GAPDH Activity and Immunogenicity of Staphylococcus aureus Recombinant GapC Protein

CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 5, May 2008 Online English edition of the Chinese language journal RESEARCH PAPER

Cite this article as: Chin J Biotech, 2008, 24(5), 754í759.

GAPDH Activity and Immunogenicity of Staphylococcus aureus Recombinant GapC Protein Hongwei Zhu1, Zhanbo Zhu1, Yudong Cui1, 2, Jing Zhang1, Fanze Piao1 1

College of Animal Science and Technology, Heilongjiang August First Land Reclamation University, Daqing 163319, China

2

College of Life Science and Technology, Heilongjiang August First Land Reclamation University, Daqing 163319, China

Abstract:

To characterize the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, immunogenicity and

immunoprotection of Staphylococcus aureus (S. aureus) surface protein GapC, gapC gene of S. aureus was amplified from strain BMSA/855/23-1 by PCR, and was inserted into pQE-30 vector subsequently. The recombinant plasmid, designated as pQE/gapC, was transformed into E. coli strain M15 (pREP4). The recombinant GapC fusion proein was successfully expressed in E. coli M15 induced with IPTG and its GAPDH activity was confirmed by GAPDH activity assay. Then, the recombinant GapC protein, inactivated S. aureus whole cell and placebo (PBS) were administrated to healthy rabbits respectively. The IgG antibody titers, concentration of IFN-J and IL-4 cytokines in immunized rabbit sera were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Finally, immunized rabbits were challenged with S. aureus strain Wood46 to evaluate the immunoprotection. The IgG antibody titers against GapC and whole cell in rabbit sera reached their peaks at day 28 after boost immunization (1:64 000). The concentrations of IL-4 and IFN-J in GapC group rabbit sera increased significantly (P<0.05) at day 14 after boost immunization, while the concentrations of those in whole cell group did not increase (P>0.05) compared with the placebo group. 4 rabbits in 5 of the protein immunized group were protected against challenge with 1×108CFU S. aureus. The results above indicate that the expressed recombinant GapC protein have high GAPDH activity and immunogenicity, can also protect against S. aureus challenge to some extent. S. aureus GapC protein could be an attractive target for further genetic engineering vaccine. Keywords: Staphylococcus aureus; GapC protein; GAPDH activity; immunogenicity; immunoprotection

Introduction Staphylococcus aureus is an important opportunistic pathogen that may cause various diseases among human and animals. Owing to the lack of common protective antigens among different S. aureus isolates, the existing vaccines have somewhat limited protection against new infection in clinical trials, emphasizing the need for the conserved antigens candidates[1]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known as a key enzyme in glycolytic pathway and it reversibly catalyses the oxidative phosphorylation of glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate.

In addition, studies show that GAPDH proteins may be used as attractive antigens in vaccines to protect against bacterial, fungal, and parasitic infections[2–6]. Two conserved proteins with GAPDH activity, GapB and GapC, were found on both human and bovine strains of S. aureus[7]. In this study, we expressed the GapC protein in E. coli via the molecular cloning technique. The protein was purified and immunized to rabbits afterwards to characterize the cellular and humoral immune level of GapC protein from an S. aureus isolate BMSA/855/23-1. Thus, it is expected to serve as an antigen in the formulation of the engineering vaccine against S. aureus.

Received: August 2, 2007; Accepted: September 3, 2007 Supported by: the Key Technologies R&D Program of Heilongjiang Province (No. GA02B501) Corresponding author: Yudong Cui. Tel/Fax: +86-459-6819290; E-mail: [email protected] Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

Hongwei Zhu et al. / Chinese Journal of Biotechnology, 2008, 24(5): 754– 759

1

Materials and methods

1.1 Materials 1.1.1 Bacterial strains and plasmid: S. aureus strain BMSA/855/23-1 was used to amplify gapC gene, and the strain Wood46 was used as gapC positive control. Escherichia coli M15(pREP4) was used as final host, whereas XLI Blue was used to facilitate cloning and sequencing. Plasmid pQE-30 was used as a vector for gene expression. 1.1.2 Primers: A pair of primers was selected on the basis of the gapC gene nucleotide sequence of S. aureus (1008 bp long; GenBank Accession No. AY356529). A 30-nucleotide forward primer, gapC Pf (5ƍ-CGCGGATCCATGGCAGTAA AAGTAGCAATT-3ƍ), corresponding to positions 1 to 22 of the gap gene, and a 34-nucleotide reverse primer, gapC Pr (5ƍ-AAATTAGGTACCTTTAGAAAGTTCAGCTAAGTAT3ƍ), corresponding to positions 1008 to 987, were selected. BamH I and Kpn I restriction enzyme sites were introduced at the 5ƍ end of the primers (as indicated underline). The primers were synthesized by Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. (Shanghai, China). 1.1.3 Reagents: Taq DNA polymerase, T4 DNA ligase, restriction enzymes, and protein molecular weight marker were purchased from Fermentas Life Sciences Inc. PIPES, DMSO, glyceraldehyde-3-phosphate (G3P), HRP-Goat Anti-Rabbit IgG, Freund's adjuvant were the products of Sigma-Aldrich Co. Tryptic Soy Broth(TSB) medium for S. aureus cultivation was obtained from Becton Dickinson Co. BioSpin plasmid preparation kit and DNA gel purification kit were obtained from BIOER Technology Co., Ltd. Goat anti-rabbit interleukin 4 and goat anti-rabbit Ȗ interferon cytokines ELISA Kit were obtained from BPB Biomedical Co. DAB Kit was the product of Wuhan Boster Biological Engineering Co., Ltd. 1.1.5 Experimental Animals: 25 male New Zealand white rabbits, about 2.0–2.5 kg in weight, were used for immunologic tests. 1.2 gapC gene amplification from S. aureus Genomic DNA from strains of S. aureus was prepared as described before[8] and used for PCR templates. gapC genes were obtained by PCR amplification using the selected primers. Briefly, the reaction mixture consisted of ~50 ng of template DNA, 1.25 U of Taq DNA polymerase, 5 ȝL of 10 × PCR amplification buffer, 1 ȝmol each primer, 0.2 mmol dNTP, 2 mmol Mg2+, and double-distilled water to a final volume of 50 ȝL. DNA was denatured at 94oC for 2 min. A total of 30 PCR cycles were run under the following conditions: DNA denaturation at 94oC for 90 s, primer annealing at 53.5oC for 1 min, and DNA extension at 72oC for 1 min. After the final cycle, the reactions were terminated by an extra run at 72oC for 10 min. PCR

products were analyzed by agarose(1%) gel electrophoresis. 1.3 Construction of recombinant GapC expression vector The obtained gapC gene was purified with Biospin DNA purification Kit, then double digested with BamH I and Kpn I restriction endonuclease. Ligation reaction was performed to the sticky-ended gapC gene and the corresponding enzymes treated pQE30 plasmid. Competent cells of E. coli strain XLI Blue and M15 (pREP4) were prepared following the Inoue’s protocol[9]. The DNA was then transformed into the XLI Blue competent cell. The recombinant plasmid, designated as pQE30/gapC, was then isolated from E. coli XLI Blue and identified with PCR, enzyme digestion, and sequencing. At last, the pQE30/gapC plasmid was transformed into the final host E. coli strain M15 (pREP4), and the host was renamed E. coli M15 (pQE/gapC+pREP4). 1.4 Expression and purification of the recombinant GapC protein 50 ȝL E. coli M15 (pQE/gapC+pREP4) was cultivated in 50 mL Luria broth (LB) medium to mid-log phase at 37oC. The expression of the 6×His-GapC proteins was induced by the addition of 1mM isopropyl-ȕ-D-thiogalactopyranoside (IPTG), and the cells were harvested 3 h, 4 h, 5 h, 6 h postinduction. Protein samples of the uninduced and induced cultures as well as E. coli M15 (pQE-30) were analyzed by SDS-PAGE, and gels were stained with Coomassie brilliant blue. For GapC purification, after SDS-PAGE, the gels were stained with KCl (250 mM), and the white-stained GapC was then carefully chipped off from the gel. The protein was detached from the gel after electrophoresis in dialysis bag. The GapC protein was purified after dialysis in PBS overnight at 4oC and concentrated in polyethylene glycol (MW. 8000). The concentration of the protein was measured in protein quantitative apparatus (Amersham Pharmacia Biotech, USA). 1.5 GAPDH assay Whole staphylococcal cells and recombinant GapC protein were assayed for GAPDH activity. Whole cells were ultrasonic-treated, frozen, and thawed for 3 times followed by centrifugation for 5 min at 10 000 rpm. The proteins were then incubated with 20 mmol G-3-P and 10 mmol (NAD+) in a final volume of 1 mL of assay buffer [40 mmol triethanolamine, 50 mmol Na2HPO4, and 5 mmol of EDTA (pH 8.6)]. After incubation for 30 min, the formation of NADH was monitored spectrophotometrically at OD340. G-3-P was omitted and used for controls. 1.6 Animals immunization A total of 25 rabbits were randomly divided into five groups of five each, among which two groups were immunized with GapC with one for challenge, two with inactivated staphylococcal bacterin with one for challenge, and a placebo group was included as well. For initial immunization, 500 ȝg protein was administered to each rabbit via subcutaneous injection, and via the

Hongwei Zhu et al. / Chinese Journal of Biotechnology, 2008, 24(5): 754– 759

intramuscular route for boost immunization (300 ȝg) 14 days later. 1 mL Staphylococcal bacterin (5×107 CFU/mL inactive S. aureus) was immunized to rabbits as per the same procedure. 2 mL of blood samples were collected every week after boost immunization and the serum samples were prepared and stored before use. 1.7 Immunogenicity of the recombinant GapC protein 1.7.1 Western blotting analysis: For Western blotting, uninduced and 4 h induced proteins separated by SDS-PAGE were transferred onto a nitrocellulose membrane and incubated with diluted anti-GapC polyclonal antisera (1:1000), and probed with HRP-conjugated goat anti-rabbit IgG as the secondary antibody. Chromogenic reaction was developed after incubation at room temperature with DAB kit. 1.7.2 IgG titers detection: 96-well ELISA plates were coated with GapC (10 ȝg per well) or whole staphylococcal cell as antigens. Serum samples were diluted and tested by indirect ELISA for the presence of IgG, with HRP-conjugated goat anti-rabbit IgG as the secondary antibody and TMB (3, 3ƍ, 5, 5ƍ-tetramethylbenzidine) as the chromogenic reagent. The absorbance (optical density) of each well was read with a plate reader (Bio-Rad Laboratories, Inc.) at OD450. The IgG titers of samples were determined at the dilution of which OD value •OD value of control sera + 3 s (standard deviation). 1.7.3 Cytokine assay: On day 14 after boost immunization, concentrations of cytokine IL-4 and IFN-Ȗ in serum samples were detected with goat anti-rabbit interleukin 4 and goat anti-rabbit interferon Ȗ ELISA kits (BPB Biomedical). Concentrations of IL-4 and IFN-Ȗ were calculated according to the standard curves after ELISA. In addition, statistical analyses of the concentrations of cytokines as well as IgG titers were performed using SPSS10.0 statistical software. The data were analyzed by T-test. 1.8 Animals challenge 50 µL of S. aureus strain Wood46 was cultivated in 5 mL TSB medium, and then the bacteria was counted and diluted to a final concentration of 1.0×108 CFU/mL. 28d after boost immunization, all three groups of rabbits (n=5) were infected by ear intravenous injection with 1mL S. aureus Wood46. The body temperatures, food intake, waterdrinking, defecation etc, were recorded twice a day to monitor possible signs of illness. 5 d after challenge, all survival rabbits were executed and dissected. S. aureus were re-isolated and identified from livers, spleens, and lymph nodes aseptically taken. The immunoprotection efficiency was evaluated accordingly.

BMSA/855/23-1 according to 1% agarose gel electrophoresis (Fig. 1). The fragments were then cloned into pMD 18-T vector, and sequenced by Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. The sequence shared 99.3% DNA similarity and 99.4% deducted amino acid similarity with gapC gene of S. aureus strain BM10 in GenBank (Accession No. AY356529). The deducted amino acid sequence was similar to NAD binding domain of Glyceraldehyde-3-phosphate dehydrogenase, according to the NCBI http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. 2.2 Construction of GapC expression vector gapC gene was cloned into pQE30 plasmid to construct an expression vector pQE/gapC; the plasmid was then identified with PCR(Fig. 2A) and restriction endonuclease digestion(Fig. 2B). Electrophoresis analysis showed that the gapC gene was amplified and could be cut from vector with BamH I and Kpn I. 2.3 Expression of recombinant GapC protein and Western blotting

Fig. 1 PCR products of S. aureus gapC gene M: DL2000 DNA marker; 1: PCR product of Wood46 strain; 2: PCR product of BMSA/855/23-1strain

Fig. 2

Identification of the recombinant pQE/gapC plasmids by PCR and restriction endonuclease digestion

2

Results

M1: DNA marker DL2 000; M2: DNA marker DL15000; A1, A2: positive clones identified by PCR; A3: negative clone; B1: pQE/gapC plasmid

2.1 Cloning and sequence analysis of gapC gene 1008 bp DNA fragments were obtained by PCR amplification from S. aureus strain Wood46 and isolate

digested with BamH I; B2: pQE/gapC digested with BamH I and Kpn I

Recombinant GapC protein was expressed when the E. coli M15 (pREP4) harboring pQE/gapC plasmid induced

Hongwei Zhu et al. / Chinese Journal of Biotechnology, 2008, 24(5): 754– 759

with IPTG, SDS-PAGE showed that the mass of the protein was in accordance with the molecular masses of the proteins deduced from the amino acid sequences (Fig. 3A); in addition, the protein could bind the polyclonal antisera as indicated by Western blotting (Fig. 3B). 2.4 GAPDH characterization of recombinant GapC The GAPDH activity of S. aureus whole cell and purified protein (rGapC) was determined as described above, and the GAPDH activity was indicated by OD340. The result indicated that the rGapC of S. aureus (0.467) had stronger GAPDH activity than that of whole cell (0.135) and control (0.012). 2.5 IgG titers Antibody IgG levels in rabbits sera were detected by indirect ELISA, and the results showed that the IgG titers could reach 1:32000 to 1:64000 two weeks after boostimmunization with the peak titers week 4 after boost-immunization (data not shown). To reflect IgG dynamic levels, indirect ELISA tests were performed to 100-times dilute sera. The results showed that the IgG in sera increased rapidly after boost-immunization and reached the peaks appropriately on day 28, and then declined slowly with time, whereas, the titers in control group did not increase significantly (Fig. 5). 2.6 Cytokines concentration in sera of rabbits Concentration of IFN-Ȗ and IL-4 cytokines in sera of each group were measured with ELISA kit. Compared with the whole cell group as well as placebo group, GapC immunized group resulted in significantly higher concentration of IL-4 and IFN-Ȗ in rabbits’ sera (P<0.05); despite the concentrations of both cytokines in whole cell group increasing, these differences were not significantly different (p>0.05) (Tab. 1).

Fig. 4

GAPDH activity assay of the recombinant GapC protein

Fig. 5 Dynamic curves of antibody IgG from the serums of boost immunized rabbits (n=5) Tab. 1 Concentrations of cytokines in sera of vaccine immunized and control rabbits (x±s) Group

n

IFN-Ȗ

IL-4

rGapC

5

182.9±12.4*

205.8±23.4*

Whole cell

5

52.4±9.9**

60.7±11.4**

Saline control

5

49.0±13.6

54.4±12.7

*P<0.05 compared with whole cell and saline control; **P>0.05 compared with saline control

Fig. 3

SDS-PAGE (A) and Western blotting assay (B) of the expressed product

A1: uninduced E. coli M15 (pQE/gapC+pREP4); A2–A5: 3 h, 4 h, 5 h, and 6 h induced E. coli M15 (pQE/gapC+pREP4) respectively; M: protein molecular weight marker; A6: 6h induced E. coli M15 (pQE-30); A7: purified GapC protein; B1: Western blotting of uninduced E. coli M15 (pQE/gapC+pREP4); B2: Western blotting of the expressed product by using the anti-GapC serum from rabbits; M: protein marker

2.7 Immune protection of the recombinant GapC 2.7.1 Clinical features of the challenged rabbits: 3 days before challenge, all rabbits were in good health status, with the body temperatures between 38.5oC and 39.5oC. 5 days after challenge with virulent S. aureus, one rabbit from GapC immunized group and two from whole cell group showed clinical symptoms, while all rabbits in control group had severe clinical symptoms with one death. All rabbits had temporary high body temperatures (39.5oC to 40.2oC), which however, declined to normal 2 days after challenge. Body temperatures of the rabbits with clinical phenomenon kept above 41.0oC for at least 3 days (data not shown). 2.7.2 Pathological changes of challenged rabbits: Anatomical examinations were conducted to the rabbits with clinical signs. The results showed that these rabbits had severely

Hongwei Zhu et al. / Chinese Journal of Biotechnology, 2008, 24(5): 754– 759

swollen kidneys with petechia on surface and sometimes tip-like necrotic lesion as well. 2.7.3 Re-isolation of S. aureus from rabbits: S. aureus were isolated and identified from the corresponding rabbits with clinical symptoms or dead, and the biochemical characteristics of the isolated S. aureus were consistent with that of challenged isolate. Therefore, according to the results of clinical signs, pathological changes of challenged rabbits, and isolation of S. aureus from sick rabbits, the immune efficiencies were evaluated: 4 rabbits of recombinant GapC immunized group(n=5) were protected against challenge of S. aureus, whereas only 3 rabbits in whole cell group(n=5) were protected.

3

Discussion

Major hurdles to overcome in the development of vaccines against S. aureus are the complexity of the bacterial components and the lack of conserved antigens that provide cross species immune protection. GapC protein of S. aureus is a highly conversed surface protein with GAPDH activity. Perez-Casal’s group found that the protein encoding gene exists in all 11 bovine mastitis derived isolates[7]. Thus, these features make GapC an attractive target antigen for vaccines against S. aureus. GAPDHs catalyze the oxidative phosphorylation of glyceraldehyde-3-phosphate (G-3-P) to 1-3 diphosphoglycerate in the presence of phosphate (Pi) and NAD+, and their activity can be measured by measuring the formation of NADH[10]. In the study, the recombinant GapC as well as whole cell was tested for their GAPDH activity, and it was apparent that the whole cell had less GapC component than purified protein, and thus had low activity in the test. Meanwhile, we demonstrated that the proteins retained their activity in SDS-denatured forms. IgG titers in sera of rabbits, as indicated by ELISA test, increased rapidly after immunization and reached their peaks in week 4 after boost immunization; these results confirmed its ability to elicit humoral immune response. However, the IgG1 with agglutination and toxolysin activities and IgG2a with opsonic activity remain to be characterized later. The cytokines IFN-Ȗ and IL-4 in sera of rabbits increased significantly (P<0.05) on day 14 after boost immunization; however, there was no significant difference in sera of the whole cell immunized groups and control group. This difference indirectly demonstrated that the GapC protein caused cytokines secreted into blood, and thus, elicited cellar immune response. The inactivated S. aureus failed to elicit increased cytokines in sera, which may attribute to the capsular polysaccharide component surrounding S. aureus. Capsular polysaccharide has anti-phagocytosis activity that prevents reorganization by neutrophils and it has weak antigenicity, unable to elicit

antibody response. In addition, the capsular polysaccharide is a T-lymphocyte independent antigen, unable to elicit cellar immune response[11]. To determine the pathogenesis of challenged rabbits, the body temperature, clinical signs, and pathological changes were monitored before and after challenge; besides, the results were then confirmed by reisolating S. aureus from suspected organs. All rabbits had raising temperatures at different degrees after receiving S. aureus infection; however, these recovered to normal in short time for the rabbits protected. Thus, we counted this transient high temperature change as well as the rabbits recovered to health states in a rather short time. Recently, it is suggested that S. aureus can be classified as an intracellular bacterium. Therefore, specific cellar immune response is critical to clear the bacteria and prevent S. aureus infection[12]. Thus, immunizations with either plasmids containing protein encoding gene alone or a combination of priming with plasmid DNA followed by a boost with the recombinant proteins may constitute an attractive strategy[13,14].

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