. coli under the control of the T7 RNase promoter. Recombinant aerolysin showed hemolytic activity and good antigenicity.
Acknowledgments
The work was supported by the Sciential Innovation Project of Chinese Academy of Sciences No. KSCX2-1-04 and the Sciential Innovation Project of Institute of Hydrobiology, Chinese Academy of Sciences No. 220317.
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