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Immunoproteomics of extracellular proteins of the Aeromonas hydrophila China vaccine strain J-1 reveal a highly immunoreactive outer membrane protein

Xiao-dan Ni, Na Wang, Yong-jie Liu, Cheng-ping Lu
DOI: http://dx.doi.org/10.1111/j.1574-695X.2009.00646.x 363-373 First published online: 1 April 2010


Aeromonas hydrophila is a gram-negative bacterium that can infect a variety of aquatic and terrestrial animals. It is essential to develop a vaccine to reduce the economic losses caused by this bacterium in aquaculture worldwide. Here, an immunoproteomic assay was used to identify the immunogenic extracellular proteins of the Chinese vaccine strain J-1. Ten unique immunogenic proteins were identified from the two-dimensional electrophoresis immunoblot profiles by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS. One protein of interest, Omp38, was detected by antisera on two-dimensional immunoblots, suggesting that it might be located both extracellularly and in the membrane. In exploring the potential of Omp38 as a vaccine candidate in fish, we found the omp38 gene to be prevalent by PCR among different (36/48) A. hydrophila isolates. The recombinant Omp38 induced a strong antibody response in rabbits, and the polyclonal antibody could recognize a band of approximately 38 kDa in the immunoblots of outer membrane protein extracts from most (24/40) of the A. hydrophila strains, including different predominant serotypes in China. These results indicated that the outer membrane antigen identified in this study could be developed as a vaccine candidate to induce protective immunity against A. hydrophila infection.

  • Aeromonas hydrophila
  • immunoproteomics
  • extracellular proteins
  • outer membrane protein
  • immunogenicity


Aeromonas hydrophila is a gram-negative bacterium that can infect a variety of aquatic and terrestrial animals, including humans. It causes hemorrhagic septicemia in fish and, as a zoonotic agent, soft tissue wound infections and diarrhea in humans (Holmberg et al., 1986; Janda & Duffey, 1988; Paniagua et al., 1990; Bi et al., 2007). The development of a vaccine is essential to reduce the economic losses caused by this bacterium in the aquaculture industry. Several studies have focused on the development of a vaccine against hemorrhagic septicemia caused by A. hydrophila, and vaccine preparations of A. hydrophila using whole cells and cell components as immunogens have been tested (Lamers et al., 1985; Loghothetis & Austin, 1994; Hernanz Moral et al., 1998; Wong et al., 1998; Chu & Lu, 2001; Chandran et al., 2002; Priebe et al., 2002; Liu & Bi, 2007). However, an effective A. hydrophila vaccine has not been developed. Killed whole-cell vaccines were reported to confer some protection, but they could not protect against challenge with heterologous strains (Karunasagar et al., 1997; Fang et al., 2004). A major problem for the development of vaccines is the antigenic diversity of A. hydrophila strains (Stevenson, 1988). This problem may be overcome using a broad protective vaccine containing common protective antigens of A. hydrophila. Recently, some approaches for broad protective vaccine development in other bacterial species have been initiated. Maione (2005) developed multiserotype vaccines against Group B Streptococcus, which is a multiserotype bacterial pathogen that causes life-threatening infections in newborns. Li (2009) identified polyvalent vaccine candidates from outer membrane proteins (OMPs) of Vibrio alginolyticus and used the expressed recombinant proteins to immunize carps. The resulting broad cross-protection has abilities to fight against infections not only caused by V. alginolyticus but also by A. hydrophila and Pseudomonas fluorescens.

Considerable work has been carried out on protein-based vaccines for A. hydrophila (Hirst & Ellis, 1994; Bricknell et al., 1999; Fang et al., 2000; Rahman & Kawai, 2000; Khushiramani et al., 2007a,; b; Maiti et al., 2009), but their protection against challenge with heterologous strains has not been evaluated sufficiently. Thus, other immunogenic epitopes should be identified in A. hydrophila for inclusion in potential vaccine candidates. The pathogenesis of A. hydrophila has been reported to involve a variety of virulence factors, including extracellular products (ECPs) (Allan & Stevenson, 1981), S-layer (Murray et al., 1988) and adhesins (Fang et al., 2000). ECPs play a vital role in the wide distribution and adaptability by A. hydrophila to environmental changes (Yu et al., 2007).

Immunoproteomics, a technique involving two-dimensional electrophoresis (2-DE), followed by immunoblotting, which can be used to identify specific and nonspecific antigens, has been widely used to identify pathogenic antigenic molecules for the development of new vaccines (Chen et al., 2004). In this study, we applied an immunoproteomic approach to survey the extracellular proteins in the Chinese A. hydrophila strain J-1, and identified a novel 38 kDa extracellular OMP (Omp38). Our results indicate that Omp38 is a strongly immunoreactive antigen in rabbits and could be a rational candidate for vaccine development.

Materials and methods

Bacterial strains, plasmids, media and culture conditions

The A. hydrophila strain J-1, used for the immunoproteomics analysis and used as a vaccine strain in China, was first isolated from dead cultured cyprinoid fish in southeastern China in 1989 (Chen & Lu, 1991). The other A. hydrophila strains used in this study are listed in Table 1. All the plasmids and their host Escherichia coli in the study were obtained from Takara (Dalian, China). All A. hydrophila strains and E. coli were routinely cultured in Luria–Bertani (LB) broth or on Luria agar plates at 28 and 37 °C, respectively. For all the strains used in this study, ampicillin (100 µg mL−1) and kanamycin (50 µg mL−1) were added as required.

View this table:
Table 1

Details of Aeromonas hydrophila strains and the distribution of Omp38

StrainsSourcesHostPCRWestern blot
J-1Jiangsu, ChinaCrucian carp++
TPS-30Zhejiang, ChinaBlunt-snout bream+/
SBS-106Zhejiang, ChinaBlunt-snout bream++
TPS49Zhejiang, ChinaSilver carp++
PBS-10Zhejiang, ChinaCrucian carp++
SPS103Zhejiang, ChinaBlunt-snout bream++
AH9617Hubei, ChinaSilver carp++
HA9Jiangsu, ChinaTurtle++
BSK-10Zhejiang, ChinaCrucian carp++
SPS-104Zhejiang, ChinaBlunt-snout bream+
L316Fujian, ChinaEel++
T8Zhejiang, ChinaBlunt-snout bream++
T1Jiangsu, ChinaTurtle++
HB07Zhejiang, ChinaMussel++
DF4-3CCJiangsu, ChinaCrucian carp++
PEG14Zhejiang, ChinaCrucian carp+
B17Jiangsu, ChinaUnknown++
HA50Jiangsu, ChinaTurtle++
Y5Zhejiang, ChinaPrawn+
HA15Zhejiang, ChinaBlunt-snout bream++
LL1/88GermanyMirror carp
MR-2Zhejiang, ChinaMarsh prawn++
HB03Zhejiang, ChinaMussel++
FBS-35Zhejiang, ChinaSilver carp+
N-1-2Shanghai, ChinaCrucian carp++
WCOO-2Fujian, ChinaEel++
ST7833Beijing, ChinaUnknown++
CHSOO-3Fujian, ChinaEel++
Z4Guangdong, ChinaCrucian carp++
SB95-1Zhejiang, ChinaBlunt-snout bream+
TK961010Zhejiang, ChinaTurtle+
DF4-2GCZhejiang, ChinaGrass carp+
SB94-7Zhejiang, ChinaBlunt-snout bream
G1Zhejiang, ChinaUnknown
SB94-5Zhejiang, ChinaBlunt-snout bream+/
G3Zhejiang, ChinaUnknown
NL-3Our laboratorySilver carp
Y1Jiangsu, ChinaBighead carp
FPK-81Zhejiang, ChinaBlunt-snout bream+
PBJS-45Zhejiang, ChinaCrucian carp
SCK-020826Zhejiang, ChinaUnknown++
MR-1Zhejiang, ChinaMarsh prawn/
LS-5Chengdu, ChinaMirror carp/
W1Nanjing,ChinaPond water/

Preparation of antisera against A. hydrophila J-1

New Zealand white rabbits were used in accordance with the guidelines established by the Nanjing Agricultural University and the Guide for the Care and Use of Laboratory Animals. One milliliter (1 × 109 cells mL−1) of a formaldehyde-inactivated whole-cell vaccine of J-1 was injected subcutaneously into rabbits (n=4) at days 1, 15 and 29, diluted with an ISA 206 adjuvant (SEPPIC, France) at a ratio of 1 : 1. Sera from rabbits were collected at 1 week before immunization and 1 week after the last immunization for immunological assays.

Precipitation of extracellular proteins

Protein precipitations were performed as described by Bumann (2002), with a few modifications. Briefly, exponential cultures were centrifuged at 10 000 g for 15 min. The supernatant was collected, centrifuged at 4 °C for 15 min and filtered through a 0.45 µm membrane to remove residual bacteria. Extracellular proteins were precipitated using trichloroacetic acid (TCA) and acetone. The filtrate was mixed with prechilled 100% TCA to a final concentration of 10% and incubated in ice water for 30 min. After centrifugation at 10 000 g for 10 min at 4 °C, the pellet was resuspended in 10 mL of prechilled acetone and washed twice. The final pellet was air-dried.

2 -DE and Western blot analysis

Precipitated extracellular proteins were dissolved in rehydration buffer (8 M urea, 2% CHAPS, 50 mM DTT, 2% IPG buffer solution and 0.002% bromophenol blue) containing 150 µg of the protein sample in a total volume of 125 µL and centrifuged at 10 000 g for 20 min at room temperature to remove the insoluble materials. Protein concentrations were determined using the Ettan™ Sample Preparation Kits and Reagents 2-D Quant Kit (Amersham Pharmacia Biotech). Proteins were absorbed onto 7 cm immobilized pH gradient (IPG) strips (Immobiline DryStrip, pH range 4–7; Amersham Pharmacia Biotech), isoelectric focusing was performed using an Ettan IPGphor III (Amersham Pharmacia Biotech) and focusing was conducted by a stepwise increase of the voltage as follows: S1: 250 V, 30 min; S2: 500 V, 30 min; S3: 4000 V, 3 h; and S4: 4000 V 25 000 V·h.

Before sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), each IPG strip was washed in 1.5 mL of equilibration buffer 1 [75 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS, 2% DTT; 0.002% bromophenol blue] for 15 min and in 1.5 mL of equilibration buffer 2 [75 mM Tris-HCl (pH 8.8), 6 M urea, 2.5% iodoracetamide, 2% SDS; 0.002% bromophenol blue] for an additional 15 min. IPG strips and SDS-PAGE molecular weight (MW) standards were loaded into homogeneous 12% polyacrylamide gels (Laemmli, 1970) and sealed with a 1% agarose solution. Electrophoresis was performed in two steps at 15 °C: 80 V per gel for 30 min and 120 V per gel until the tracking dye reached the bottom of the gels. All gels were stained with Coomassie brilliant blue G-250. Three replicates were run for the sample.

The immunoblotting procedure was performed according to Zhang & Lu (2007). Proteins on the gels were transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia Biotech) and blocked with 5% w/v skimmed milk in TBS (pH 7.4) containing 0.05%v/v Tween-20 (TBST) for 2 h at room temperature. Subsequently, the membranes were probed with antisera to whole cells (1 : 1000 dilution) for 90 min at room temperature. Membranes were washed three times with TBST for 10 min and incubated with staphylococcal protein A-HRP (SPA-HRP, Boster, Wuhan, China; 1 : 2000 dilution) for 1 h at room temperature. The membranes were then washed with TBST, followed by development using a DAB kit (Tiangen, Beijing, China) until an optimum color development was observed. Three replicates were performed for each sample.

Protein identification and database searches

Protein spots of interest were excised from the two-dimensional (2-D) gels and sent to Shanghai GeneCore BioTechnologies for tryptic in-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS. The peak lists of each protein spot were analyzed with the aid of ‘PMF’ and ‘MS/MS Ion Search’ engines of mascot software (http://www.matrixscience.com/) and/or a local mascot server (http://www.proteomics.cn/mascot) for sequence matches. Proteins that were in similar locations on gels and on PVDF membranes were coded with the same number. The probability score for the match, MW, isoelectric point (pI) and number of peptide matches were analyzed for confident spot identification. Sequences of identified proteins were submitted to a blast server (http://www.ncbi.nlm.nih.gov/BLAST/) to find similar sequences. The SignalP webserver (http://www.cbs.dtu.dk/services/SignalP/) was used for signal peptide prediction.

PCR amplification and cloning of the omp38 gene

The gene encoding Omp38 of A. hydrophila (accession number CP000462) was retrieved from GenBank. The sequence was analyzed using the SignalP 3.0 server and the amino acids 1–22 were identified as the signal peptide. To amplify the gene fragment encoding the polypeptide with better antigenicity, the following primers were designed: omp38-F (5′-TCT GAA TTC CAG ACC GTC AAC GAG CAG-3′) and omp38-R (5′-GCT CTC GAG TTA GAA ATC CTC GCT GTT GCT-3′), containing the EcoRI and XhoI restriction enzyme sites (underlined), respectively. PCR conditions were as follows: initial denaturation at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 45 s and extension at 72 °C for 45 s. An extension step of 10 min at 72 °C was carried out following the last cycle in order to ensure full-length synthesis of the fragment. The PCR product was visualized on 1% agarose gels stained with ethidium bromide, purified using a gel extraction kit (Takara) and cloned into the pMD19-T simple vector and sequenced. PCR amplification was further used to investigate the distribution of omp38 in the A. hydrophila strains listed in Table 1.

Sequence analysis of omp38

The omp38 gene was amplified from genomic DNA of A. hydrophila J-1 and sequenced. A comparison of deduced amino acid sequences with their counterparts was performed using the blast program (http://www.ncbi.nlm.nih.gov/blast). The protein domain was predicted using the simple modular architecture research tool (http://www.smart.emblheidelberg.de/). megalign from DNAStar and the clustalw multiple alignment program (http://www.ebi.ac.uk/clustalw/) was used to construct multiple sequence alignments of omp38. The phylogenetic tree was constructed based on amino acid sequence alignments using the dnastar software.

Expression, purification and Western blot analysis of Omp38

The PCR-amplified product of omp38 from A. hydrophila J-1 was cloned into the pET28a expression vector and transformed into E. coli BL21 competent cells (Takara). The recombinant transformants were selected using kanamycin (50 µg mL−1) on LB agar plates, followed by identification by PCR and restriction enzyme digestion. The recombinant clones of omp38 were inoculated into LB broth (200 mL) containing kanamycin (50 µg mL−1). Once an OD600 nm of the cultures reached 0.6–0.8, cells were induced by 1 mM isopropyl-β-d-thiogalactoside. After 4 h, whole cells were harvested by centrifuging at 10 000 g for 10 min. The cell pellet was then washed three times with phosphate-buffered saline (PBS) (pH 7.4) to remove the broth. The bacterial cells were resuspended in 20 mL PBS and sonicated on ice 90 times at 5-s intervals, with 5-s rest periods between each pulse, in an ultrasonic disintegrator (JY92-II, Xinzhi Scientific Equipment Institute of Ningbo, China). The inclusion bodies containing most of the Omp38 protein were further dissolved in 8 mL binding buffer (8 M urea, 20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4), filtered using 0.45-µm-pore filters and purified as described in the manuals for HisTrap™ HP (GE Healthcare, NJ). The resultant proteins were separated electrophoretically by SDS-PAGE according to the method of Laemmli (1970) and visualized with Coomassie brilliant blue R-250. The purified protein was refolded using a linear 6.0–0.0 M urea gradient in PBS (pH 7.4). The protein concentrations were determined using the Bradford Protein Quant Kit (Tiangen).

Bacterial cells (uninduced recombinant and induced recombinant E. coli) harvested from LB and purified recombinant proteins were processed on SDS-PAGE gels. Then, the proteins were electrically transferred to PVDF membranes (Amersham Pharmacia Biotech) in a blotting buffer (39 mM glycine, 48 mM Tris base, 20% methanol and 0.037% SDS) using a semi-dry blotting apparatus (TE77, Amersham Pharmacia Biotech). The electrotransfer time was 2 h with a current/area of 0.8 mA cm−2. Sera from rabbits vaccinated with A. hydrophila J-1 were used as the primary antibody at a 1 : 500 dilution. The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG (Boster) and used at a 1 : 2000 dilution. The antibody-bound proteins were then visualized using the DAB kit (Tiangen).

Preparation of antiserum against recombinant Omp38

Rabbits were immunized with purified recombinant proteins (300 and 500 µg) by a subcutaneous injection along with an ISA 206 adjuvant (SEPPIC) at days 1, 15 and 29. The sera were collected from rabbits at 1 week before immunization and 1 week after the last immunization.

Antibody titers of the preimmunized and immunized sera were determined using an enzyme-linked immunosorbent assay (ELISA) as described by Engvall & Perlman (1971), with a few modifications. Briefly, 100 µL of carbonate–bicarbonate buffer (pH 9.6) containing 5 µg mL−1 purified recombinant proteins were added to microtiter plate wells and incubated overnight at 4 °C. The plates were washed three times with PBS containing 0.05% Tween-20 (PBST) and blocked for 2 h at 37 °C with blocking buffer (5% w/v skimmed milk in PBST). After the plates were washed three times, sera from immunized and control rabbits, at a starting dilution of 1 : 50 in PBST, were titrated through a twofold dilution series. Plates were incubated at 37 °C for 1 h and washed four times with PBST, and then probed with 50 µL horseradish peroxidase-conjugated goat anti-rabbit IgG (1 : 20 000) (Boster) for 1 h at 37 °C. After washing the plates four times with PBST, the reaction was developed with the 3,3,5,5-tetramethylbenzidine (Tiangen) substrate with H2O2. The plates were incubated at room temperature for 15 min, and the reaction was stopped with 100 µL of 2 M H2SO4. OD450 nm was measured using a Model 680 microplate reader (Bio-Rad). Positive sera were determined by OD450 nm values that exceeded that of the negative sera by 2.1 times, and the sera with high titers were selected for use in the following experiments.

Preparation of OMPs of A. hydrophila and Western blot analysis

OMPs from whole cells of 40 different A. hydrophila isolates were prepared with Sarkosyl as described by Filip (1973). Briefly, different strains of A. hydrophila were incubated overnight in LB broth (200 mL) at 28 °C. After centrifugation at 10 000 g for 10 min, the cell pellets were washed three times by Tris-HCl (0.02 mo1 L−1, pH 7.5), resuspended in 20 mL of Tris-HCl and sonicated in the ultrasonic disintegrator (600 W). The cells were removed by a single centrifugation step at 7000 g for 30 min. The supernatant was centrifuged at 100 000 g for 30 min and the pellet was resuspended into 20 mL of 0.5% sodium lauroy sarcosine and incubated overnight at 4 °C. Then, the sodium lauroy sarcosine was removed by a centrifugation at 100 000 g for 30 min, and OMPs (in the pellet) were resuspended in 1.5 mL of ddH2O and frozen at −20 °C.

The OMPs were electrophoresed by SDS-PAGE, which contained a 5% stacking gel and a 12% separating gel. Afterwards, the proteins were electrically transferred to PVDF membranes with a blotting buffer using a semi-dry blotting apparatus. The electrotransfer time was 2 h with a current/area of 0.8 mA cm−2. The sera from rabbits vaccinated with recombinant Omp38 were used as the primary antibody at a 1 : 500 dilution. The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG (Boster) used at a 1 : 2000 dilution. The antibody-bound proteins were then visualized using the DAB kit (Tiangen).


Immunoreactive proteins

Supernatant proteins of strain J-1 were separated on a 2-DE gel covering a pH range of 4–7 (IPG linear gradient; Fig. 1a). The 2-DE was repeated three times, and the patterns were highly reproducible. A total of approximately 850 spots were detected on the Coomassie G-250-stained gels using the image master 2d elite software [version 6.0, GE Healthcare (China) Ltd, China], with most proteins having MWs between 14.4 and 116 kDa.

Figure 1

2-DE profile of extracellular proteins of the virulent strain J-1 (a) and Western blot analysis of the proteins on the 2-D gel using the rabbit serum against J-1 (b). The spots on these gels were encoded using the strain name, followed by the protein number, which was assigned based on their similar locations on different gels/membranes.

A total of 18 immunoreactive proteins from J-1 were observed on the immunoblots (Fig. 1b), consistent with our observations on the duplicated 2-D gel (Fig. 1a). The 18 spots in duplicate gels were then excised and characterized by MALDI-TOF-MS or MALDI-TOF-TOF-MS, and the data were compared with those in the NCBI sequence database. The probability score for the match, MW, pI and number of peptide matches were used for confident spot identification. The 15 successfully identified immunoreactive spots corresponded to 10 individual proteins. Among the 15 protein spots, nine were analyzed by MALDI-TOF-MS and six were identified by MALDI-TOF-TOF-MS. However, spots J15, J16 and J20 were unsuccessfully identified by MALDI-TOF-MS or MALDI-TOF-TOF-MS. The results are summarized in Table 2. One protein of interest, OMP (named Omp38), corresponding to spot J17 was identified.

View this table:
Table 2

Protein spots of Aeromonas hydrophila identified by MALDI-TOF-MS or MALDI-TOF-TOF-MS

Identified protein
Spot numbermascot resultsblast results/speciesMr (kDa)/pImascot scoreNumber of peptides matched
J11gi|117619940Nuclease/A. hydrophila ssp. hydrophila ATCC 7966113 473/4.8313315
J12gi|117619940Nuclease/A. hydrophila ssp. hydrophila ATCC 7966113 473/4.7815718
J13gi|117619940Nuclease/A. hydrophila ssp. hydrophila ATCC 7966113 473/4.7211114
J14gi|117618809Hypothetical protein AHA_0616/A. hydrophila ssp. hydrophila ATCC 796649 267/6.0119612
J17gi|117621254Outer membrane protein/A. hydrophila ssp. hydrophila ATCC 796638 443/4.9022915
J18gi|56682611Flagellin A/Aeromonas punctata31 356/5.0316111
J19gi|117617709Hcp-2 hemolysin-coregulated protein/A. hydrophila ssp. hydrophila ATCC 796619 001/5.241966
J21gi|117618936Chitin-binding protein/A. hydrophila ssp. hydrophila ATCC 796653 605/5.721287
J22gi|122938436Hemolysin/A. hydrophila52 116/4.751009
J23gi|1827608Chain A, Pro aerolysin/A. hydrophila ssp. hydrophila ATCC 796651 899/4.831538
J24gi|122938436Hemolysin/A. hydrophila52 116/5.511279
J25gi|6319150Elastase/A. hydrophila62 690/5.7730710
J26gi|60458507Hemolysin/A. hydrophila68 898/6.061227
J27gi|117618575Putative metalloprotease/A. hydrophila ssp. hydrophila ATCC 796685 160/6.5016515
J28gi|117618575Putative metalloprotease/A. hydrophila ssp. hydrophila ATCC 796685 160/6.8013112

Cloning of the omp38 gene and sequencing analysis

The Omp38 protein, consisting of 357 amino acids with a calculated molecular mass of 38 464 kDa, is encoded by an ORF of 1074 nucleotides. The nucleotide sequence data of Omp38 have been submitted to GeneBank with the accession number GU134620.

Secondary structure analysis points toward the putative porin nature of Omp38. The amino acid composition of Omp38 is typical of nonspecific porins, and there is a final Phe in the carboxyl-terminal region that is present in all members of the bacterial porin superfamily (Jeanteur et al., 1991). This observation suggests that this protein is the Aeromonas analog of the well-characterized porins. Analysis by multiple alignment of similar sequences (Fig. 2) showed that Omp38 is closest to the OMP of A. hydrophila ssp. hydrophila ATCC 7966 (YP_854940.1), with a 99% similarity in the amino acid composition. The omp38 gene did not exhibit any homology with other known omp genes detected in Aeromonas isolates. However, Omp38 exhibited significant homology with the OMP of Vibrio harveyi HY01 (ZP_01986243.1, 60% identity), as well as with the OmpC of Vibrio fischeri ES114 (YP_205178.1, 53%). blast analysis of the deduced amino acid sequence revealed that Omp38 has a low homology with members of other nonspecific porin families from gram-negative bacteria. Distance relatedness was observed to the OMP of Edwardsiella tarda (AAL82724.1, 33%), E. coli UMN026 porin OmpN (YP_002412976.1, 33%), Klebsiella pneumoniae OmpK36 (ACM07444.1, 33%), Salmonella enterica ssp. enterica serovar Schwarzengrund strain SL480 OmpC (ZP_02661500.1, 28%), Aeromonas veronii Omp38 protein precursori (AAP40343.2, 27%), A. hydrophila OmpTS (AAF87725.2, 27%), Aeromonas salmonicida ssp. salmonicida A449 porin II (OmpK40) (YP_001141380.1, 27%), A. hydrophila porin II (AAD56398.1, 26%), A. hydrophila major adhesin Aha1 (ABC54614.1, 26%) and A. hydrophila major adhesion protein (ABM69028.1, 25%).

Figure 2

Neighbor-joining tree for Omp38. The sequences included in the analysis are abbreviated as Ah-OmpTS (Aeromonas hydrophila, AAF87725.2); Ah-Aha1 (A. hydrophila, ABC54614.1); Ah-porin II (A. hydrophila, AAD56398.1); As-OmpK40 (Aeromonas salmonicida ssp. salmonicida A449, YP_001141380.1); Av-Omp38 (Aeromonas veronii, AAP40343.2); Ah-adhesion (A. hydrophila, ABM69028.1); Ah-Omp (A. hydrophila ssp. hydrophila ATCC 7966, YP_854940.1); Ah-Omp38 (A. hydrophila); Vh-Omp (Vibrio harveyi HY01, ZP_01986243.1); Vf-OmpC (Vibrio fischeri ES114, YP_205178.1); Kp-OmpK36 porin (Klebsiella pneumoniae, ACM07444.1); Se-Porin OmpC (Salmonella enterica ssp. enterica serovar Schwarzengrund strain SL480, ZP_02661500.1); Et-Omp (Edwardsiella tarda, AAL82724.1); Ec-Porin OmpN (Escherichia coli UMN026,YP_002412976.1).

Distribution of the omp38 gene in different A. hydrophila isolates

The omp38 fragment could be PCR amplified from 75% (36/48) of the A. hydrophila strains tested in our study (Table 1), suggesting that omp38 is widespread in the A. hydrophila strains.

Expression and immunogenicity of the Omp38 recombinant protein

The PCR primers used in this study amplified an 852-bp fragment of the omp gene in strain J-1 (Fig. 3), which encodes 284 amino acids with a calculated molecular mass of 30 567 kDa. The amplicon was cloned into the pET28a vector using E. coli BL21 host cells. After kanamycin selection, colonies were screened for the insert using the same primers and PCR conditions. A novel protein band corresponding to 35 kDa was detected in the induced E. coli harboring pET28a-Omp38 (Fig. 4, lanes 2–3), while no protein was found at the same position in the uninduced E. coli harboring pET28a-Omp38 (Fig. 4, lane 1) by SDS-PAGE. The MW of the recombinant protein was identical to the predicted value. The highest proportion of the recombinant protein in the total bacterial protein was about 47.5%, and most of the recombinant proteins were in inclusion body form. After purification using HisTrap™ HP, the protein purity obtained was about 80%.

Figure 3

PCR amplification of the omp38 gene. Lane M, MW marker (5-kb ladder); lanes 1–3, Aeromonas hydrophila J-1; lane 4, negative control.

Figure 4

SDS-PAGE and Western blot analysis of the recombinant Omp38 expressed in Escherichia coli BL21. Lane M, MW marker; lane 1, pET28a-Omp38 in E. coli BL21, uninduced; lanes 2–3, pET28a-Omp38 in E. coli BL21, 1 mM isopropyl-β-d-thiogalactoside-induced for 4 h; lane 4, purified recombinant Omp38; lane 5, Western blot of purified recombinant Omp38; lane 6, Western blot of unpurified recombinant Omp38.

By Western blot analysis, a single band at the 35 kDa region was observed with the purified recombinant protein and the induced recombinant E. coli cell lysate (Fig. 4, lanes 5–6). Serum samples obtained from the immunized rabbits with the recombinant Omp38 were titrated for antibody quantification by sandwich ELISA. After three booster doses, a mean titer of 1 : 25 600 was observed in the rabbit sera.

Conservation of Omp38 among A. hydrophila strains

Western blot of the total OMP extracts from 40 different A. hydrophila strains showed that, in 60% of the strains, the protein at the approximate location of 38 kDa strongly reacted with our specific antiserum against Omp38 (Table 1). The results also showed that this polyclonal antibody reacted with some other very faint bands. It is not clear whether these bands resulted from a specific reaction or whether they were just nonspecific bands. Figure 5 shows an SDS-PAGE gel (Fig. 5a) of OMP preparations from eight typical Aeromonas isolates of domestic major serotypes and the corresponding immunoblot (Fig. 5b) using the sera from rabbits vaccinated with the recombinant Omp38 as the primary antibody. The various strains shown are J-1 and BSK10 belonging to the serogroup O5, while the isolates FBS35, HA50, AH9617 and PEG14 included are from the serogroup O9; the serogroups of MR-2 and L316 are unknown. OMPs from the eight strains of different serogroups reacted with anti-Omp38, indicating that the protein is conserved among typical A. hydrophila strains from a wide variety of geographic areas. The strength of the antibody reaction appeared to be distinctive, possibly due to differences in epitopes or protein expression levels. In addition, the Western blot results did not agree well with PCR detection for omp38. Omp38 was detected in 60% of A. hydrophila strains by Western blot, but 75% by PCR (Table 1), suggesting that some strains with omp38 gene do not express this protein or expression is too low to even be detected.

Figure 5

SDS-PAGE (a) of OMPs of different Aeromonas hydrophila serotype strains and immunoblot analysis (b) of these OMPs and serum of purified recombinant Omp38. Lane M, MW marker; lanes 1–8, OMPs extracts of AhJ-1 (O5), FBS35 (O9), BSK10 (O5), L316 (unknown), HA50 (O9), AH9617 (O9), MR-2 (unknown) and PEG14 (O9).


In this study, the secreted proteins of China vaccine strain A. hydrophila J-1 were profiled on 2-D gels, and further analyzed by immunoblotting with rabbit sera against A. hydrophila J-1. The immunoblot assays showed that rabbit sera reacted strongly with many antigens of ECPs. Eighteen of the protein dots recognized by immune sera were identified on a duplicated 2-D gel and analyzed by MS. The 15 successfully identified immunoreactive spots corresponded to 10 individual proteins, including nuclease, hypothetical protein aha_0616, OMP, hcp-2 hemolysin-coregulated protein, chitin-binding protein, proaerolysin chain A, hemolysin, elastase, putative metalloprotease and flagellin A (Table 2). This study identified three spots corresponding to nuclease, three to hemolysin and two to putative metalloprotease. The genes encoding these proteins mostly showed high identities with their homologs from A. hydrophila ssp. hydrophila ATCC 7966, whose complete genome has been published (Seshadri et al., 2006). Most of the above proteins have been reported as virulence factors in some A. hydrophila isolates (Cascón et al., 2000; Yu et al., 2005). A large number of putative virulence genes have been identified, which would be useful for development of effective vaccines, diagnostics and novel therapeutics against animal and human infection caused by A. hydrophila. Recently, the subunit vaccines for this bacterium were mainly focused on OMPs. The outer membrane of gram-negative pathogenic bacteria, which functions in enabling the bacteria to adhere to host tissues and take up nutrients from the hosts, play an important role in the interaction between bacteria and hosts (Seltman, 2002). Similarly, the components of the outer membrane can easily interact with the host immune system. It has been shown that some OMPs of bacteria can confer protective immunity. Rahman & Kawai (2000) demonstrated that OMPs of A. hydrophila are immunogenic and may be useful for developing vaccines using OMPs as antigens in fish. It was shown that recombinant OmpTS of A. hydrophila was highly immunogenic in Indian major carp and could protect fish from infection by A. hydrophila (Karunasagar et al., 1997). Maiti (2009) identified and characterized the 22 kDa recombinant OmpW in Aeromonas spp., and the PCR-based method targeting the ompW gene could be used for the rapid detection of Aeromonas spp. up to the genus level, indicating the significant utility of recombinant OmpW both in diagnostics and in vaccine development. The above subunit vaccines showed greater protective immunogenicity in animals. The development of vaccines utilizing OMPs against infections had been reported with other bacteria, such as A. salmonicida (Lutwyche et al., 1995), A. veronii (Vázquez-Juárez et al., 2005), V. harveyi (Li et al., 2008), Vibrio parahaemolyticu (Mao et al., 2007) and E. tarda (Kawai et al., 2004). However, whether they confer protection against a challenge with heterologous strains was not evaluated.

Interestingly, we found that J17, which was identified as an OMP, could also be recognized by antisera on 2-D immunoblots that were used to analyze the extracellular proteins of A. hydrophila J-1. This result suggested that the protein may be located both extracellularly and associated with the membrane (Hoekstra et al., 1976; Loeb & Kilner, 1978; Gamazo & Moriyón, 1987; Vázquez-Juárez et al., 2003). Extracellular localization of OMPs has been reported previously in many gram-negative bacteria, including A. hydrophila and A. salmonicida (MacIntyre et al., 1980; Vázquez-Juárez et al., 2003). During exponential growth of bacteria, as the peptidoglycan layer of bacteria is synthesized more slowly than the outer membrane contributing to the formation of surface blebs, the OMPs are formed and released (Loeb & Kilner, 1978). The release of OMP is assumed to be a method for delivery of Helicobacter pylori virulence factors in addition to classical secretion pathways (Fiocca et al., 1999); although this mechanism remains to be further clarified, it may also apply to other gram-negative bacterial pathogens including A. hydrophila (Vázquez-Juárez et al., 2003).

In the present study, omp38 of A. hydrophila was cloned and expressed in E. coli. The sequence analysis indicated that Omp38 is a member of the porin superfamily. Porins are highly immunogenic because of exposed epitopes on bacterial surfaces (Vázquez-Juárez et al., 2003). Because of the considerable homology at their primary and secondary structures, porins are suitable candidates for developing vaccines against gram-negative bacterial infections (Jeanteur et al., 1991; Tabaraie et al., 1994; Lutwyche et al., 1995). Merino (2005) demonstrated that A. hydrophila AH-3 porin II was an important molecule for fish immune protection against either A. salmonicida or A. hydrophila strains.

In order to explore the potential application of Omp38 as a vaccine candidate for fish, the present study investigated the distribution of omp38 in different A. hydrophila isolates. Furthermore, we analyzed the expression and immunogenicity of the Omp38 protein. The results demonstrated that the omp38 gene is prevalent among A. hydrophila isolates (36/48). The Western blot analysis showed that rabbit polyclonal antibodies against A. hydrophila J-1 could react with the purified recombinant Omp38 protein, suggesting that antigenicity is maintained. When rabbits were vaccinated with the recombinant Omp38 of A. hydrophila J-1, a strong antibody response was induced. Using the sera from rabbits vaccinated with recombinant Omp38 as the primary antibody, we performed the Western blot analysis of OMP extracts of 40 A. hydrophila isolates, including different predominant serotypes in China. The polyclonal antibody to the purified recombinant Omp38 could recognize a band of approximately the same molecular mass when immunoblots were performed with most of the above-mentioned A. hydrophila strains (24/40). Our findings indicate that the expressed protein is reasonably conserved among A. hydrophila strains. Considering that 40% A. hydrophila strains could not detect Omp38, one can expect that this could be a problem in vaccine development because the antibodies will not be protective against the strains. However, Western blot analysis revealed that the antiserum against Omp38 could react with some bands at the other MWs in these strains. If these bands resulted from a specific reaction, it suggested that Omp38 may have several antigenic epitopes and may share similar antigenic determinants with other OMPs. Therefore, regardless of the origin and virulence of the strains, the polyclonal antibodies against several epitopes of Omp38 could confer cross-protection against the infections of heterogenous strains.

In conclusion, the present study demonstrates the usefulness of immunoproteomics in understanding the immunogenicity of A. hydrophila extracellular proteins. The OMP expressed in the study shows promise as a vaccine candidate in aquaculture. The vaccine candidate is being further evaluated based on their protective efficacy against challenge with both homologous and heterologous strains of A. hydrophila in animal models. The role of this protein in the pathogenesis of A. hydrophila will also be investigated.

Authors' contribution

X.-d.N. and N.W. contributed equally to this work.


This research was supported by the Program for New Century Excellent Talents in University (NCET-07-0440), Special Funding of Public Sector Agricultural Research Project from the Chinese Ministry of Agriculture (200803013), the Jiangsu Provincial Natural Science Foundation of China (BK2007155) and the International Foundation for Science (A/4108-1).


  • Editor: Jacques Schrenzel


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