Antigenic and functional profiles of a Lawsonia intracellularis protein that shows a flagellin-like trait and its immuno-stimulatory assessment
© The Author(s) 2018
Received: 14 November 2017
Accepted: 3 January 2018
Published: 15 February 2018
The obligate intracellular Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), is an economically important disease in the swine industry. Due to extreme difficulty of in vitro culture of the pathogen, molecular characterization of protein components of LI that are targets of the immune system, is difficult; thus, the scientific evidence to drive the development of preventive measures is lacking. In this work, we investigated the antigenic and functional characteristics of a putative flagellar-associated protein, LI0570, using in silico computational approaches for epitope prediction and an in vitro protein-based molecular assay. The amino acid sequence of LI0570 exhibited similarities to flagellar-associated proteins in four different bacterial strains. The presence of B cell linear confirmative epitopes of the protein predicted by a bioinformatics tool was validated by western blot analysis using anti-LI mouse hyperimmune serum, which implied that LI0570 induced production of antigen-specific antibodies in vivo. Further, TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling were observed in HEK-Blue™-hTLR5 cells stimulated with LI0570. This result indicates that the LI0570 protein can trigger an innate immune response followed by a T-cell-related adaptive immune response in an infected host. Collectively, the data presented here support that the LI0570 protein which shows the antigenic potential could be a useful component of a recombinant vaccine against PE, providing progress toward an effective prevention strategy.
Porcine proliferative enteropathy (PE) is caused by Lawsonia intracellularis (LI), an obligate intracellular bacterium, commonly occurring in swine herds. The infection results in thickening of the intestinal epithelium due to enterocyte proliferation. Clinical features such as chronic enteritis, lethargy, retarded growth rate, diarrhea, and acute hemorrhagic enteritis causing sudden death are exhibited. Indeed, Porcine PE is responsible for severe economic loss in the swine industry worldwide . Proliferative enteropathy (PE) has also been diagnosed in a variety of animals such as horses, rabbits, rats, guinea pigs, dogs, chickens, sheep, deer, and non-human primates . Despite high herd prevalence of LI infection in growing-finishing pigs, its pathogenic mechanisms remain speculative due to difficulty of in vitro culture of this obligate intracellular bacterium.
Intracellular motility of LI ultimately leads to cell dissemination, which enhances LI’s ability to penetrate mucous layers and colonize the intestinal tract. Other enteroinvasive bacteria pathogens such as Listeria and Shigella are randomly detected in the intestinal cell cytoplasm , while LI is largely present in the apical region of enterocytes . Thus, molecular mechanisms of actin-based motility by which other intracellular bacterial pathogen spread in the infected cells may differ from those adapted by LI . Interestingly, some enteric bacterial pathogens have mechanisms to penetrate the mucus layer to reach epithelial cells via flagella-driven motility which plays a role in the initial phase of infection [5, 6]. The presence of a single flagellar motor on LI, which is one of the main phenotypic elements of the pathogen, was observed in the supernatant of an infected cell culture in vitro  and also a preliminary analysis of the LI genome sequence (PHE/MN1-00; NCBI accession #NC_008011) exhibits genes responsible for flagellar assembly. It is also known that the bacterial protein flagellin effectively induces an innate immune response of the host that is mediated by its ability to bind to toll-like receptor 5 (TLR5). Flagellin, acting via TLR5, leads to activation of MyD88-dependent signaling and the proinflammatory transcription factor, NF-κB, which induces an intense innate and adaptive immune response against flagellated bacteria . Although other bacterial flagellin has been widely used as an adjuvant molecule and as an antigen in vaccinology , the ability of LI flagellin to trigger the activation of immuno-modulatory pathway has not been demonstrated.
In this work, we initiated the study on the functional characteristics of LI0570 annotated by the United States National Center for Biotechnology Information (NCBI) as a putative flagellin and related hook-associated protein and its antigenic traits. We putatively defined LI0570 as Lawsonia flagellin (LFliC). Antigenic characteristics of LFliC were assessed by bioinformatics tools for in silico B-cell prediction. Further, we investigated whether the LI0570 retained immuno-adjuvant characteristics. To elucidate the role of the TLR5 agonist, TLR5-stimulating activity and IL-8 expression by the purified flagellin protein were measured using HEK293 cells.
Materials and methods
Sequence analysis of the Lawsonia flagellin protein LI0570 was performed using the NCBI BLAST programs  and FASTA program . Sequence similarity analysis was assessed by creating multiple sequence alignments using CLUSTALW . The antigenicity index (linear B-cell epitopes) for the LFliC protein was estimated in silico using the BepiPred 2.0 web server . BepiPred 2.0 employs the hidden Markov model combined with amino acid propensity scales to predict epitope data derived from crystal structures by assessing surface accessibility, helix probability, sheet probability, and coil probability .
Construction of the protein expression system
Bacterial strains and plasmids used in this study
F-ompT hsdSB (rB- mB-) dcm galλ (DE3) pLysS Cmr
E. coli BL21(DE3) pLysS expressing LfliC via pET28a (+) system
IPTG-inducible, T7 expression vector, C-terminal 6×His tag, KanR
pET28a derivative containing LfliC
Western blot analysis was performed to evaluate immunoreactivity of the LFliC protein in vitro using anti-LI hyperimmune serum obtained from the mice immunized with an L. intracellularis modified-live vaccine (Enterisol® Ileitis, Boehringer Ingelheim, Ingelheim am Rhein, Germany). The mice were inoculated with 5 × 106.9 50% tissue culture infectious doses (TCID50) via an oral route twice at 2-week intervals. The purified protein (10 μg/μL) was subjected to 15% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with phosphate-buffered saline (PBS) buffer containing 3% bovine serum albumin (BSA) for 1 h, followed by incubation with the anti-LI mouse hyperimmune serum at a 1:300 dilution for 2 h. The antigen–antibody interaction was detected with a 1:5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotechnology, Birmingham, AL, USA; diluted 1:5000).
In vitro characterization of LFliC protein
Determination of TLR5 bioactivity with the putative Lawsonia flagellin was conducted using HEK-Blue™-hTLR5 cells (InvivoGen, San Diego, CA, USA). The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), zeocin (50 μg/mL), and blasticidin (1 μg/mL). Cells (1 × 106 cells per well) that had been incubated overnight in a 96-well plates were stimulated with the LFliC protein (10 μg) in duplicate for various time periods (0, 1, 3, 4, and 7 h). The expression of TLR5 on the stimulated cells was assessed by fluorescence-activated cell sorting (FACS) analysis using an anti-TLR5 monoclonal antibody (1 μg/mL; Anti-hTLR5-IgG2a, Thermo Fisher Scientific, Waltham, MA, USA) as previously described . Furthermore, expression of IL-8 cytokine produced by signaling through TLR5 was measured in the stimulated cells by reverse transcription-polymerase chain reaction (RT-PCR). The primers for the IL-8 gene were obtained previously . Briefly, 1 × 106 HEK cells were treated with two concentrations of LFliC (10 and 100 ng/mL) in duplicate. The treated cells were incubated at 37 °C in a 5% CO2 incubator for 24 h and the transcription level of IL-8 was determined by RT-PCR as described previously .
In vitro cytotoxicity assay
The cytotoxic potential of the LFliC protein was assessed by cell viability assay with thiazolyl blue tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT) . Cells from a rat intestinal epithelial cell line (IEC-18) were plated at a concentration of 1 × 106 and were stimulated with various concentrations of LFliC (0.1, 1, 5, and 10 ng/mL) in duplicate. The pulsed cells were incubated at 37 °C in a 5% CO2 incubator for 24 h, and then the cells were incubated for an additional 4 h with 5 mg/mL MTT. The bromide salt of MTT was absorbed by the cells and was reduced to a product called formazan within the mitochondria, which accumulated in the cell. The resulting blue formazan crystals was solubilized by dimethyl sulfoxide (DMSO), and the absorbance value was measured at 560 nm using a microplate spectrophotometer.
Non-parametric Mann–Whitney test was used to evaluate the statistical difference using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). P-values less than 0.05 were considered statistically significant.
Characterization of LFliC as a putative antigen
Homologues of LI0570 in the amino acid sequence of flagellin associated protein searched by the BlastP program
Query cover (%)
Flagellar filament structural protein/Escherichia coli str. K-12 substr. MG1655
B-type flagellin/Pseudomonas aeruginosa PAO1
Flagellin C/Clostridioides difficile 630
In vitro and in vivo characterization of immunogenic proteins
In vitro cytotoxicity assay
Porcine PE caused by L. intracellularis is a common intestinal disease affecting susceptible pigs and poses a substantial economic loss to swine industries. Due to the obligate intracellular life style of the pathogen, conventional laboratory approaches to reveal molecular genetic traits such as antibiotic resistance have been restricted, and development of prevention strategies has been hampered . Despite the completion of LI genome sequencing, protein immunogens against LI infection have yet to be characterized. Proteomic and comparative transcriptional analyses were recently conducted to identify functions related to the pathogenic mechanisms of the LI proteins [7, 21].
In this study, we predicted functional properties and the antigenic potential of the putative LI flagellin protein LI0570. By BLASTP analysis, the amino acid sequence of LI0570 displayed a certain degree of similarity to the amino acid sequence of a flagellin-associated protein in D. alaskensis, E. coli K-12, P. aeruginosa, and C. difficile. Particularly, the B-type flagellin of P. aeruginosa showed 40% similarity of sequence identity to LI0570 and partially contributed to effective production of a protective antibody against P. aeruginosa infection . Immunotherapy with IgG antibodies specific to the N-terminus of the B-type flagellin protein also resulted in effective reduction of mortality and morbidity in a pathogen-infected murine burn model . Given that LI0570 showed substantial similarity to the N-terminal region of P. aeruginosa (Figure 1), the putative LFliC could hypothetically be considered a target for the host immune system.
In this work, the capacity of LFliC as a potential immunogen was confirmed by in silico analysis for linear B-cell epitope prediction and in vitro immunoblotting (Figures 2, 3). Given that an antigen–antibody interaction is fundamental to inducing a humoral immune response to invading pathogens , prediction of B-cell epitopes is necessary to evaluate its potential antigenic property. To elucidate whether the predicted B-cell epitope within the LFliC protein reflected its actual antigenicity, western blot analysis was performed using mouse hyperimmune serum raised against LI infection (Figure 3). An immuno-reactive band corresponding to the predicted size of the LFliC protein (33 kDa) was recognized with mouse polyclonal antibodies against LI, which indicated that the LFliC protein may possess potential antigenic determinants. However, the epitope recognition by the mouse anti-LI serum may not guarantee the recognition by a swine convalescent serum, which is still needed to be confirmed in the further study.
Bacterial flagellin, a pathogen-associated molecular pattern (PAMP), binds to TLR5 on the surface of dendritic cells (DC) or epithelial cells  and directly or indirectly induces innate and adaptive immune responses . The D1 and D2 domains of bacterial flagellin, two highly conserved regions, are crucial for the recognition of TLR5 . Following activation with the TLR5 ligand flagellin, TLR5-dependent secretion of IL-8, which is controlled by activation of the transcription factor NF-κB and MAPK, necessitates stimulating a signal transduction cascade via the TLR5 pathway . In this study, LFliC protein effectively induced enhanced expression of TLR5 on HEK cells (Figure 4). Further, the transcriptional level of chemokine IL-8 mRNA, which is a TLR5-mediated proinflammatory gene, also increased in the stimulated HEK cells (Figure 5). The results imply that LI0570 possesses intrinsic immuno-stimulatory characteristics involved in inducing innate immunity. Further, cell-mediated immunity is an important feature involved in host defense against intracellular bacterial infection . Given that previous studies reported that bacteria flagellin functions as stimulators for differentiation of CD4+ T cell via DC activation , more work is needed to understand the involvement of the LFliC protein in T-cell immunity mediated by DC.
Taken together, the results presented suggest that the LI0570 protein annotated by NCBI as a putative flagellin and related hook-associated protein of LI shows immuno-antigenicity in silico and in vitro, providing evidence that the protein could produce antigen-specific antibodies in vivo. Further, the capacity of LI0570 to induce increased expression of TLR5 suggests that the prominent antigens trigger innate immunity and prime antigen-specific adaptive immunity. Currently, a licensed avirulent live vaccine strain against LI is commercially available and has the potential for reactogenicity . The structural and functional findings of LI0570 in this study could aid in the production of safe recombinant vaccines, and these results will serve as an important groundwork for improving the prevention of PE more generally.
The authors declare that they have no competing interests.
GW conducted the experiments; JHL designed the experiments and JHL and GW wrote the paper. Both authors read and approved the final manuscript.
This work was supported by the Technological Innovation R&D Program (S2448723) funded by the Small and Medium Business Administration (SMBA, Korea).
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