Abstract
Surface display of heterologous peptides and proteins such as receptors, antigens, and enzymes on live bacterial cells is of considerable value for various biotechnological and industrial applications. In this study, a series of novel cell surface display systems were examined by using Vibrio anguillarum outer membrane protein and outer membrane lipoprotein as anchoring motifs. These display systems consist of (i) the signal sequence and first 11 N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first 9 N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and (ii) transmembrane domains of V. anguillarum outer membrane proteins Omporf1, OmpU, or Omp26La. In order to assay the translocation efficiency of constructed display systems in bacteria, green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on the E. coli surface. For assaying its potential application in live bacteria carrier vaccines, an excellent display system Wza-Omporf1 was fused with the major capsid protein (MCP) of large yellow croaker iridovirus and introduced into attenuated V. anguillarum strain MVAV6203, and subsequent analysis of MCP surface localization proved that the novel display system Wza-Omporf1 could function as a strong tool in V. anguillarum carrier vaccine development.
The display of heterologous proteins or peptides on the surface of prokaryotic or eukaryotic cells, especially bacterial and yeast cells (8), enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications, including live vaccine development, antibody production, peptide library screening, the use of environmental bioadsorbents, and whole-cell biocatalysis (12). A variety of surface anchoring motifs have been used to establish display systems, including various outer membrane proteins, lipoproteins, autotransporters, subunits of surface appendages, and S-layer proteins (12, 16). The efficiency of surface display systems are highly related to the characteristics of the carrier protein, passenger protein, host cell, and fusion method (17).
Lpp-OmpA is an efficient surface display system developed by Georgiou et al. (9), which has been used successfully to anchor a variety of proteins, including some enzymes, onto the cell surface (10, 21). This display system allows C-terminal fusion of the passenger proteins and consists of two key anchoring motifs: (i) the signal sequence and the first nine amino acids of E. coli lipoprotein (Lpp), to target the proteins to the inner face of the outer membrane, and (ii) the transmembrane region (amino acids 46 to 159) of E. coli outer membrane protein A (OmpA), to conduct the proteins across the outer membrane. Since the Lpp-OmpA mode combines the anchoring capabilities of lipoprotein and outer membrane protein, it has outstanding advantages in high surface display efficiency and strong adaptability to passenger proteins varying in size (21).
V. anguillarum is an important bacterial fish pathogen, responsible for both marine and freshwater fish epizootics throughout the world (3). In our previous work, several attenuated V. anguillarum strains derived from wild-type V. anguillarum strain MVM425 had been constructed by recombinant DNA technology and proved to be excellent live vaccine candidates against Vibrio pathogens, including V. anguillarum and V. alginolyticus, in vaccination tests (unpublished results). For developing potential multivalent recombinant vaccines based on attenuated V. anguillarum live vaccine, efficient surface display systems are needed to display protective antigens onto the surface of live carrier. In this study, according to the model of Lpp-OmpA system, a series of novel display systems were investigated based on the outer membrane anchoring elements of V. anguillarum. Green fluorescence protein (GFP) was used by C-terminal fusion to investigate the expression and display pattern of recombinant protein via these surface display systems in E. coli. Based on the system of Wza-Omporf1, we also displayed the major capsid protein (MCP) of large yellow croaker iridovirus (LYCIV) on the surface of an attenuated V. anguillarum MVAV6203.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions.
E. coli Top10 [F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 araD139Δ(ara-leu)7697 galU galK rpsL(Strr)endA1 nupG] (Invitrogen, Carlsbad, CA) was used as the host for recombinant plasmid construction and GFP surface display. An attenuated V. anguillarum strain MVAV6203 was used as the host for MCP surface display. Plasmid mTn5gusA-pgfp12 carrying the gfpuv gene, a gift from Chuanwu Xi (23), provided the gene source of GFP. Plasmid pTX101, a gift from George Georgiou, was used as the gene source of Lpp-OmpA fusion (7). Plasmid pUC18 (TaKaRa, Shiga, Japan) was used as a parent plasmid for construction of display hybrids. Plasmids used in the present study are listed in Table 1.
TABLE 1.
Plasmids used in this study
| Plasmid | Relevant characteristicsa | Size (kb) | Source or reference |
|---|---|---|---|
| pUC18 | Apr; lac promoter expression vector | 2.7 | TaKaRa |
| pUC18ΔE | Apr; pUC18 derivative; silent point mutation EcoRI site | 2.7 | This study |
| pTX101 | Apr Cmr; lpp-ompA-bla trifusion | 12 | 7 |
| pTXG | Cmr; pTX101 derivative; lpp-ompA-gfp trifusion | 12.7 | This study |
| pG | Apr, pUC18 derivative; plac-gfp fusion | 3.4 | This study |
| pL-O-G | Apr, pUC18ΔE derivative; plac-lpp-ompA-gfp fusion | 3.9 | This study |
| pL-orf1-G | Apr, pUC18ΔE derivative; plac-lpp-omporf1-gfp fusion | 3.7 | This study |
| pW-orf1-G | Apr, pUC18ΔE derivative; plac-wza-omporf1-gfp fusion | 3.7 | This study |
| pL-U-G | Apr, pUC18ΔE derivative; plac-lpp-ompU-gfp fusion | 3.7 | This study |
| pW-U-G | Apr, pUC18ΔE derivative; plac-wza-ompU-gfp fusion | 3.7 | This study |
| pL-26La-G | Apr, pUC18ΔE derivative; plac-lpp-omp26La-gfp fusion | 3.8 | This study |
| pW-26La-G | Apr, pUC18ΔE derivative; plac-wza-omp26La-gfp fusion | 3.8 | This study |
| pW-orf1-M | Apr, pUC18ΔE derivative; plac-wza-omporf1-mcp fusion | 4.3 | This study |
| pM | Apr, pUC18ΔE derivative; plac-mcp fusion | 4 | This study |
Cmr, chloramphenicol resistance; Apr, ampicillin resistance.
The E. coli Top10 strains harboring plasmids were grown in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) supplemented with ampicillin to a final concentration of 100 μg/ml. Cells were grown in 250-ml flask with a 50-ml working volume in a shaker at 200 rpm and 37°C. For expression, freshly inoculated culture was grown at 37°C to an optical density (OD600) of 0.6 and was induced by adding 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) for further 12 h culture at 25, 30, or 37°C.
The V. anguillarum attenuated strains harboring plasmids were grown in LBS medium (1% tryptone, 0.5% yeast extract, 2% NaCl) supplemented with ampicillin to a final concentration of 200 μg/ml. Cells were grown in 250-ml flask with a 50-ml working volume in a shaker at 200 rpm and 30°C. For expression, overnight cultures were subcultured at 1:100 and grown at 30°C for 24 h.
Plasmid construction.
The 720-bp gfpuv1 fragment was amplified from plasmid mTn5gusA-pgfp12 by PCR and inserted into the EcoRI/BamHI-digested plasmid pTX101 to generate pTXG. Then, with pTXG as a template, a 1.2-kb SacI/BamHI lpp-ompA-gfp trifusion fragment was amplified by PCR and cloned into the SacI/BamHI-digested vector pUC18 to get the GFP surface display plasmid pL-O-G as a positive control.
By silent point mutation, the EcoRI restriction site of pUC18 was eliminated to get pUC18ΔE. Based on E. coli lipoprotein Lpp (L), V. anguillarum lipoprotein Wza (W), and three V. anguillarum outer membrane proteins Omporf1 (orf1), OmpU (U), and Omp26La (26La), a series of GFP (G) display plasmids pL-orf1-G, pW-orf1-G, pL-U-G, pW-U-G, pL-26La-G, and pW-26La-G were constructed with pUC18ΔE as the parent plasmid. Moreover, the GFP cytosolic expression plasmid was also constructed by fusing the GFP gene into the multiple cloning site of pUC18 with EcoRI and BamHI, resulting in pG as a negative control. The gene encoding the major capsid protein (MCP [M]) was cloned from the chromosome of LYCIV, and the MCP display plasmid pW-orf1-M and the MCP cytosolic expression plasmid pM were constructed based on pW-orf1-G and pUC18, respectively (for more details, see the supplemental material).
Cell fractionation.
Bacterial cell fractionation was performed according to a method described elsewhere with minor modifications (11). Each E. coli display strain was cultured at 37°C, and when the OD600 reached 0.6, IPTG was added to a final concentration of 0.5 mM and for further 12 h of culture, while each V. anguillarum display strain was cultured at 30°C for 24 h without IPTG induction. All of the harvested cells were centrifuged at 10,000 × g for 2 min, washed with phosphate-buffered saline (PBS) three times, and resuspended in 1.5 ml of Tris-HCl-NaCl buffer (50 mM [pH 8.0], containing 0.3% NaCl). The cell suspension was then treated with an ultrasound sonication for 5 min on ice. To remove unbroken cells and debris, the whole-cell lysate was centrifuged at 10,000 × g for 5 min. The supernatant was pelleted by centrifugation at 20,000 × g and 4°C for 1 h to obtain total membrane fraction. The supernatant was regarded as the soluble cytoplasmic/periplasmic fraction. For further outer membrane fractionation, the pellet (total membrane fraction) was resuspended with 0.4 ml of HEPES buffer (10 mM [pH 7.4], containing 1% sodium lauroyl sarcosine) for solubilizing inner membrane and incubated at room temperature for 30 min, and then the outer membrane fraction was repelleted by ultracentrifugation at 20,000 × g and 4°C for 1 h. The supernatant was regarded as the inner membrane fraction, and after a repeat for outer membrane fractionation, the pellet was regarded as the outer membrane fraction.
The harvested cytoplasmic/periplasmic fraction and outer membrane fraction were all diluted to the same OD value (OD280 = 1.0) to ensure an identical total protein level in either cytoplasmic/periplasmic fraction or outer membrane fraction, and equal volumes of each fractionated sample were stored for further display analysis.
Enzyme-linked immunosorbent assay (ELISA).
Microtiter plate wells were coated with 50 μl of each fraction (cytoplasmic/periplasmic and outer membrane) by overnight incubation at 4°C. Excess protein was discarded, and wells were blocked with 200 μl of PBS containing 3% bovine serum albumin for 1 h at 37°C. After removing the blocking solution and washing three times, the wells were incubated for 1.5 h at 37°C with rabbit anti-GFP antibody (Proteintech Group) at a dilution of 1:3,000 (vol/vol) or with rabbit anti-MCP antibody (YingJi Technology, Shanghai, China) at a dilution of 1:100,000 (vol/vol). After three washes, the cell-antibody complex was incubated for 1.5 h at 37°C with horseradish peroxidase-conjugated goat anti-rabbit antibody (Jackson Immunoresearch Laboratories) at a dilution of 1:5,000 (vol/vol). Finally, the wells were washed three times with PBS-T (PBS buffer [pH 7.2] containing 0.05% Tween 20), and TMB solution (TIANGEN Biotech, Beijing, China) was added as a color-developing substrate. After addition of 1 M H2SO4 for termination of the reaction, the absorbance at 450 nm was recorded by using a Bio-Rad model 550 microplate reader (Bio-Rad, Hercules, CA).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.
Equal volume of each fraction (cytoplasmic/periplasmic and outer membrane) of the cells containing GFP or MCP was mixed with sample loading buffer, boiled for 5 min, and resolved by 12% (wt/vol) discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For Western blot analysis, gels were electroblotted onto polyvinylidene-fluoride (PVDF) membranes by using a Mini Protean 3 Cell (Bio-Rad) at 100 V for 3 h. For GFP analysis, the monoclonal mouse anti-GFP antibody (1:200 [vol/vol]; NeoMarkers For Lab Vision) and goat anti-mouse immunoglobulin G-horseradish peroxidase (1:2,500 [vol/vol]) conjugate (Jackson Immunoresearch Laboratories) were used. For MCP analysis, rabbit anti-MCP antibody (1:100,000 [vol/vol]; YingJi Technology) and horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000 [vol/vol]; Jackson Immunoresearch Laboratories) were used.
Protease accessibility.
E. coli cells were induced with IPTG and cultured at 37°C for additional 8 h, while each V. anguillarum display strain was cultured at 30°C for 12 h without IPTG induction. For protease accessibility tests, collected cells were suspended in a buffer (pH 7.8) with 1% (wt/vol) NaCl and 15 mM Tris-HCl to OD600 of 10. Cell suspensions were incubated with 0.5 mg of proteinase K/ml at 30°C for 0.5 h, and the reaction was stopped by adding phenylmethylsulfonyl fluoride to a final concentration of 1 mM. Finally, the reaction mixture was filtered through a 0.22-μm-pore-size membrane and subjected to Western blot analysis.
RESULTS
Design and construction of surface display system.
To develop the novel systems for displaying heterologous proteins on bacterial surface, with reference to the successful mode of Lpp-OmpA, two lipoproteins of E. coli Lpp and V. anguillarum Wza and three V. anguillarum outer membrane proteins of Omporf1, OmpU, and Omp26La were selected for the design and construction of display systems. Wza is a conserved surface-located outer membrane lipoprotein in bacteria (4, 6); Omporf1 is a putative outer membrane protein, required for biofilm formation on scales in V. anguillarum (4); OmpU is a major outer membrane porin, involved in environmental adaptation in V. anguillarum (22); and Omp26La is an outer membrane protein related to drug resistance in V. anguillarum (18).
The secondary structures, transmembrane helices, and anchoring elements of the above membrane-related proteins were analyzed with ExPASy Proteomics tools (TMHMM Server v.2.0 [http://kr.expasy.org/tools/]) and SoftBerry (data not shown), respectively, and the regions, highly related to the outer membrane anchoring, were separately cloned and combined into six potential display systems, consisting of (i) the signal sequence and first 11 N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza or the signal sequence and first nine N-terminal amino acids of Lpp to mediate proper localization, and (ii) residues 6 to 50 of Omporf1, residues 180 to 238 of OmpU, or residues 80 to 158 of Omp26La to guide the transmembrane location; and (iii) the entire mature GFP or MCP (Fig. 1). In our constructs, these fusion proteins were expressed from IPTG-inducible lac promoter of pUC18, and the molecular sizes of Lpp-OmpA-GFP, Lpp-Omporf1-GFP, Lpp-OmpU-GFP, Lpp-Omp26La-GFP, Wza-Omporf1-GFP, Wza-OmpU-GFP, Wza-Omp26La-GFP, and Wza-Omporf1-MCP are estimated to be 43.7, 35.8, 37.8, 40.3, 36, 38.1, 40.6, and 58.9 kDa, respectively, and the predicted conformations of all of the fusions, indicated by bioinformatics analysis, was illustrated in Fig. 2.
FIG. 1.
Gene maps of recombinant plasmids harboring anchoring motif-GFP/MCP fusion constructs. Plasmid pUC18 was used as parent vector for constructing these fusions. plac, lac promoter; lpp and wza, outer membrane-anchoring sequences of E. coli outer membrane lipoprotein Lpp and V. anguillarum outer membrane lipoprotein Wza; ompA, omporf1, ompU, and omp26La, the encoding gene of transmembrane regions of E. coli outer membrane protein OmpA and V. anguillarum outer membrane proteins Omporf1, OmpU, and Omp26La; gfp, the encoding gene of UV-optimized GFP; mcp, the encoding gene of major capsid protein from LYCIV.
FIG. 2.
Scheme of the expected structures of the Lpp/Wza-Omporf1-GFP, Wza-Omporf1-MCP (A), and Lpp/Wza-OmpU/Omp26La-GFP (B) in the outer membrane. Gray rectangles represent membrane-spanning β-strands of Omporf1, OmpU, or Omp26La based on the predicated structures. aa, amino acid(s).
Expression and surface localization of GFP fusion protein.
To investigate the feasibility of Lpp/Wza-Omporf1/OmpU/Omp26La as anchoring motifs for targeting heterologous proteins, it is essential to verify the location and activity of those fusion proteins. All of the GFP expression plasmids were transformed into E. coli top10 to result in Top10/pL-O-G, Top10/pG, Top10/pL-orf1-G, Top10/pL-U-G, Top10/pL-26La-G, Top10/pW-orf1-G, Top10/pW-U-G, and Top10/pW-26La-G for further studies.
Harvested cells were fractionated by ultracentrifugation, and the GFP in various fractions (cytoplasmic/periplasmic and outer membrane) was assayed by ELISA (Fig. 3). Since all of the samples were diluted to the same OD280 value (OD280 = 1), the value (OD450) of ELISA represented the relative amount of GFP fusions per unit OD of total protein in either cytoplasmic/periplasmic fraction or outer membrane fraction. In cytosolic expressing strain Top10/pG, GFP was localized predominantly in the cytoplasm fraction, with small amounts detected in the outer membrane fraction. Considering minor contamination of subcellular fractions is absolutely inevitable in the fractionation process, the small amounts of GFP detected in the outer membrane fraction of Top10/pG probably represent the background contamination of the cytoplasm fraction. In contrast, there was a pronounced shift to the outer membrane fraction in all of the surface display strains except strains Top10/pL-orf1-G and Top10/pW-26La-G in which little or no GFP could be detected in cytoplasmic/periplasmic and outer membrane fractions.
FIG. 3.
ELISA analysis for the outer membrane fraction (□) and the cytoplasmic/periplasmic fraction (▪) of different E. coli display strains. Cells were grown in LB medium at 37°C and harvested after 12 h induction. CP, cytoplasmic/periplasmic fraction; OM, outer membrane fraction.
The presentation of fusion proteins on the cell surface was additionally demonstrated by Western blot analysis. GFP signal was detected only in cytoplasmic/periplasmic fraction of negative control strain Top10/pG (Fig. 4A, lanes 1 and 2). The bands approximately corresponding to 38.1 kDa (W-U-G), 36 kDa (W-orf1-G), 40.3 kDa (L-26La-G), 37.8 kDa (L-U-G), and 43.7 kDa (L-O-G) were detected in cytoplasmic/periplasmic fraction and outer membrane fraction (Fig. 4A, lanes 3 to 12). This was assumed that part of the GFP fusions were successfully displayed on the cell surface. Compared to the Lpp-fusion proteins, the Wza-fusion protein W-U-G showed some degraded GFP fusion bands only in outer membrane fraction (Fig. 4A, lane 4), probably due to the instability of GFP fusions on surface. Interestingly, the Wza-fusion proteins of W-U-G and W-orf1-G also showed a larger GFP fusion band only in outer membrane fractions (Fig. 4A, lanes 4 and 6), and the reason for this remains unknown.
FIG. 4.
Western blot analysis for the cytoplasmic/periplasmic fraction and outer membrane fraction of Top10/pG (lanes 1 and 2), Top10/pW-U-G (lanes 3 and 4), Top10/pW-orf1-G (lanes 5 and 6), Top10/pL-26La-G (lanes 7 and 8), Top10/pL-U-G (lanes 9 and 10), and Top10/pL-O-G (lanes 11 and 12) (A) and protease accessibility analysis of the fusion proteins Top10/pW-orf1-G (lane 1), Top10/pL-U-G (lane 2), and Top10/pG (lane 3) (B). CP, cytoplasmic/periplasmic fraction; OM, outer membrane fraction. Lanes M, protein molecular weight marker (MBI Fermentas).
Since proteinase K could not readily diffuse across the cell membrane, it was applied in a protease accessibility assay to further ascertain the surface localization of GFP. Two better display systems, pL-U-G and pW-orf1-G, were selected, and their cells were subjected to protease accessibility tests as described in Materials and Methods. As shown in Fig. 4B, after the cell suspensions were incubated with proteinase K for 0.5 h, a 27-kDa band, corresponding to GFP, was detected in the supernatants of Top10/pL-U-G and Top10/pW-orf1-G but not in that of the GFP cytosolic expression strain Top10/pG. The result of protease accessibility strongly proved that the surface display systems could successfully display GFP on the cell surface.
Effect of induction temperature on cell growth and GFP surface display.
Temperature is a key factor in cell growth and protein expression. In order to investigate the effect of induction temperature on the surface location of GFP, two better display systems, pL-U-G and pW-orf1-G, were selected in the present study. The culture of E. coli display strain was divided into three groups in parallel, and the induction temperature was set at 25, 30, and 37°C, respectively. After 12 h of incubation, cells were fractionated and GFP in various fractions was analyzed by ELISA. Simultaneously, the growth kinetics of cells carrying pL-U-G, pW-orf1-G, and pG were compared at three temperatures. As shown in Fig. 5, the ELISA values (OD450), which represent the relative amount of displayed GFP fusions per OD unit of total outer membrane protein, obviously increased from 0.05 at 25°C to 0.5 at 37°C in Top10/pL-U-G and from 0.27 at 25°C to 0.55 at 37°C in Top10/pW-orf1-G, suggesting that a higher induction temperature facilitated the display of GFP, either by elevating the translocation efficiency or by increasing the GFP expression rate. Meanwhile, the initial cell growth (before 5 h of culture or 3 h of induction) remained almost the same for different strains at three induction temperatures, and the subsequent cell growth revealed obvious growth inhibition in the display strains (Top10/pL-U-G and Top10/pW-orf1-G) at 37°C (Fig. 5A). However, with the decreased induction temperature and the deduced GFP display, the cell growth of Top10/pW-orf1-G was partly recovered at 30°C (Fig. 5B) and completely recovered at 25°C (Fig. 5C), while the cell growth of Top10/pL-U-G remained to be inhibited at three induction temperatures (Fig. 5), probably because the deduced GFP display at low induction temperature was still too high to release its inhibition to growth.
FIG. 5.
Effect of induction temperature on outer membrane display and cell growth of strains Top10/pL-U-G, Top10/pW-orf1-G, and Top10/pG. OM, outer membrane fraction. Cells were cultured in LB medium and induced at 37°C (A), 30°C (B), or 25°C (C) for 12 h and followed by fractionation.
Time course of GFP surface display.
The better display systems pL-U-G and pW-orf1-G were selected, and their time courses of GFP surface localization during cell growth were investigated. According to the growth profiles at 37°C, cultures of Top10/pL-U-G and Top10/pW-orf1-G were sampled at 1, 3, 9, 13, and 21 h of induction, and the samples were fractioned for ELISA analysis. As shown in Fig. 6, the GFP signal appeared in the cytoplasmic/periplasmic fractions of Top10/pL-U-G and Top10/pW-orf1-G after 1 h of induction and reached the peak at 13 h, while in the outer membrane fraction, the GFP signal was not detected until 3 h induction and then kept increasing until the end of the experiment.
FIG. 6.
Time course of GFP surface display in Top10/pL-U-G (A) and Top10/pW-orf1-G (B) analyzed by ELISA. Cells were cultured in LB medium and induced at 37°C, and different samples of cytoplasmic/periplasmic fraction (▪) and outer membrane fraction (□) were harvested at different time. CP, cytoplasmic/periplasmic fraction; OM, outer membrane fraction.
The fractions of Top10/pL-U-G and Top10/pW-orf1-G at 1, 3, 9, 13, and 21 h of induction were subjected to Western blot for further analysis (Fig. 7). Coinciding with the results in Fig. 6, the GFP fusion band appeared in the cytoplasmic/periplasmic fraction after 1 h of induction and appeared in the outer membrane fraction after 3 h of induction, and the translocation was obviously lagged behind the protein expression. Consistently, a larger GFP fusion band was also observed in the outer membrane fraction of Top10/pW-orf1-G as mentioned above (Fig. 4, lane 6). Interestingly, as shown in Fig. 7B (lanes 4, 6, 8, 10, and 12), the Wza-fusion protein W-orf1-G in the outer membrane fraction was gradually converted from the 36-kDa GFP fusion to a larger GFP fusion when the induction time was prolonged.
FIG. 7.
Time course of GFP surface display in Top10/pL-U-G (A) and Top10/pW-orf1-G (B) analyzed by Western blotting. Cells were cultured in LB medium and induced at 37°C, and different fraction samples were harvested after 21 h. OM, outer membrane fraction; CP, cytoplasmic/periplasmic fraction. Lanes M, protein molecular weight marker (MBI Fermentas).
Expression and surface localization of Wza-Omporf1-MCP fusion protein.
For developing potential multivalent V. anguillarum live carrier vaccine, the feasibility of these novel surface display systems as anchoring motifs for targeting protective antigenic proteins on the surface of an attenuated V. anguillarum strain was preliminarily investigated.
Iridoviruses were the causative agents of serious systemic diseases among fishes (15), and their MCP was reported to be an important protective antigen (1). In the present study, the gene of MCP from LYCIV (2) was fused with the display system pW-orf1 to result in W-orf1-M. The MCP display plasmid pW-orf1-M and the MCP cytosolic expression plasmid pM were introduced into a recombinant V. anguillarum strain MVAV6203 which was attenuated by 10,000 times and resulted in AV/pW-orf1-M and AV/pM.
After 24 h of culture, the cells were harvested, and subcellular fractionation was performed to separate equal volumes of the outer membrane and cytoplasmic/periplasmic fractions for Western blot assay. The cytosolic MCP band (50 kDa) expressed by negative control strain AV/pM was found only in the cytoplasmic/periplasmic fraction and not in the outer membrane fraction at all (Fig. 8A, lanes 1 and 2). Moreover, the fusion protein of W-orf1-M (58.9 kDa) could be found in both the cytoplasmic/periplasmic fraction and the outer membrane fraction (Fig. 8A, lanes 3 and 4). Although MCP was successfully displayed on MVAV6203 cell surface, the fusion proteins showed signs of degradation (Fig. 8A, lane 3) only in the outer membrane fraction, and this indicated the instability of MCP fusion on the surface. An additional protease accessibility test was performed to ascertain the surface localization of MCP, as shown in Fig. 8B, and a 50-kDa band, corresponding to MCP, was detected in the supernatant of AV/pW-orf1-M but not in that of the MCP cytosolic expression strain Top10/pM, which demonstrated that the system pW-orf1-M could successfully display MCP on the cell surface of V. anguillarum.
FIG. 8.
Surface display of MCP by pW-orf1-M in attenuated V. anguillarum MVAV6203. (A) Cells were cultured in LBS medium at 30°C, and different fraction samples were harvested after 24 h for Western blot analysis. Lanes 1 and 2, OM and CP fractions of negative control AV/pM; lanes 3 and 4, OM and CP fractions of AV/pW-orf1-M. (B) Protease accessibility analysis of fusion proteins. Lane 1, AV/pM; lane 2, AV/pW-orf1-M; lane M, protein molecular weight marker (MBI Fermentas).
DISCUSSION
Several systems have been established for expressing proteins or peptides on the surface of bacteria, although many are limited in the translocation capability and the size of passenger protein. Lpp-OmpA is an efficient hybrid display system developed by Georgiou et al. (9). The system combines the outer membrane-targeting function of E. coli lipoprotein (Lpp) and the transmembrane function of E. coli OmpA and succeeded in anchoring a variety of proteins, including some enzymes, onto the cell surface (10, 21). In the present study, according to the successful mode of Lpp-OmpA, two lipoproteins (E. coli Lpp and V. anguillarum Wza) and three V. anguillarum outer membrane proteins (Omporf1, OmpU, and Omp26La) were selected, and their outer membrane targeting regions, indicated by bioinformatics analysis, were separately cloned and combined into six hybrid display systems Lpp/Wza-Omporf1, Lpp/Wza-OmpU, and Lpp/Wza-Omp26La to mediate proper localization of passenger protein via C-terminal fusion strategy.
By fusion with GFP, the display systems of Wza-Omporf1, Lpp/Wza-OmpU and Lpp-Omp26La could successfully target GFP onto the outer membrane of E. coli under the control of lac promoter and exceed the Lpp-OmpA system in translocation capabilities (Fig. 3), whereas the systems Lpp-Omporf1 and Wza-Omp26La showed a very low display level, probably caused by the low expression level (Fig. 3). Moreover, the Lpp-fused GFP proteins were detected as single bands in all of the fractions (Fig. 4A), and the Wza-fused GFP proteins showed some extra bands in the outer membrane fractions but not in the cytoplasmic/periplasmic fractions (Fig. 4A), suggesting some structural instability of the Wza fusions displayed onto the cell surface of E. coli. In addition, the protease accessibility experiments (Fig. 4B) proved that GFP was indeed on the cell surface of E. coli.
According to the estimated amount ratio of displayed protein to total expressed protein, the translocation efficiencies of the systems were as low as ca. 0.1 to 0.3%. However, this low translocation efficiency may root in the space hindrance on the outer membrane. As shown in Fig. 4A, when the cytoplasmic/periplasmic and outer membrane fractions were equally diluted to same level (OD280 = 1), the GFP fusions in both fractions were presented at a similar level. This means that the displayed GFP fusions accounted for a percentage of total outer membrane fraction similar to that of the cytoplasmic/periplasmic GFP fusions in the total cytoplasmic/periplasmic fraction. Therefore, the translocation capability of the systems may not really be as poor as indicated by the translocation efficiency, since it is easy to understand that a limited cell surface cannot be anchored by so many heterologous proteins because of the saturated position.
Similar to some reported anchoring motifs for GFP display onto the cell surface of E. coli (5, 7), the display of GFP by Wza-Omporf1 and Lpp-OmpU also resulted in a negative influence on cell growth. As shown in Fig. 5A, compared to the control strain Top10/pG the display strains Top10/pL-U-G and Top10/pW-orf1-G showed a significant growth inhibition after 3 h of induction at 37°C. However, with a low induction temperature, the display level of GFP was deduced, and the cell growth of Top10/pL-U-G and Top10/pW-orf1-G was accordingly recovered to some extent (Fig. 5B and C). A high induction temperature facilitated the display of GFP, but a high display level inhibited cell growth. It was assumed that the display of heterologous protein on cell surface might lead to destabilization of the membrane integrity and therefore affected cell growth (20). This membrane destabilization was reported to be a general consequence of heterologous proteins display (8, 20, 21). In our view, it is better for the bacterial cells to express and display heterologous protein moderately so that the cells could retain high viability with a stable membrane.
Based on the system of Wza-Omporf1, we also successfully displayed the MCP of LYCIV, a 50-kDa antigenic protein, on the surface of the attenuated V. anguillarum MVAV6203 (Fig. 8). In contrast to the E. coli Top10 strain with lac promoter strictly induced by IPTG, the expression of heterologous proteins in V. anguillarum strains was independent of IPTG induction (19). The IPTG-independent expression and display of heterologous proteins by Wza-Omporf1 in V. anguillarum will be useful for simplifying the cell culture process and important in the application of carrier vaccine. In addition, the possibility of using various bacterial species as a host for heterologous passenger proteins means that the surface display systems constructed in this study have enormous biotechnological application potential in various fields, including whole-cell biocatalysis, peptide library screening, and the use of environmental bioadsorbents (13, 14, 24-26).
Supplementary Material
Acknowledgments
We thank Chuanwu Xi (F. A. Janssens Laboratory of Genetics, Belgium) for the gift of plasmid mTn5gusA-pgfp12 and George Georgiou (University of Texas) for the gift of plasmid pTX101.
This study was supported by the National Natural Science Foundation of China (no. 30600460), the National High Technology Research and Development Program of China (2006AA100310), and the Major State Basic Research Development Program (2006CB101800).
Footnotes
Published ahead of print on 16 May 2008.
Supplemental material for this article may be found at http://aem.asm.org/.
REFERENCES
- 1.Caipang, C. M., T. Takano, I. Hirono, and T. Aoki. 2006. Genetic vaccines protect red seabream, Pagrus major, upon challenge with red seabream iridovirus (RSIV). Fish Shellfish Immunol. 21:130-138. [DOI] [PubMed] [Google Scholar]
- 2.Chen, X. H., K. B. Lin, and X. W. Wang. 2003. Outbreaks of an iridovirus disease in maricultured large yellow croaker, Larimichthys crocea (Richardson), in China. J. Fish Dis. 26:615-619. [DOI] [PubMed] [Google Scholar]
- 3.Crosa, J. H. 1980. A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron-sequestering system. Nature 284:566-568. [DOI] [PubMed] [Google Scholar]
- 4.Croxatto, A., J. Lauritz, C. Chen, and D. L. Milton. 2007. Vibrio anguillarum colonization of rainbow trout integument requires a DNA locus involved in exopolysaccharide transport and biosynthesis. Environ. Microbiol. 9:370-382. [DOI] [PubMed] [Google Scholar]
- 5.Daugherty, P. S., M. J. Olsen, B. L. Iverson, and G. Georgiou. 1999. Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng. 12:613-621. [DOI] [PubMed] [Google Scholar]
- 6.Drummelsmith, J., and C. Whitfield. 2000. Translocation of group 1 capsular polysaccharide to the surface of Escherichia coli requires a multimeric complex in the outer membrane. EMOB J. 19:57-66. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Francisco, J. A., C. F. Earhart, and G. Georgiou. 1992. Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc. Natl. Acad. Sci. USA 89:2713-2717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Georgiou, G., C. Stathopoulos, P. S. Daugherty, A. R. Nayak, B. L. Iverson, and R. Curtiss. 1997. Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat. Biotechnol. 15:29-34. [DOI] [PubMed] [Google Scholar]
- 9.Georgiou, G., D. L. Stephens, C. Stathopoulos, H. L. Poetschke, J. Mendenhall, and C. F. Earhart. 1996. Display of β-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp′-OmpA′-β-lactamase fusions. Protein Eng. 9:239-247. [DOI] [PubMed] [Google Scholar]
- 10.Isoda, R., S. P. Simanski, L. Pathangey, A. E. Stone, and T. A. Brown. 2007. Expression of a Porphyromonas gingivalis hemagglutinin on the surface of a Salmonella vaccine vector. Vaccine 25:117-126. [DOI] [PubMed] [Google Scholar]
- 11.Lattemann, C. T., J. Maurer, E. Gerland, and T. F. Meyer. 2000. Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter. J. Bacteriol. 182:3726-3733. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Lee, S. Y., J. H. Choi, and Z. Xu. 2003. Microbial cell-surface display. Trends Biotechnol. 21:45-52. [DOI] [PubMed] [Google Scholar]
- 13.Martineau, P., A. Charbit, C. Leclerc, C. Werts, D. O'Callaghan, and M. Hofnung. 1991. A genetic system to elicit and monitor antipeptide antibodies without peptide synthesis. Bio/Technology 9:170-172. [DOI] [PubMed] [Google Scholar]
- 14.Nakajima, H., N. Shimbara, Y. Shimonishi, T. Mimori, S. Niwa, and H. Saya. 2000. Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides. Gene 260:121-131. [DOI] [PubMed] [Google Scholar]
- 15.Qin, Q. W., C. Shi, K. Y. Gin, and T. J. Lam. 2002. Antigenic characterization of a marine fish iridovirus from grouper, Epinephelus spp. J. Virol. Methods 106:89-96. [DOI] [PubMed] [Google Scholar]
- 16.Samuelson, P., E. Gunneriusson, P. A. Nygren, and S. Stahl. 2002. Display of proteins on bacteria. J. Biotechnol. 96:129-154. [DOI] [PubMed] [Google Scholar]
- 17.Sandkvist, M., and M. Bagdasarian. 1996. Secretion of recombinant proteins by gram-negative bacteria. Curr. Opin. Biotechnol. 7:505-511. [DOI] [PubMed] [Google Scholar]
- 18.Satoru, S., K. Yasue, and R. Kusuda. 1998. A porin like outer membrane protein (Omp26La) appears to increase in an oxytetracycline resistant strain of marine fish pathogen Vibrio (Listonella) anguillarum. Microb. Environ. 13:197-202. [Google Scholar]
- 19.Shao, M., Y. Ma, Q. Liu, and Y. Zhang. 2005. Secretory expression of recombinant proteins in an attenuated Vibrio anguillarum strain for potential use in vaccines. J. Fish Dis. 28:723-728. [DOI] [PubMed] [Google Scholar]
- 20.Shi, H., and W. W. Su. 2001. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme Microb. Technol 28:25-34. [DOI] [PubMed] [Google Scholar]
- 21.Wan, H. M., B. Y. Chang, and S. C. Lin. 2002. Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli. Biotechnol. Bioeng. 79:457-464. [DOI] [PubMed] [Google Scholar]
- 22.Wang, S. Y., J. Lauritz, J. Jass, and D. L. Milton. 2003. Role for the major outer-membrane protein from Vibrio anguillarum in bile resistance and biofilm formation. Microbiology 149:1061-1071. [DOI] [PubMed] [Google Scholar]
- 23.Xi, C., M. Lambrecht, J. Vanderleyden, and J. Michiels. 1999. Bi-functional gfp- and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies. J. Microbiol. Methods 35:85-92. [DOI] [PubMed] [Google Scholar]
- 24.Xu, Z., and S. Y. Lee. 1999. Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Appl. Environ. Microbiol. 65:5142-5147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Yang, C., N. Cai, M. Dong, H. Jiang, J. Li, C. Qiao, A. Mulchandani, and W. Chen. 2008. Surface display of MPH on Pseudomonas putida JS444 using ice nucleation protein and its application in detoxification of organophosphates. Biotechnol. Bioeng. 99:30-37. [DOI] [PubMed] [Google Scholar]
- 26.Zhu, K. L., Z. M. Chi, J. Li, F. L. Zhang, M. J. Li, H. N. Yasoda, and L. F. Wu. 2006. The surface display of haemolysin from Vibrio harveyi on yeast cells and their potential applications as live vaccine in marine fish. Vaccine 24:6046-6052. [DOI] [PubMed] [Google Scholar]
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