GapC Protein Research Articles

Prokaryotic expression of the GapC protein of Streptococcus uberis and prediction, identification of its B-cell epitopes

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide 266AANDSYGYTEDPIVSSD282 reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.

RETRACTED ARTICLE: TaWRKY40 transcription factor positively regulate the expression of TaGAPC1 to enhance drought tolerance

BackgroundsDrought stress is one of the major factors that affects wheat yield. Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that plays the important role in abiotic stress and plant development. However, in wheat, limited information about drought-responsive GAPC genes has been reported, and the mechanism underlying the regulation of the GAPC protein is unknown.ResultsIn this study, we evaluated the potential role of GAPC1 in drought stress in wheat and Arabidopsis. We found that the overexpression of TaGAPC1 could enhance the tolerance to drought stress in transgenic Arabidopsis. Yeast one-hybrid library screening and EMSA showed that TaWRKY40 acts as a direct regulator of the TaGAPC1 gene. A dual luciferase reporter assay indicated that TaWRKY40 improved the TaGAPC1 promoter activity. The results of qRT-PCR in wheat protoplast cells with instantaneous overexpression of TaWRKY40 indicated that the expression level of TaGAPC1 induced by abiotic stress was upregulated by TaWRKY40. Moreover, TaGAPC1 promoted H2O2 detoxification in response to drought.ConclusionThese results demonstrate that the inducible transcription factor TaWRKY40 could activate the transcription of the TaGAPC1 gene, thereby increasing the tolerance of plants to drought stress.

Cytosolic GAPDH as a redox-dependent regulator of energy metabolism

BackgroundPlant cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GapC) displays redox-dependent changes in its subcellular localizations and activity. Apart from its fundamental role in glycolysis, it also exhibits moonlighting properties. Since the exceptional redox-sensitivity of GapC has been suggested to play a crucial role in its various functions, we here studied its redox-dependent subcellular localization and the influence of the redox-state on GapC protein interactions.ResultsIn mesophyll protoplasts from Arabidopsis thaliana, colocalization of GapC with mitochondria was more pronounced under reducing conditions than upon oxidative stress. In accordance, reduced GapC showed an increased affinity to the mitochondrial voltage-dependent anion-selective channel (VDAC) compared to the oxidized one. On the other hand, nuclear localization of GapC was increased under oxidizing conditions. The essential role of the catalytic cysteine for nuclear translocation was shown by using the corresponding cysteine mutants. Furthermore, interaction of GapC with the thioredoxin Trx-h3 as a candidate to revert the redox-modifications, occurred in the nucleus of oxidized protoplasts. In a yeast complementation assay, we could demonstrate that the plant-specific non-phosphorylating glyceraldehyde 3-P dehydrogenase (GapN) can substitute for glucose 6-P dehydrogenase to generate NADPH for re-reduction of the Trx system and ROS defense.ConclusionsThe preferred association of reduced, glycolytically active GapC with VDAC suggests a substrate-channeling metabolon at the mitochondrial surface for efficient energy generation. Increased occurrence of oxidized GapC in the nucleus points to a function in signal transduction and gene expression. Furthermore, the interaction of GapC with Trx-h3 in the nucleus indicates reversal of the oxidative cysteine modification after re-establishment of cellular homeostasis. Both, energy metabolism and signal transfer for long-term adjustment and protection from redox-imbalances are mediated by the various functions of GapC. The molecular properties of GapC as a redox-switch are key to its multiple roles in orchestrating energy metabolism.

Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein

The GapC protein of Staphylococcus aureus (S. aureus) is a surface protein that is highly conserved among Staphylococcus strains, and it can induce protective humoral immune responses. However, B-cell epitopes in S. aureus GapC have not been reported. In this study, we generated a monoclonal antibody (mAb2A9) targeting S. aureus GapC. Through a passive immunity test, mAb2A9 was shown to partially protect mice against S. aureus infection. We screened the motif 236PVATGSLTE243 that is recognized by mAb2A9 using a phage-display system. The motif sequence exactly matched amino acids 236–243 of the S. aureus GapC protein. Then, we identified the key amino acids in the motif using site-directed mutagenesis. Site-directed mutagenesis revealed that residues P236, G240, L242, and T243 formed the core of the 236PVATGSLT243 motif. In addition, this epitope was proven to be located on the surface of S. aureus, and it induced a protective humoral immune response against S. aureus infection in immunized mice. Overall, our results characterized a conserved B-cell epitope, which will be an attractive target for designing effective epitope-based vaccines against S. aureus infection.

Identification of a protective B-cell epitope of the Staphylococcus aureus GapC protein by screening a phage-displayed random peptide library.

The impact of epidemic Staphylococcus aureus (S. aureus) on public health is increasing. Because of the abuse of antibiotics, the antibiotic resistance of S. aureus is increasing. Thus, there is an urgent need to develop new immunotherapies and immunoprophylaxes. Previous studies showed that the GapC protein of S. aureus, which is a surface protein with high glyceraldehyde 3-phosphate dehydrogenase activity, transferrin binding activity, and other biological activities, is highly conserved. GapC induces an effective humoral immune response in vivo. However, the B-cell epitopes of S. aureus GapC have not been well identified. Here we used the bioinformatics tools to analyze the sequence of GapC, and we generated protective anti-GapC monoclonal antibodies (mAbs). A protective mAb (1F4) showed strong specificity to GapC and the ability to induce macrophages to phagocytose S. aureus. We screened the motif 272GYTEDEIVSSD282, which was recognized by mAb 1F4, using a phage display system. Then, we used site-directed mutagenesis to identify key amino acids in the motif. Residues G272 D276 E277 I278 and V279 formed the core of the 272GYTEDEIVSSD282 motif. In addition, we showed that this epitope peptide induced a protective humoral immune response against S. aureus infection in immunized mice. Our results will be useful for the further study of epitope-based vaccines against S. aureus infection.

Immunogenicity of amino acids 1-150 of Streptococcus GapC displayed on the surface of Escherichia coli.

Streptococcus is one of the main pathogens that cause bovine mastitis. They includes into S.agalactiae, S.dysgalactiae, and S.uberis. The GapC protein is a virulence factor that is expressed on the surface of Streptococcus species. GapC is highly antigenic and immunization with GapC confers cross-protection against all three species. Our previous data showed that amino acids 1-150 of GapC (GapC1-150) of S.dysgalactiae conferred similar immunoprotection compared to full-length GapC. Thus, the present study aimed to construct a recombinant Escherichia coli XL1-Blue strain that displayed GapC1-150 on its surface, and to investigate the immunogenicity of the surface-localized GapC1-150. To do so, the ompA gene of the E.coli XL1-Blue strain was replaced with the lpp'-ompA-gapC11-150 or lpp'-ompA genes by λ Red recombination, the former of which fused GapC1-150 to an Lpp lipoprotein signal peptide and amino acids 1-159 of OmpA; the recombinant strains were named XL1-Blue/LOG76 and XL1-Blue/LO11, respectively. GapC1-150 was confirmed to localize to the surface of the XL1-Blue/LOG76 strain by an indirect enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sorter analysis, and laser-scanning confocal microscopy. Then, ICR mice were immunized intramuscularly with the XL1-Blue/LOG76 or XL1-Blue/LO11 strains, or recombinant GapC1-150. The sera of the immunized mice were collected and the anti-GapC1-150 antibody levels were detected by ELISA. Lymphocytes secreting interleukin (IL)-4 and interferon-γ were detected by an enzyme-linked ImmunoSpot assay, as was the level of IL-17A level in the supernatant of cultured splenic lymphocytes. The mice immunized with the XL1-Blue/LOG76 strain or GapC1-150 exhibited better cellular and humoral immunity. Lastly, the immunized mice were challenged with S.uberis, S.dysgalactiae, and S.agalactiae strains, and mice that were immunized with the XL1-Blue/LOG76 strain were better protected than those that were immunized with the XL1-Blue/LO11 strain. These results indicate that it is feasible to display GapC1-150 on the E.coli surface as a vaccine against Streptococcus species.

Identification and characterization of CD4+ T-cell epitopes on GapC protein of Streptococcus dysgalactiae

The GapC protein is highly conserved surface dehydrogenase among Streptococcus dysgalactiae (S. dysgalactiae) and is shown to be involved in bacterial virulence. Immunization of GapC protein can induce specific CD4+ T-cell immune responses and protect against S. dysgalactiae infection. However, there are no studies to identify immunodominant CD4+ T-cell epitopes on GapC protein. In this study, in silico MHC affinity measurement method was firstly used to predict potential CD4+ T-cell epitopes on GapC protein. Six predictive 15-mer peptides were synthesized and two novel GapC CD4+ T-cell epitopes, GapC63–77 and GapC96–110, were for the first time identified using CD4+ T-cells obtained from GapC-immunized BALB/c (H-2d) and C57BL/6 (H-2b) mice spleen based on cell proliferation and cytokines response. The results showed that peptides containing 63–77 and 96–110 induced significant antigen-specific CD4+ T-cells proliferation response in vivo. At the same time, high levels of IFN-γ and IL-17A, as well as moderate levels of IL-10 and IL-4 were detected in CD4+ T-cells isolated from both GapC and peptide-immunized mice in vivo, suggesting that GapC63–77 and GapC96–110 preferentially elicited polarized Th1/Th17-type responses. The characterization of GapC CD4+ T-cell epitopes not only helps us understand its protective immunity, but also contributes to design effective T-cell epitope-based vaccine against S. dysgalactiae infection.

Identification of a Conserved Linear B-Cell Epitope of Streptococcus dysgalactiae GapC Protein by Screening Phage-Displayed Random Peptide Library.

The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif 48DTTQGRFD55 shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of 48DTTQGRFD55. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection.

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched 30TRINDLT36 of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of 30TRINDLT36, and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope 30TRINDLT36 showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines.

Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope

Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97–103 of the S. dysgalactiae GapC. In addition, the epitope 97TGFFAKK103 showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of 97TGFFAKK103, and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine.

Role of GapC in the pathogenesis of Staphylococcus aureus

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating cows, sheep and goats. S. aureus produces a wide arsenal of cell surface and extracellular proteins involved in virulence. Among these are two conserved proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity named glyceraldehyde-3-phosphate dehydrogenase-B (GapB) and -C (GapC). In this study, we used the S. aureus wild type strain RN6390 and its isogenic gapC mutant H330 in in vitro and in vivo studies and determined that the S. aureus GapC protein plays a role on adherence to and internalization into bovine mammary epithelial (MAC-T) cells. In addition, we found that S. aureus H330 did not caused mastitis after an experimental infection of ovine mammary glands. Together, these results show that GapC is important in the pathogenesis of S. aureus mastitis.

GAPDH Activity and Immunogenicity of Staphylococcus aureus Recombinant GapC Protein

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-γ 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-γ 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 1×10 8CFU 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.

Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase

Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and GapC proteins share considerable homology to the GapB and GapC products of human strains of S. aureus. These two proteins could be distinguished by their different GAPDH activities and binding to bovine transferrin properties. Both gapB and gapC genes were conserved in 11 strains tested, and the GapC protein was present on the surface of all S. aureus strains.

A GapC chimera retains the properties of the Streptococcus uberis wild-type GapC protein

The GapC products of Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis share considerable homology at the DNA and amino acid levels. The high similarity at the protein level suggests that one GapC protein might be used as a single antigen to protect dairy cows against infections with the contagious S. agalactiae and the environmental S. dysgalactiae and S. uberis strains. Despite their similarities, immunization with the S. dysgalactiae GapC did not protect dairy cows from a challenge with S. uberis, suggesting the presence of regions in GapC that are involved in species-specific protection. To produce a single antigen that can be used to protect against all streptococcal mastitis infections, we constructed a GapC chimeric protein using the S. uberis GapC product as the backbone followed by non-conserved peptide regions from the S. agalactiae and S. dysgalactiae GapC proteins. We report that the chimeric GapC protein retains the enzymatic activity of the S. uberis GapC protein. In addition, we fused the chimera to the OmpF and LipoF transport sequences of Escherichia coli and the GapC chimeras were present in membrane fractions of E. coli. These extracts could be the basis of an antigen preparation for use in mastitis vaccines.

A novel system for efficient gene expression and monitoring of bacteria in aquatic environments.

In a previous study, we reported the identification of Escherichia coli genes with increased expression in an aquatic environment. Here, we describe the use of one of these genes, gapC, as an expression system in freshwater habitats. We have identified the transcriptional start site of gapC and analysed the synthesis of the GapC protein during incubation in aquatic medium. The promoter of gapC was used to construct fusions to the reporter genes lacZ and gfp. Analysis of these fusions indicates the potential of gapC as a valuable tool for the detection of E. coli in freshwater habitats, as well as for expressing other genes in aquatic environments.

Cloning, sequencing and functional overexpression of the Streptococcus equisimilis H46A gapC gene encoding a glyceraldehyde-3-phosphate dehydrogenase that also functions as a plasmin(ogen)-binding protein. Purification and biochemical characterization of the protein.

We previously identified DNA sequences involved in the function of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A, a human serogroup C strain known to express this gene at a high level. As a prerequisite to understanding possible mechanisms that control the balance between the plasminogen activating and plasmin(ogen) binding capacities of H46A, we describe here its gapC gene encoding glyceraldehyde-3-phosphate dehydrogenase (GraP-DH, EC 1.2.1.12), a glycolytic enzyme apparently transported to the cell surface where it functions as a plasmin(ogen).binding protein. The gapC gene was cloned and sequenced and found to code for a 336-amino-acid polypeptide (approximately 35.9 kDa) exhibiting 94.9% sequence identity to the Plr protein from Streptococcus pyogenes shown by others to be capable of plasmin binding [Lottenberg, R., Broder, C. C., Boyle, M. D., Kain, S. J., Schroeder, B. L. & Curtiss, R. III (1992) J. Bacteriol. 174, 5204-5210]. To study the properties of the GapC protein, its gene was inducibly overexpressed in Escherichia coli from QIAexpress expression plasmids to yield the authentic GapC or (His)6GapC carrying a hexahistidyl N-terminus to permit affinity purification. Both proteins were functionally active, exhibiting specific GraP-DH activities of about 80 kat/mol (approximately 130 U/mg) after purification. Their binding parameters [association (ka) and dissociation (kd) rate constants, and equlibrium dissociation constants (Kd = kd/ka)] for the interaction with human Gluplasminogen and plasmin were determined by real-time biospecific interaction analysis using the Pharmacia BIAcore instrument. For comparative purposes, the commercial GraP-DH from Bacillus stearothermophilus (BstGraP-DH), a nonpathogenic organism, was included in these experiments. The Kd values for binding of plasminogen to GapC, (His)6GapC and BstGraP-DH were 220 nM, 260 nM and 520 nM, respectively, as compared to 25 nM, 17 nM and 98 nM, respectively, for the binding to plasmin. These data show that both the zymogen and active enzyme possess low-affinity binding sites for the gapC gene product and that the hexahistidyl terminus does not affect its function. Prior limited treatment with plasmin enhanced the subsequent plasminogen binding capacity of all three GraP-DHs, presumably by the exposure of new C-terminal lysine residues for binding to the zymogen.