Identification and characterization of a novel stress-responsive outer membrane protein Lip40 from Actinobacillus pleuropneumoniae

Background Actinobacillus pleuropneumoniae, a Gram-negative bacterium, is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal disease. Because current vaccines confer limited protection against A. pleuropneumoniae infection, the development of more effective vaccines is urgently required. The identification of immunogenic and protective antigens, such as an outer-membrane lipoprotein, will advance this purpose. Results Sixty putative lipoproteins were predicted from the genomic sequence of A. pleuropneumoniae using multiple algorithms. Here, we focused on the characteristics of the putative lipoprotein Lip40 from A. pleuropneumoniae strain SLW01 (serovar 1). Lip40 shares sequence similarity with many bacterial lipoproteins, and the structural prediction of Lip40 suggests that it is similar to A. pleuropneumoniae TbpB. The N-terminus of Lip40 contains an interesting tandemly repeated sequence, Q(E/D/P)QPK. Real-time RT–PCR indicated that the expression of lip40 was significantly upregulated at 42 °C, at 16 °C, and under anaerobic conditions. Recombinant Lip40 (rLip40) produced in Escherichia coli BL21(DE3) was specifically recognized by porcine convalescent serum directed against A. pleuropneumoniae. Lip40 was confirmed to localize at the bacterial outer membrane, and its expression was significantly stimulated when A. pleuropneumoniae was cultured under various stress conditions. Lip40 also protected 75 % of mice from fatal virulent A. pleuropneumoniae infection. Conclusions The immunogenic outer-membrane protein Lip40 is stress responsive, protects mice against infection, and might be a virulence determinant. Further investigation of Lip40 should expedite vaccine development and provide insight into the pathogenesis of A. pleuropneumoniae. Electronic supplementary material The online version of this article (doi:10.1186/s12896-015-0199-8) contains supplementary material, which is available to authorized users.


Background
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a severe and often fatal respiratory disease of swine, which is associated with significant economic losses in industrialized pig production worldwide [1]. To date, 16 serovars of A. pleuropneumoniae have been identified [2]. The pathogenesis of A. pleuropneumoniae infection is associated with several virulence factors, including but not limited to exotoxins. Other factors, such as capsular polysaccharides, lipopolysaccharides, adhesins, proteases, outer-membrane proteins, and transcriptional regulators, are also reported to be involved in its pathogenesis [3][4][5][6].
Bacterial lipoproteins are a set of membrane-associated proteins characterized by a conserved lipid-modified cysteine, which play important roles in bacterial physiological processes [7]. The involvement of lipoproteins in the infection processes of many pathogens has received wide attention [8]. Lipoproteins play a key role in the bacterium's adhesion to the host cell, are involved in antibiotic resistance, and regulate the host immune response [8]. They have been reported to be pathogen-associated molecular patterns, which are recognized and captured by Toll-like receptors or other pattern-recognition receptors, inducing the activation of the immune cells and initiating the inflammatory processes [9]. Many immunogenic bacterial lipoproteins have been developed as candidate vaccines [8].
A putative lipoprotein, Lip40, containing the tandemly repeated sequence Q(E/D/P)QPK was observed among the 60 putative lipoproteins predicted from the published genomic sequence of A. pleuropneumoniae strain JL03. The lip40 gene from an A. pleuropneumoniae field isolate, SLW01 (serovar 1), was cloned and characterized, including its sequence features, predicted structure, immunogenicity, subcellular localization, expression after stimulation, and protective efficiency in mice, extending our understanding of this lipoprotein.

Expression and purification of Lip40 from E. coli
The mature protein-coding sequence of Lip40 was cloned from the A. pleuropneumoniae genomic DNA using primers P7 and P8 ( Table 1). The PCR products were then digested with BamHI/HindIII and ligated into the prokaryotic expression vector pGEX-KG [19], generating the recombinant plasmid pGEX-lip40. Recombinant Lip40 (rLip40) was then expressed from pGEX-lip40 in E. coli BL21(DE3). rLip40 was collected from the E. coli lysate and purified with a glutathione (GST)-Sepharose 4B column (Amersham Biosciences, Buckinghamshire, England). The immunoreactivity of the purified rLip40 was verified with western blotting, using porcine convalescent serum directed against A. pleuropneumoniae as the primary antibody.

Production of polyclonal antibodies against rLip40
Two female New Zealand white rabbits (~2.5 kg; purchased from the Centre for Disease Control and Prevention [CDC] of Hubei Province, China) were used to produce polyclonal antibodies directed against rLip40. All the animal experiments in the present study were approved by the Animal Experiment Committee of Central China Normal University. Each rabbit was first injected intradermally with purified rLip40 (650 μg). The purified protein (0.5 ml) was emulsified with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, USA). Animals were subsequently injected three times, at 15-day intervals, with the same immunogen emulsified with incomplete Freund's adjuvant with the same regimen. Antisera were collected 10 days after the third booster immunization.
The titers and specificity of the polyclonal antibodies were evaluated with a Lip40 enzyme-linked immunosorbent assay (ELISA). Briefly, flat-bottomed 96-well polystyrene ELISA plates (Haimen Shengbang, Haimen, China) were coated with 0.17 μg of purified rLip40 diluted in 100 μl of coating buffer (50 mM sodium carbonate, pH 9.6). The coated plates were washed three times with phosphate-buffered saline (PBS) plus 0.05 % Tween 20 (PBST), blocked at 37°C for 1 h with blocking buffer (5 % skimmed milk in PBST), and then washed three times with PBST. For the ELISA, 2 μl samples and 198 μl of PBST were added to each well in the first line of wells in the plate and 100 μl of PBST was added to the rest of the wells. The samples were diluted and incubated at 37°C for 40 min. After four washes with PBST, 100 μl of horseradish peroxidase (HRP)-conjugated secondary antibody (Southern Biotechnology Associates, Birmingham, USA), diluted 1:5000 in PBST, was added to each well, and the plates were incubated at 37°C for 30 min. After five washes, 100 μl of 3,3′,5,5′-tetramethylbenzidine color development solution (Biotime Biotech, Haimen, China) was added to each well. The plates were incubated at room temperature in the dark for approximately 10 min, and the catalytic reaction was then stopped with 50 μl of 1 % SDS. The optical density was read at 630 nm (OD 630 ) in an ELISA reader (Power-Wave XS, Bio-Tek, Winooski, USA).

Subcellular localization of Lip40
The subcellular fractionation of A. pleuropneumoniae was performed as described previously [20]. In brief, 1 l of bacterial culture was pelleted and resuspended in 10 ml of solution A (0.2 M Tris-HCl [pH 8.0], 1 M sucrose, 1 mM EDTA). Lysozyme was then added to the cell suspension to a final concentration of 1 mg/ml, vortexed, and incubated at room temperature for 5 min. dH 2 O (40 ml) was added to the swirling mixture before it was placed on ice. The cells were centrifuged at 200,000 × g for 45 min at 4°C. The supernatant contained the periplasmic fraction. The pellet was resuspended in 7.5 ml of ice-cold solution B (10 mM Tris-HCl [pH 7.5], 5 mM EDTA, 0.2 mM DTT), supplemented with 50 μl of DNase (1 mg/ml). The cells were broken by two passes at 10 8 Pa. The unbroken cells were pelleted by centrifugation at 4000 × g for 10 min at 4°C. The supernatant was then centrifuged at 280,000 × g for 4 h at 4°C. The supernatant contained the cytoplasmic fraction and the pellet contained the crude membranes, which were collected separately. The crude membrane pellet was resuspended in 9 ml of solution C (50 mM Tris-HCl [pH 8.0], 2 % [v/v] Triton X-100, 10 mM MgCl 2 ), and centrifuged at 85,000 × g for 30 min at 4°C. The supernatant contained the cytoplasmic membrane fraction. The pellet, containing the outer membrane, was washed in 1 ml of solution C, centrifuged at 85,000 × g for 20 min at 4°C, washed three times with 500 μl of dH 2 O, and stored at −20°C. The extracellular proteins were precipitated from the filtered culture supernatant with trichloroacetic acid at 4°C overnight, followed by centrifugation (17,700 × g, 1 h, 4°C), and were washed six times with 1 ml of 96 % ethanol. The pellet was dried and redissolved in 400 μl of solution D (7 M urea, 2 M thiourea, 4 % CHAPS [w/v], 30 mM Tris-HCl, [pH 8.0]), and stored at −20°C. The subcellular localization of the Lip40 protein was determined with western blotting using rabbit hyperimmune anti-rLip40 serum (1:400) as the primary antibody.

Expression of Lip40 protein under stress conditions
Actinobacillus pleuropneumoniae, cultured in TSB under normal conditions (aerobically at 37°C) for 3 h, was divided into four equal parts and incubated under anaerobic condition, or at 42°C, 16°C, 37°C for another 3 h, respectively . The cells were harvested by centrifugation, and the outer-membrane fractions were extracted as described above. The concentration of all outer-membrane samples prepared under different culture conditions were determined with a BCA protein assay kit (GenStar BioSolutions, Beijing, China). Equal volumes of the samples (66 μg) were separated with 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Western blotting was performed with rabbit hyperimmune anti-rLip40 serum (1:400) as the primary antibody. The expression of Lip40 was determined in triplicate. The expression levels of Lip40 in the outer-membrane samples were determined according to the density of the bands, with the Quantity One software.

Vaccination and challenge of mice
The protective efficacy of rLip40 was tested in a mouse vaccination/challenge model. Forty-eight 6-week-old specific-pathogen-free BALB/c mice (CDC, Hubei Province, China) were randomly allocated to four groups of 12 animals. ApxI toxin was obtained from the supernatant of A. pleuropneumoniae strain 13039 (serovar 10) cultures, as described previously [21], and inactivated with 0.4 % formaldehyde. Group 1 was inoculated with PBS and used as the negative control; group 2 was vaccinated with 150 μg of rLip40; group 3 was vaccinated with 150 μg of inactivated ApxI; and group 4 was vaccinated with a commercial trivalent inactivated vaccine (containing serovars 1, 2, and 7; purchased from Wuhan Keqian Biotech, China). For groups 2 and 3, the antigens were emulsified separately with complete Freund's adjuvant for the first immunization, and with incomplete Freund's adjuvant for the booster immunization. Serum samples were collected before each immunization and before the mice were challenged, and assayed with the Lip40-ELISA, as described above, and an ApxI-ELISA, as described previously [22]. Each group was intraperitoneally challenged with 2.5 × 10 6 colony-forming units of log-phase A. pleuropneumoniae SLW01 14 days after the second vaccination. The numbers of surviving mice were recorded every day for 5 days after the challenge.

Sixty putative A. pleuropneumoniae lipoproteins
With the 'majority vote' approach [17], 60 of the 89 identified putative lipoproteins from A. pleuropneumoniae JL03 were predicted as lipoproteins, including some previously reported lipoproteins, such as TbpB (gene locus APJL_0250) [23] and PalA (gene locus APJL_0317) [24], as well as several previously reported putative lipoproteins [25], indicating that our prediction method and results are reliable. The sequence characteristics of the 60 lipoproteins are listed in Additional file 1: Table S1. Putative lipoprotein Lip40, containing a special N-terminal tandemly repeated sequence, was further investigated in the present study.

Lip40 has a conserved lipoprotein domain
The lip40 gene from A. pleuropneumoniae SLW01 was cloned and sequenced (GenBank accession number: KP119767). The Lip40 protein was predicted to have the typical characteristics of lipoproteins with the LipoP 1.0 program, and contains the recognition sequence for signal peptidase II (LVITA ↓ CGSKN). The lipid modification site of Lip40 was predicted to be located at Cys20. A multiple sequence alignment of Lip40 and lipoproteins from Aggregatibacter actinomycetemcomitans and Cardiobacterium hominis showed that Lip40 is homologous to those lipoproteins (similarity > 33 %; Additional file 2: Figure S1). It also displayed similarity with several transferrin-binding proteins (Tbps) from pathogenic bacteria (similarity > 34 %; Additional file 3: Figure S2). The multiple sequence alignment showed that Lip40 is homologous to both the N-and C-lobes of A. pleuropneumoniae TbpB (Fig. 1a). Structural prediction revealed that the Lip40 protein consists of an eight-stranded β barrel accompanied by a domain made up of four β strands (Fig. 1b). Superimposition of Lip40 onto the C lobe and N lobe of TbpB (PDB: 3HOL) indicated that they have high structural similarity ( Fig. 1c and d). We also noted that Lip40 contains a tandemly repeated sequence, Q(E/D/P)QPK, at its Nterminus. Actinobacillus pleuropneumoniae strain SLW01 Lip40 has nine tandem repeats. The numbers of repeats in the Lip40 homologues from other A. pleuropneumoniae strains were also calculated, and according to the available genome sequences of these strains, the repeat numbers differ among the different strains (8-27) ( Table 2).

Transcription of lip40 is stimulated by stress
The expression of the lip40 gene was analyzed under in vitro growth conditions mimicking the environment during infection, including anaerobic conditions and high temperature (42°C). The expression of lip40 at low temperature (16°C) was also evaluated. As shown in Fig. 2, the transcription of the lip40 gene was significantly upregulated under stress relative to its transcription under normal culture conditions (37°C, aerobic), and heat shock most strongly increased lip40 expression (45-fold). These results indicate that lip40 is responsive to temperature and anaerobic stress, and might be involved in the bacterial infection process.

rLip40 is immunoreactive
To express the Lip40 protein, the lip40 gene without the signal peptide was cloned from A. pleuropneumoniae SLW01, restricted, and ligated into the prokaryotic expression plasmid pGEX-KG. Escherichia coli cells containing pGEX-lip40 were induced with isopropyl-β-Dthiogalactopyranoside. The recombinant Lip40 protein was confirmed to be present in the soluble supernatant of the cell lysate with SDS-PAGE, as shown in Fig. 3a. The purification of rLip40 is shown in Fig. 3b. Western blotting revealed a band with a molecular weight of~56 kDa for rLip40, whereas no signal was detectable for GST (Fig. 3c). This result confirms that porcine convalescent serum directed against A. pleuropneumoniae recognized and bound specifically to the rLip40 protein.

Lip40 elicits a strong humoral immune response in rabbits
Rabbits were immunized with four doses of purified rLip40 by intradermal injection. Serum samples were collected before each immunization and before euthanasia and the antibodies against rLip40 were tested with an ELISA, to verify the generation of a Lip40-specific immune response in rabbits. Lip40-specific antibodies appeared two weeks after the first immunization, and then increased quickly after the second and third injections (Additional file 4: Figure S3A). At the end of the immunization regimen, titration of the rabbit sera (at 55 days) showed the highest reactivity, with an increase of up to 2 × 10 5 -fold (Additional file 4: Figure  S3B), suggesting that rLip40 stimulates a strong antibody response in rabbits. The same amount of purified GST was used to coat 96-well plates, and antibody directed against GST was evaluated with a GST-ELISA, as described for the Lip40-ELISA. The GST-specific IgG titer in the hyperimmune serum was only 1:3200 (Additional file 4: Figure S3B), much lower than that of the Lip40-specific antibody. Therefore, it appears that Lip40 played a major role in the elicitation of rabbit antibodies directed against the GST-Lip40 fusion protein.

Lip40 is located in the outer membrane
Although bioinformatic analysis showed that Lip40 is located on the outer membrane, experimental verification  was required (data not shown). Subcellular fractions of A. pleuropneumoniae cells were extracted carefully, including the cytoplasmic proteins, periplasmic proteins, cytoplasmic membrane proteins, outer-membrane proteins (OM), and extracellular proteins (Fig. 4a). A band corresponding to Lip40 (~30 kDa) was observed in the outer-membrane fraction with western blotting, as shown in Fig. 4b. No Lip40 signal was observed in the other subcellular fractions. Our results confirm that Lip40 localizes to the outer bacterial membrane.

Enhanced expression of Lip40 protein under stress
To test whether the expression of Lip40 protein in the outer-membrane fraction is stimulated by stress conditions, A. pleuropneumoniae cells cultured under anaerobic condition or high and low temperatures were collected, and their outer-membrane fractions were extracted and quantified. Equal amounts of samples (66 μg) were separated by SDS-PAGE, and the expression levels were determined with western blotting (Fig. 5). In general, the expression of Lip40 protein was enhanced under stress conditions, especially at 42°C, and the Lip40 signal increased more than three-fold under heat shock. These results indicate that both the transcription of the lip40 gene and the translation of the Lip40 protein increased in response to stress. Therefore, Lip40 may be involved in the adaptation of A. pleuropneumoniae to environmental changes.

Lip40 is protective in mice
Serum samples from mice immunized with different antigens were analyzed with ELISAs (Fig. 6). All the mice were negative for rLip40 and ApxI before immunization. Serum samples from the mice inoculated with PBS (group 1) or ApxI (group 3) showed no detectable antibody against Lip40 during this study (Fig. 6a), whereas the sera from group 2, vaccinated with rLip40, were positive for Lip40 antibodies 14 days after the first vaccination, and the titer had increased by the second vaccination. The Lip40 antibody titers in group 2 were significantly higher than those in the other groups (P <0.01, Student's t test; Fig. 6a). The mice in groups 1 and 2 were also free of ApxI antibodies, but the mice in group 3 showed a significantly stronger ApxI antibody response than the other groups (P < 0.01). As shown in Fig. 6b, group 4, vaccinated with commercial vaccine, also showed a strong ApxI antibody response after vaccination. The mice were challenged with virulent A. pleuropneumoniae SLW01 14 days after the second immunization, and the survival rates were recorded for 5 days after challenge (Fig. 7). No mice inoculated with PBS survived the challenge, and the protective efficacies of rLip40, ApxI, and commercial vaccine were 75 % (9/12), 100 % (12/12), and 91.7 % (11/12), respectively. These results show that rLip40 protected more than half the animals (75 %) from the challenge. Thus, the protective efficacy of rLip40 was significantly higher than that of the negative control (0 %), indicating that rLip40 is immunoprotective and provides effective protection against A. pleuropneumoniae infection in mice.

Discussion
Lipoproteins are required for the virulence of some pathogenic bacteria. They play a variety of roles in host- pathogen interactions, from surface adhesion and the initiation of inflammatory processes to the translocation of virulence factors into the host cytoplasm [8]. However, the pathogenesis of A. pleuropneumoniae lipoproteins has rarely been examined. Most such studies have focused on their immunogenicity and vaccine potential, including studies of TbpB [26][27][28] and OmlA [29,30]. These proteins were shown to be immunoprotective and important vaccine components (PleuroStar APP, Novartis Animal Health Inc., Switzerland). However, PalA was reported to inhibit the protective efficacy of ApxI and ApxII [24]. In the present study, a putative lipoprotein, Lip40, which contains an interesting tandemly repeated sequence, Q(E/D/P)QPK, was characterized. Previous studies have demonstrated the relationship between coding tandem repeat (CTR) sequences and virulence in Chlamydia pecorum [31,32]. CTRs at the incA locus encode a variable number of repeated motifs, and the number of repetitions in incA is related to the virulence of C. pecorum strains [32]. Similarly, the number of tandem repeats in Lip40 varies within its homologues in different serovars of A. pleuropneumoniae strains. Therefore, the possible role of Lip40 in bacterial pathogenesis requires further characterization.
Oxygen deprivation and high temperature are two common stresses that A. pleuropneumoniae encounters during infection [5,33]. Transcriptional profiles under anaerobic conditions [33] and acute disease [34] have been analyzed with microarray chips to discover potential virulence factors, which are usually considered to be upregulated under in vivo conditions or in a mimicked infection. This genome-wide transcriptional screening of the differentially expressed genes of A. pleuropneumoniae revealed no Lip40 homologue. In this study, we confirmed that Lip40 is upregulated at both the transcriptional and translational levels by stress. The mechanisms involved in the adaptive responses of A. . c Intensity of the bands was quantified with the Quantity One software. Relative expression levels were obtained by normalizing the intensity of stress-treated samples to that of untreated samples in each assay. Double asterisks (**) indicate a significant difference (P < 0.01) compared with the stress-treated samples (Student's t test) Fig. 6 Humoral responses in mice. Antibodies against rLip40 (a) and ApxI (b) were evaluated at different time points with ELISAs. ELISA titers are expressed as the logarithm (log10) of the reciprocal of the highest dilution of serum with an OD 630 value above that of the cutoff value for each mouse. Double asterisks (**) indicate P < 0.01, calculated with Student's t test Fig. 7 Protection conferred by different antigens against A. pleuropneumoniae challenge in mice (survival rates). Numbers of surviving mice in each group was monitored for 5 days after challenge. Numbers of surviving mice did not change 72 h after infection pleuropneumoniae remain to be investigated. The effects of cold shock were tested as a typical environmental stress. Reports have suggested that the autumn-winter transition is one of the outbreak peaks of porcine pleuropneumonia in China [35], and the environmental temperature is always far lower than the normal growth temperature for mesophilic bacteria during this period. It is possible that the adaptation of A. pleuropneumoniae to cold stress is responsible for the infections observed at this time of year. Our results indicate that both the transcriptional and translational levels of Lip40 were elevated at 16°C compared with those at 37°C, suggesting that Lip40 is a factor in the responsiveness of A. pleuropneumoniae to low temperature. Therefore, we hypothesize that Lip40 is a potential virulence factor and involved in pathogen-environment and/or pathogenhost interactions.
Commercial vaccines, including whole-cell bacterins and second-generation subunit vaccines, only confer partial protection against A. pleuropneumoniae infection [29]. Therefore, it is essential that more effective vaccines be developed. The identification of conserved immunogenic antigens, especially outer-membrane-located proteins, will advance this purpose. Several outermembrane-associated proteins have been verified separately as vaccine components or candidate vaccines with various investigation methods, including TbpB, OmlA, OmpP2, OmpA, and OmpW [25,36,37]. In addition to their location on the outer bacterial membrane, many lipoproteins of Gram-negative bacteria are considered to be efficient candidate vaccines [38]. The putative outermembrane proteins and lipoproteins have been identified in the genome of Pasteurella multocida, a great step forward in the development of a protective vaccine against fowl cholera [39,40]. Similar research was undertaken in A. pleuropneumoniae, and several lipoproteins were cloned and their protective efficacies were investigated [25]. Here, we report a putative lipoprotein, Lip40, confirmed to be an outer-membrane-localized stress-responsive factor. Purified rLip40 induced strong antibody responses when it was administered to rabbits. Mice vaccinated with rLip40 displayed a survival rate of 75 %, which was significantly higher than that of the negative control group (0 %), indicating that rLip40 can be used as a component of vaccine against A. pleuropneumoniae infection. Our results provide additional confirmation of the importance of bacterial outer-membrane lipoproteins in vaccine development.