Construction and use of a prokaryotic expression system for Helicobacter pylori AhpC

Background Helicobacter pylori is an important pathogen responsible for human gastric problems like inflammation, ulcers and cancer. It is widely prevalent in developing countries with low socioeconomic status. Since the infection remains asymptomatic in most individuals, efforts for efficient diagnostic markers to identify high risk patients are warranted. In this study, we constructed an expression vector that overexpresses the H. pylori AhpC protein as a glutathione S-transferase fusion protein. We furthermore examined whether this recombinant fusion protein retained immunogenicity and thus would be useful as a diagnostic marker. Findings The full-length tsaA gene from H. pylori strain G27, which encodes AhpC, was cloned in plasmid vector pGEX-6P-2 to create the recombinant plasmid vector pGEX-tsaA. The nucleotide sequence of the clone showed 100% homology with corresponding published sequence of original gene. Over-expression of the target protein GST-AhpC was achieved in E. coli BL21 (DE3) cells by induction with isopropyl-beta-D-thiogalactoside (IPTG). GST-AhpC was extracted and identified using SDS-PAGE as a 52 kDa protein. Western blotting results using commercial antibodies against whole cell H. pylori showed that the fusion protein retained immunogenecity. Conclusion A recombinant prokaryotic expression system was successfully established with high expression efficiency for target fusion gene pGEX-tsaA. The expressed GST-AhpC protein showed immunoreactivity against commercial anti-H. pylori antibodies. This recombinant fusion protein can be developed as a diagnostic marker for screening patients with chronic H. pylori infections.


protein, AhpC
Findings Background Helicobacter pylori has been recognized as a human gastric pathogen able to colonize in the stomachs of around half of the world's population [1]. Most infected individuals remain asymptomatic, however, the infection may cause acute and chronic gastritis or peptic ulceration, besides being a risk factor for development of gastric adenocarcinoma, mucosa-associated lymphoid tissue (MALT) lymphoma and primary gastric non-Hodgkin's lymphoma [2][3][4][5].
H. pylori infection is acquired in early childhood. Like all developing countries, the prevalence of H. pylori infection in Pakistan is very high in children. Results of urea breath test in infants from suburbs of Karachi revealed that 80% were positive for H. pylori [6]. H. pylori is able to colonize human stomach for life, if not eradicated. Persistent colonization requires H. pylori to avoid damage from by-products of oxygen metabolism and oxidative host responses. H. pylori has an impressive array of antioxidant proteins. The bacterium protects itself against such oxidative damage by expressing enzymes like superoxide dismutase SodB [7], catalase KatA [8] and KatA-associated protein KapA [9]. The activities of alkyl hydroperoxide reductase AhpC [10], thiol peroxidases Bcp and Tpx [11] have also been reported to protect H. pylori against organic peroxides. NADPH quinone reductase MdaB [12] and the iron-binding protein NapA [13] were also found involved in resistance to oxygen stress.
H. pylori AhpC is a thioredoxin (Trx)-dependent AhpC and a member of the 2-Cys peroxiredoxin family . AhpC is one of the major proteins for antioxidant defense in H. pylori and plays an important role in gastric colonization by the microbe [10]. The tsaA gene was originally annotated as HP1563 in H. pylori 26695 [14] and HP1471 in H. pylori J99 [15], however Chalker et al. (2001) annotated the gene as ahpC, because this nomenclature is more usual for bacterial alkyl hydroperoxide reductase enzymes [16]. AhpC has been reported in many bacteria, like Escherichia coli and Salmonella typhimurium [17]. H. pylori AhpC was reported much near to eukaryotic Prxs unlike reductases found in many other bacterial species and indeed, could act like a molecular chaperone similar to Prxs present in yeast and human [18]. Recently, AhpC was found to be consistently expressed in higher amounts in H. pylori strains isolated from gastric cancer patients than in patients presenting gastritis only [19]. The 26-kDa protein first reported as an antigenically conserved H. pylori species specific protein, is now being predicted to be a useful diagnostic marker for detection of H. pylori infection [20]. It was also found associated with a specific antibody response in patients with adenocarcinoma [21].
In the present study, a recombinant expression plasmid containing whole tsaA gene from H. pylori G27 was constructed. The plasmid was cloned in E. coli BL21 cells and recombinant fusion protein was expressed, extracted, identified and analyzed for immunoreactivity with commercial anti H. pylori antibodies. The results of this initial work provide a basis for future studies using this fusion protein to develop a specific diagnostic marker for detection of advanced stage diseases like peptic ulcer, gastric cancer and adenocarcinoma due to chronic H. pylori infection in Pakistani population.

Polymerase chain reaction, cloning and transformation
The full-length tsaA gene was amplified as described in the methods, digested with restriction enzymes and ligated with pGEX-6p-2 that had been cut with the same enzymes to generate pGEX-tsaA. This plasmid places the GST coding sequences N-terminal to the TsaA/AhpC coding sequences and thus should generate rGST-AhpC fusion protein (Figure 1). pGEX-tsaA was used to transform E. coli DH10B to ampicillin resistance, and the correct nature of pGEX-tsaA was verified using PCR and DNA sequencing ( Figure 2).

Expression of target fusion protein
We next examined whether pGEX-tsaA would overexpress rGST-AhpC. For this experiment we transformed pGEX-tsaA into the E. coli strain BL21 (DE3) that encodes a chromosomal T7 RNA Polymerase under the control of a tac promoter. Under IPTG induction, the tac promoter gets activated that drives expression of the rGST-AhpC. We thus added IPTG at concentrations of 0.1 and 1 mmol/L to mid-log phase cultures of pGEX-tsa-BL21 (DE3) grown at 37°C, and collected whole cell proteins for SDS-PAGE gel analysis. We found that pGEX-tsaA expressed robust amounts of rGST-AhpC that migrated at the expected size of 52 kDa ( Figure 3). Further analysis demonstrated that this rGST-AhpC was mainly present as inclusion bodies, based on the observation that the protein was not soluble after cell lysis (Data not shown).

Immunoreactivity of GST-AhpC
Although it is well known that antibodies against AhpC are generated during H. pylori infection, we did not know if such antiserum would recognize a rGST-AhpC fusion protein. We thus tested whether a commercial rabbit antibody that had been generated against whole cell H. pylori would recognize our recombinant rGST-AhpC. We obtained robust recognition, confirming that this recombinant protein still displays critical antigenic determinants ( Figure 4).

Purification of fusion protein
Target fusion protein rGST-AhpC was extracted from large volume LB-Amp culture (1 L) using repeat sonication and washing followed by centrifugation after each step. The samples were checked on 10-12% SDS-PAGE gel for protein quality. Pellet after third sonication and centrifugation step (Pellet-III) showed best results with a good intense band and little degradation/cleavage products. Pellet-III sample was dialyzed and the supernatant, after dialysis was concentrated to approx. 3 ml. Right sized band for fusion protein was observed in case of both dialyzed and concentrated protein samples with no apparent degradation product as compared to pellet-III sample without dialysis and concentration ( Figure 5).

Discussion
The study of virulence factors and important antigens of H. pylori is important to understand bacterial mechanisms involved in persistent colonization and inflammation. Prokaryotic expression systems for several important H. pylori virulence factors have been successfully constructed and recombinant fusion proteins or their purified proteins used in seroprevalence studies or screened as H. pylori vaccine candidates [22][23][24], however, little attention has been paid towards protein products of housekeeping genes like tsaA (ahpC).  reported that mutant H. pylori strains defective in AhpC were more sensitive to oxidative stress conditions compared to wild-type cells [25]. Also, Olczak et al., (2003) reported that ahpC/tsaA mutant strains were unable to colonize mouse stomach [26]. An important feature of AhpC is that it seems to promote the activity of other proteins. Specifically, loss of AhpC resulted in partial inactivation of catalase, suggesting that AhpC is required for proper catalase activity [27].
At least two studies reported that antibodies against AhpC were found only in H. pylori infections that resulted in disease. Wang et al., 2004 found antibodies against AhpC in sera of peptic ulcer, cancer and adenocarcinoma patients infected with H. pylori [21]. In a recent report, Haung et al., 2011 examined protein expression levels of AhpC in H. pylori from different clinical manifestations and observed 5-fold increase in AhpC protein expression in strains isolated from gastric cancer patients than those from gastritis [19]. This describes the potential of AhpC as an efficient biomarker to identify and risk patient population presenting different stages of dseases associated with chronic H. pylori infections. Another important H. pylori antigen associated with disease is CagA. Patients infected with cagA + H. pylori have higher risk of developing gastric cancers than those infected with cagAstrains [28,29]. Various studies have reported that 60 to 70% of H. pylori strains isolated from European and North American populations carried the cagA gene [30][31][32], whereas over 90% of the isolates from Asia-Pacific populations were found to be cagA + [33][34][35]. The fact that nearly all strains of H. pylori from countries like Taiwan and China as well as a high proportion in western countries are cagA positive makes it difficult to use cagA only as a marker for screening high-risk patients in these populations.
The tsaA gene from H pylori strain G27, cloned in this study, showed 100% nucleotide homology with published corresponding sequences. In the present study, SDS-PAGE demonstrated that the constructed expression systems pGEX-6p-2-tsaA-BL21(DE3) efficiently produced the target recombinant GST fusion protein, however mainly presented in the form of inclusion body. However, we successfully obtained free of inclusion body fusion protein using repeat sonication and centrifugation protocol which can be extended to subsequent refolding and cleavage from GST tag. The commercial rabbit antiserum against the whole cell of H. pylori recognized and combined with rGST-AhpC as confirmed by Western blot, indicating that the fusion protein had high immunoreactivity. However, we could not explain presence of lower intensity bands other than that of the right sized band, both in SDS-PAGE ( Figure 3B) and their subsequent recognition in Western Blot (Figure 4). Perhaps these are E. coli BL21 (DE3) proteins which crossreacted with the commercial rabbit antiserum against the whole cell of H. pylori. Several other approaches have been used for construction and use of prokaryotic expression system for potential H. pylori vaccine candidates like flaA, flab, ureB and cagA by other groups [22][23][24].
Limitations of present study include use of GST fusion protein itself and not its cleaved purified product, and not testing the sera of potential patient groups to demonstrate the effectiveness of this approach due to some constraints.

Conclusions
We have successfully constructed an efficient expression system for ahpC, an important gene of H. pylori in pGEX-6p-2. This construct was successfully transformed in E. coli BL21 (DE3) that gave a recombinant fusion protein (rGST-AhpC), mainly as inclusion body. Immunoreactivity against rGST-AhpC was tested against commercial anti-H. pylori antibodies raised in rabbit serum. The fusion protein was then processed to get free from inclusion body. This recombinant fusion protein can serve as a potential diagnostic marker for screening advanced clinical manifestations due to chronic H. pylori infections. Such strategies are need of the time to identify high risk patients keeping in view the genetic diversity of H. pylori regarding a well accepted biomarker CagA across the globe.

Bacterial strains
Bacterial strains used are given in Table 1. E. coli strain DH10B was used for cloning and BL21 (DE3) used for protein expression.

DNA extraction
Genomic DNA from 2-3 days culture of H. pylori strain G27 was extracted using Wizard genomic kit (Promega) according to manufacturer's instructions.

Polymerase chain reaction
PCR reaction mixture contained 100 ng of H. pylori G27 genomic DNA, 2.5 μl of dNTPs, 2.5 μl of 30 mM MgCl 2 PCR buffer, 200 ng of each primer and 1 μl of Taq polymerase. Final volume was made to 25 μl with sterile water. Genomic DNA of H. pylori 26695 in same concentration was used as positive control. PCR conditions were: Initial melting at 94°C for 3 minutes, then 30 cycles of repeat melting at 94°C for 40 sec, annealing at 52°C for 40 seconds and initial extension at 72°C for 30 seconds, then final extension at 72°C for 3 minutes and storage at 4°C until run on the gel. The results of PCR were observed under UV light after electrophoresis on 1.5% agarose gel stained with ethidium bromide. The expected size of tsaA amplification fragment was 600 bp.

Sequencing
Sequencing of PCR products was done by the DNA sequencing facility, UC Berkeley, CA. A sample containing 100 ng gel purified DNA and 0.8 pg of forward or reverse primer was adjusted to a total volume of~13 μl in dH 2 O. Sequences obtained were subjected to nucleotide BLAST feature of National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) for homology analyses with published tsaA sequence of G27.

Cloning of tsaA gene into plasmid vector
Gel purified tsaA whole gene was cloned into plasmid vector pGEX-6p-2. Both the tsaA PCR product and plasmid were digested with restriction endonucleases BamHI and EcoRI (New England Biolabs) at 37°C overnight. Digested DNA and plasmid were gel purified and concentration was determined by Nanodrop. The plasmid sample was treated with Shrimp alkaline phosphatase (New England BioLabs). Ligation was done with 0.5 μl T4 ligase enzyme (New England Biolabs) by incubating the ligation mix at 16°C for overnight. This plasmid was transformed into E. coli DH10B using electroporation; ampicillin-resistant colonies were screened using direct colony PCR using the original tsaA primers. One colony containing the correct plasmid was used to purify plasmid for sequencing.

Expression and identification of target recombinant protein
pGEX-Recombinant plasmid was transformed through electroporation into E. coli BL21 (DE3) (Promega). One BL21 (DE3) colony of successful transformant containing pGEX-tsaA was cultured in LB-Amp broth at 37°C until OD 600 of 0.5, after which IPTG was added at concentrations of 0.1 and 1.0 and mmol/L. Cultures were incubated for the times indicated in Figure 3A. Cells were then collected by centrifugation at 6000 X g force. The bacterial pellet was resuspended in 200-300 μl of 2x Laemmli sample buffer (1.52 g Tris-Base, 2 g SDS, 20 ml 100% glycerol, 2 ml β-merceptoethanol and 1 mg bromophenol blue in dH 2 O to make 100 ml), and resuspended pellets were boiled for 5 minutes on water bath for denaturing proteins. SDS-PAGE (10%) was used to measure the molecular mass and output of the target recombinant fusion protein (rGST-AhpC).

Immunoreactivity of recombinant fusion protein
Immunoreactivity of the fusion protein was then determined. Briefly, PVDF membrane (BioRad) cut to the size of the gel was pre-wet in 100% Methanol. Gel, sponge pads, 4 pieces of Whatman filter paper and MeOH-

Isolation of fusion protein from inclusion bodies
The fusion protein was isolated from inclusion bodies by using an online protocol (http://www.its.caltech.edu/ bjorker/Protocols) with some modifications. Briefly, culture from single colony of successful transformant grown in 1 L LB-Amp broth was centrifuged (Sorvall RC5C Plus, Thermo Fischer Scientific, Rockford, USA) at 7000 X g for 20 minutes at 4°C. Resultant supernatant was carefully decanted and pellet was resuspended in 13 ml of solution buffer (50 mM Tris-HCl, 25% sucrose, 1 mM NaEDTA, 0.1% Sodium azide and 10 mM DTT) on ice and sonicated (Sonic Dismembrator, Fischer Scientific, Rockford, USA) at 50% amplitude for 4-5 times on ice. Then 100 μl Lysozyme, 250 μl DNase I and 50 μl MgCl 2 were mixed with 12.5 ml of lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 1% Na deoxycholate, 100 mM NaCl, 0.1% Sodium azide and 10 mM DTT) and sample was incubated for 45 minutes at room temperature. In next step, 350 μl of NaEDTA was added to the sample and freezed in liquid nitrogen. The sample was then thawed at 37°C for 30 minutes. 200 μl MgCl 2 solution was added to the solution and placed at room temperature for an hour. Then 350 ml NaEDTA was added again to the solution and placed on ice. This solution was centrifuged at 11000 X g at 4°C for 20 minutes and supernatant was discarded in a tube. The pellet was washed with 10 ml washing buffer (50 mM Tris-HCl, 0.5% Triton X-100, 100 mM NaCl, 1 mM NaEDTA, 0.1% Sodium azide and 1 mM DTT) and sonicated on ice. Solution was spun again at 11000 X g for 20 minutes at 4°C and supernatant was discarded in a tube. Pellet was again resuspended in 10 ml washing buffer but this time without Triton X-100 and sonicated. The same procedure was repeated for third time. Samples from both supernatant and pellet after each centrifugation step were retained for checking protein quality. In the last step, pellet was dissolved in 5 ml of 8 M Guanidinium solution (pH 8.0) in the presence of 4 mM DTT and shaken at room temperature till complete dissolution. The dissolved sample was stored at −80°C.

Dialysis and concentration
Solution was diluted to 50 ml with minimal protein buffer (150 mM NaCl and 50 mM Tris-HCl at pH 8.0) and dialyzed against 1 L of MPB using SnakeSkin dialysis tubing (Thermo Fischer Scientific, Rockford, USA) with gentle shaking at 4°C. After 2 hours of shaking, the buffer was changed and then dialyzed overnight. Solution was pelleted at 3000 X g for 20 minutes at 4°C. The resultant supernatant was concentrated to about 3 ml volume using Amicon Ultra-15 concentrator (Millipore, USA) following manufacturer's instructions. Both concentrated and dialyzed pellets were analyzed for proteins on SDS-PAGE. Both the pellet and concentrate were lyophilized (FreeZone 2.5 Freeze Dry Systems, Labconco, Kansas, USA) for long term storage and further use.