Biological Activity of Recombinant Accessory Cholerae Enterotoxin (Ace) on Rabbit Ileal Loops and Antibacterial Assay

Headnote

Abstract

Objective: Vibrio choierae (V. choierae) causes a potentially lethal disease named cholera. The cholera enterotoxin (CT) is a major virulence factor of V. choierae. In ad- dition to CT, V. choierae produces other putative toxins, such as the zonula occludens toxin (Zot) and accessory cholera enterotoxin (Ace). The ace gene is the third gene of the V. choierae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli (E. coli) and determination of some characteristics of the recombinant Ace protein.

Materials and Methods: In this experimental study, the ace gene was amplified from V. choierae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli (DH5 a) host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-ß-D-galctoside (IPTG) at a dif- ferent concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa).

Results: The recombinant Ace protein with a molecular weight of 18 kDa (dimeric form) was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. choierae poly- clonal antibody. The Ace protein had antibacterial activity at a concentration of >200 pg/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test.

Conclusion: This study described an E. coli cloning and expression system (E. coli BL21- pET-28a-ace) for the Ace protein of V. choierae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein.

Keywords: Vibrio choierae, Accessory Choierae Enterotoxin, pET28a, E. coli

Cell Journalofakhteh), Vol 14, No 3, Autumn 201 2, Pages: 209- 21 4_

Citation: Anvari Sh, Najar Peerayeh Sh, Behmanesh M, Boustanshenas M. Biological activity of recombinant accessory choierae enterotoxin (Ace) on rabbit Ileal loops and antibacterial assay. Cell J. 2012; 14(3): 209-214.

Introduction

Vibrio choierae (V cholera), is a major human intestinal pathogen that causes significant mor- bidity and mortality in developing regions of the world. Cholera is endemic in Southern Asia and parts of Africa and Latin America, with 5 million cases annually. This disease is characterized by severe diarrhea caused by toxigenic V choierae which colonize in the small intestine ( 1 -4).

The cholera toxin (CT) is responsible for severe dehydration that results from diarrhea associated with V. cholerae.

V. cholerae produces other putative toxins, such as zonula occludens (Zot) and accessory cholera toxin (Ace). The genes encoding these toxins are located on a 4.5 kb region called the "core region" or virulence cassette, which is flanked by two copies of a repeat sequence (1, 2,5).

The Ace toxin is an integral membrane protein that consists of 96 amino acids (9-11.3 kDa) (6, 7). This toxin increases transcellular ion trans- port, causing FA in ligated rabbit ileal loops (4). The predicted amino acid sequence of the Ace protein shows a striking similarity to that of a family of eukaryotic ion transporting ATPases, including the human plasma membrane calcium pump, the calcium-transporting ATPase from rat brains, and the product of the cf gene (3, 6). The Ace protein acts synergistically with a Ca2+- dependent acetylcholine analog (carbachol) and stimulation secretion has been shown to be de- pendent on extracellular and intracellular Ca2+ (8). The Ace protein also shows a sequence sim- ilarity with a virulence protein of Salmonella dublin, SpvB, which is virulent in mice (6). Ace is an amphipathic molecule, which when insert- ed into the eukaryotic cell membrane creates an ion-permeable pore located on the 26-residue § toxin of Staphylococcus aureus (S. aureus) (6, 9). This study describes cloning, overexpression of Ace toxins in Escherichia coli (E. coli), and determination of some characteristics of the re- combinant Ace protein.

Materials and Methods

Bacterial strains and vectors

In this experimental study, V. cholerae strain 62013 was obtained from Pasteur Institute of Iran. E. coli DH5a and BL21 (PlysS) were used for clon- ing and expression experiments (Invitrogen and Novagen, USA). Plasmid pET-28a+ (Novagen) was the expression vector. Bacteria were cultured in LB broth or on agar (Merck, Germany) with or without 30 pg kanamycin/ml (Sigma, USA).

Preparation of DNA template and PCR

Genomic DNA of the V. cholerae strain 62013 was extracted using the Bioneer Kit (South Korea). The concentration and purity of extracted DNA were determined by spectrophotometer. Specific primers were designed according to ace gene sequences of V. cholerae from NCBI. The sequence of the for- ward primer with an endonuclease site of Ndel was 5'-GCTCCATATGCTTATGATGGACACCCTT- TATGAC-3', and for the reverse primer with an en- donuclease site of EcoRI, it was 5'- TAGAATTCT- CATAGGTTTAACGCTCGCAGGGC-3'. The PCR reaction mixture contained 0.5 pM of each primer, 10 pi 5X prime STAR buffer, 0.2 mM of each dNTP, 2.5 U of prime STAR DNA polymerase (Takara, Ja- pan), and 200 ng genomic DNA for a final volume of 50 pi. PCR amplification was performed with an initial dénaturation at 98°C for 4 minutes, followed by 35 cycles at 98°C for 10 seconds, 63°C for 15 seconds, and 72°C for 90 seconds, with a final ex- tension for 10 minutes at 72°C. PCR products were analyzed by electrophoresis on 1% (w/v) agarose gel (Fermentas, USA). The desired fragments were recovered from the gel by a PCR purification kit (Bi- oneer, South Korea).

Cloning, expression and purification of Ace

The PCR product and pET-28a (Novagen, United States) expression vector were digested by Ndel and EcoRI and purified from in agarose gel. The resultant fragment was ligated by T4 DNA ligase (Fermentas, USA). The recombinant pET-28a was transfonned to competent E. coli DH5a and the transformants were selected on LB agar plates that contained 30 pg/ml kanamycin.

The selected clones were confirmed by restric- tion enzyme digestion and PCR, and sequenced by a commercial facility using universal forward and reverse T7-promoter and T7-terminator primers (TAG Copenhagen A/S Symbion, Denmark). The result was compared to the sequence of the ace gene in the database with NCBI Blast Software. The recombinant plasmids were retransformed to an E. coli BL21 (plysS) expression host. Several conditions for the expression were tested, such as the temperature of induction and the concentration of isopropythio-ß-D-galctoside (IPTG). Bacterial cells grew in the presence of kanamycin (30 pg/ ml) at 37°C with shaking (220 rpm) until an opti- cal density at 600 nm of 0.6-1 was reached. IPTG (Sigma, USA) was added to a final concentration of 1 mM, followed by an additional 4 hours culture period at 37°C with vigorous shaking. Cells were harvested by centrifugation at 10000 g for 10 min- utes at 4°C to precipitate the pellet, after which the pellet was frozen at -20°C.

The bacterial pellets were lysed using a lysis buffer (8 M urea) until the solution cleared. After centrifuga- tion, the supernatants were examined by SDS-PAGE to verily the expressed recombinant protein.

Ace protein was purified by Ni-NTA affinity chromatography under a combination of denatur- ing and native conditions by binding, washing, and eluting steps according to the manufacturer's pro- tocol (Invitrogen). In this protocol, proteins were finally eluted in 20 mM buffer that contained imi- dazole, and then the eluted proteins were immedi- ately dialyzed against PBS (pH= 7.4) for removal of imidazole. Protein concentrations were detennined by Bradford and nanodrop analysis and purity by SDS-PAGE. Because Ace is an acidic protein (pi 4.26), it did not stain with standard Coomassie blue staining, thus we used silver nitrate staining ac- cording to the standard protocol (2).

Anti-Ace polyclonal antibody production

The overnight culture of V. cholerae 62013 (toxigenic) was exposed to fonnalin (1.5%) at 4°C overnight. After washing and defonnalization, the culture was heated at 65°C for 1 hour. The lysate was injected subcutaneously into a white New Zealand rabbit that weighed about 2 kg. The injec- tion mixture contained approximately 109 bacteria per ml of physiological serum and 1 ml complete adjuvant for the first injection, from which 0.5 ml was subcutaneously injected into the shoulder. We used incomplete adjuvant as the booster on days 14, 28, and 42. Bleeding was perfonned prior to each injection, and the serum was separated and stored at -20°C until use (10).

Western blot analysis

The proteins separated by SDS-PAGE were blotted onto 0.45 pm pore size PYDF membrane (Hi-bond Amersham Biosciences, USA) by using a semidry blotter unit (Bio-rad, USA). The membrane was blocked by 3% (w/v) skim milk according to standard procedures. Rabbit polyclonal anti- V cholerae serum was diluted 1:500 in phosphate-buffered saline (PBS) and 0.1% (v/v) Tween 20, then incubated for 3 hours at 4°C with shaking. The blocked membranes were washed with PBS-Tween 20, and then incubated with affinity purified goat anti-rabbit immunoglobulin G (heavy and light chain) horseradish peroxidase (HRP) conjugate antibody (Bio-Rad), at a 1:2500 dilution in PBS-Tween20. The membranes were then washed three times with PBS-Tween 20 and developed using DAB solution (Sigma, USA) (11).

Ligated rabbit ileal loop assay

New Zealand rabbits that weighed 2-2.5 kg were starved for 48 hours before the experiments. Rab- bits were anesthetized by subcutaneous injection of a mixture of ketamine (50 mg/g) and acepromazine (0.5 mg/kg), and the small intestines were tied off. A total of 1 ml (500 pg/ml) of the purified Ace protein was injected into the two intestinal segments. In this experiment, 1 ml (10s cfu/ml) of V cholerae 62013 was the positive control and sterile PBS was the negative control. Each test was undertaken on two rabbits and all rabbits were sacrificed after 18 hours. The enterotoxic response was detennined by measur- ing the FA ratio, which is the ratio of the volume of fluid accumulated in the intestinal loop to the length of the loop. A ratio of greater than 1.0 is indicative of a strong positive response, while a negative response is defined as a ratio of less than 0.5(11-16).

Antibacterial assay

We used bacterial strains S. aureus (ATCC25923), Pseudomonas aeruginosa (P aer- uginosa; ATCC27853), and E. coli (ATCC25922) to assay the antibacterial activity of the Ace pro- tein. Cultures of each bacteria were grown over- night in LB broth, then inoculated into fresh media and grown at 37°C until the OD595 reached 0.6. The cells were harvested and washed with 10 mM sodium phosphate (pH=7.4), then diluted in media to obtain a final density of 103 cfu/ml per assay sample. Bacteria were incubated with increasing concentrations of Ace protein (0, 50, 100, and 200 pg/ml) at 37°C for 18 hours in an incubator. The cell growth was detennined by colony count (7).

Results

Amplification of ace gene and construction of pET28a-ace

The amplified ace gene produced a single 299 bp band (Fig 1). The PCR product was purified and digested with Ndel and EcoRI, then sub- cloned into the expression vector pET28a. The result of the double enzyme digestion and PCR amplification confirmed that the ace gene was ex- actly inserted into the PET28a vector (Fig 2). The ace gene nucleotide sequence in the recombinant plasmid vector of pET28a-ace was consistent with that of V cholerae ace as published in the gene bank. The homologies of the nucleotide sequences in the pET-32a-ace compared with the published ace gene sequences were 99.8%.

Expression and purification of target recombi- nant protein

E. coli BL21 (DE3) plysS competent cells were transformed with the confirmed recom- binant vector, pET28a-ace. IPTG at 1 mmol/L efficiently induced the expression of ace fusion protein with predicated molecular masses of 18 KD (Fig 3A). Ace protein stained poorly with Coomassie blue stain, but stained efficiently with silver stain. Large scale culture and induc- tion was performed and the resultant protein was purified by Ni2+ affinity chromatography under denatured and native conditions (Fig 3B).

Western blot analysis

We performed Western blot analysis to detect immunogenicity of the expressed Ace protein, which was recognized by the rabbit polyclonal an- tibody against V cholerae (Fig 4).

Rabbit ileal loop test with recombinant protein

The purified Ace protein (500 pg) induced sig- nificant FA (ratio: 1.25 ± 0.2) in segmented rabbit ileal loops (Fig 5). FA of PBS (negative control) was not significant (ratio: 0.5 ± 0.005), while FA of the positive control was significant (ratio: 2 ± 0.2).

Antibacterial assay

We tested for antibacterial activity of purified the recombinant Ace protein to S. aureus, E. coli, and P. aeruginosa. There was inhibition and decreased bacterial growth at the 200 pg/ml concentration of Ace protein.

Discussion

Ace is a third toxin of V. cholerae (1) that causes milder cholera symptoms. It may con- tribute to an early phase of intestinal secretion in infections by V. cholerae, which can occur prior to the onset of secretion stimulated by the cholera toxin (8). Non-enterotoxigenic V. chol- erae cells that lack ace, zot, and ctx genes do not cause diarrhea in volunteers (14). Ace toxin increases the potential difference (PD) across the intestinal epithelium, alters ion transport, and increases the short-circuit current in rabbit ileal tissues that have been mounted in Ussing chambers (1-4, 14, 15). In this study the cloning of the ace gene was confirmed by colony-PCR, enzymatic digestion, and sequencing in E. coli BL21-pET-28a-ace. The nucleotide sequence of the ace gene in plasmid pET28a was 100% homologous with the ace gene reported in the gene bank ( Z22569.1).

The quantity of Ace protein produced in wild type V. cholerae was estimated to be 0.6 mg/ lit, which was 10000 fold lower than produced in our expression system (5 mg/lit). The pro- duction of the recombinant Ace protein in a concentration of 7 mg/lit in a yeast system was reported previously by Trucksis et al. (2). How- ever, because of E. coif s ability to grow rapidly and at a high density on inexpensive substrates, the prokaryotic expression system has remained very attractive for the production of recombi- nant proteins (15). According to other studies, expression of the recombinant Ace protein was successful in E. coli (LMG194), but in E. coli TOP 10, the Ace protein was not successfully produced (1,4).

Ace protein was purified by affinity chromatog- raphy with Ni-NTA resin. The predominant form of the Ace toxin was an 18 kD dimeric form. The Ace protein was not completely denatured and per- sisted as a dimer and multimer in the gel (1). The Ace protein was detected in both monomer and di- meric forms by Trucksis et al. (2). Chatterjee et al. (7) reported only the dimeric fonns of this protein. Our recombinant Ace protein did not stain with Coomassie blue, thus we used silver stain, as re- ported previously (1, 3, 15). The immunogenicity of this protein was confirmed by polyclonal anti- body against V. cholerae, by enterotoxicity in rab- bit ileal loops, and its antimicrobial effects showed it is active biologically. Ace protein has recently been used to treat cystic fibrosis (CF). CF involves insufficient chloride transport and loss of luminal sodium and water, leading to damage of the bron- chial connective tissue. The administration of Ace increases the level of secretion of chloride in the lungs and leads to an increase in the amount of air- way surface water in the lumen of the lungs (9). Therefore, recombinant production of the Ace pro- tein can be useful for medical applications.

Conclusion

Our highly efficient expression system (E. coli BL21- pET-28a-ace) can be used for conducting various biological experiments and has facilitated the production of pure proteins free from other Vi- brio antigens for investigation as potential vaccine candidates.

Acknowledgments

This work was financially supported by a grant from Tarbiat Modares University in Tehran. There is no conflict of interest in this article.

References

References

1. Somarny WM, Mariana NS, Rozita R, Raha AR. Cloning and expression of Vibrio Cholerae virulence gene acces- sory cholera enterotoxin(ace). Southeast Asian J Trop Med Public Health. 2004; 35(4): 856-862.

2. Trucksis M, Conn TL, FasanoA, Kaper JB. Production of Vibrio cholera accessory cholera enterotoxin (Ace) in the Yeast Pichia pastoris. Infect Immun. 1997; 65(12): 4984-4988.

3. Trucksis M, Galen JE, Michalski J, Fasano A, Kaper JB. Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholera virulence cassette. Proc Natl Acad Sei USA. 1993; 90(11): 5267-5271.

4. Somarny WM, Mariana NS, Neela V, Rozita R, Raha AR. Optimization of parameters for accessory cholera entero- toxin (Ace) protein expression. Med Sei. 2002; 2: 74-76.

5. Johnson JA, Morris JG Jr, Kaper JB. Gene encoding zonula occludens toxin (zot) does not occur independently from cholera enterotoxin genes (ctx) in Vibrio cholera. J Clin Microbiol. 1993; 31(3): 732-733.

6. 1 Kaper JB, Morris G Jr, Levine MM. Cholera. Clin Micro- biol Rev. 1995; 8(1): 48-86.

7. Chatterjee T, Mukherjee D, Dey S, Pal A, Hoque KM, Chakrabarti P. Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding. Biochemistry. 2011; 50(14): 2962-2972.

8. Trucksis M, Conn TL, Wasserman SS, Sears CL. Vi- brio cholerae ACE stimulated Ca (2+)- dependent Cl(-)/ HCO(3) (-) secretion in T84 cells in vitro. Am J Physiol cell physiol. 2000; 279(3): C567- 577.

9. Sears CL, Kaper JB. Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev. 1996; 60(1): 167-215.

10. Halliday LC, Artwohl JE, Hanly WC, Bunte RM, Bennett BT. Physiologie and behavioral assessment of rabbits im- munized with Freund's complete adjuvant. Contemp Top Lab Anim Sei. 2000; 39(5): 8-13.

11. Burnette WN. Western Blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detec- tion with antibody and radioiodinated protein A. Anal Bio- chem. 1981; 112(2): 195-203.

12. Begum K, Ahsan CR, Ansaruzzaman M, Dutta DK, Ah- mad QS, Talukder KA. Toxin(s), Other than cholera toxin, produced by environmental non 01 non 0139 Vibrio chol- era. Cell Mol Immunol. 2006; 3(2): 115-121.

13. Bidinost C, Saka HA, Aliendro O, Sola c, Panzetta-Duttari G, Carranza P, et al. Virulence factors of non-01 non- 0139 vibrio cholera isolated in cordoba, Argentina. Rev Argent Microbiol. 2004: 36(4); 158-163.

14. Davis BM, Waldor MK. Filamentous phages linked to viru- lence of Vibrio cholerae. Curr Opin Microbiol. 2003; 6(1): 35-42.

15. Baneyx F. Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol. 1999; 10(5): 411-421.

16. SyngkonA, Elluri S, KoleyH, Rompikuntal PK, Saha DR, Chakrabarti MK, et al. Studies on a Novel serine protease of a AhapAAprtV Vibrio cholerae 01 strain and its role in hemorrhagic response in the rabbit ileal loop model. pLoS One. 2010; 5(9): 13122.