Veterinary Microbiology 205 (2017) 57–61

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Original article

Identification of Lawsonia intracellularis putative hemolysin protein A and MARK

characterization of its immunoreactivity
⁎
Jehyung Kim1, Gayeon Won1, Suyeon Park, John Hwa Lee
College of Veterinary Medicine, Chonbuk National University, Iksan Campus, Gobong-ro 79, Iksan, 54596, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Despite the recent global increase in fatal endemic outbreaks of proliferative enteropathy (PE) caused by the
Lawsonia intracellularis obligate intracellular bacterium Lawsonia intracelluralis (LI) in the swine industry, development of effective
Proliferative enteropathy prevention strategies or immunodiagnostic tests has been delayed due to the difficulty of cultivating this
Lawsonia hemolysin A pathogen in vitro. Although several genetic analyses have been performed at the level of gene transcription after
the complete genome sequence of LI was made available, the mechanism of LI infection and virulence genes
remain unidentified. In the present study, we assessed the antigenic features of the LI0004 protein, which we
putatively defined as Lawsonia hemolysin A (LhlyA), by employing bioinformatics tools and in vivo and in vitro
protein-based molecular assays. The amino acid sequence of LhlyA showed approximately 60% homology to the
hemolysin-like proteins of Bilophila wadsworthia and Desulfovibrio piger. Presence of computationally predicted
linear antigenic B-cell epitopes on the LhlyA protein was demonstrated by immunoblotting; a band with a
molecular mass corresponding to the predicted size of the protein was strongly recognized by sera collected from
artificially infected mice. Further, in an in vivo cytotoxicity assay, no splenomegaly was observed in mice
inoculated with purified LhlyA. Collectively, the data presented here suggest that the LhlyA protein is a highly
immuno-reactive antigen of L. intracellullaris and can potentially be used to develop effective protection
strategies against PE.

1. Introduction production, difficulties related to in vitro cultivation of LI have hindered

Lawsonia intracellularis (LI) is a gram-negative, microaerophilic,
obligate, intracellular bacterial pathogen and a causative agent of
proliferative enteropathy (PE) (McOrist et al., 1995). The pathogen
has a tropism for intestinal epithelial cells and only replicates in the
cytoplasm of infected intestinal crypt, which induces enterocyte pro-
liferation (Jacobson et al., 2010). PE poses a substantial economic
burden in the swine industry worldwide and is occasionally reported in
a variety of other animals including horses, dogs, rabbits, rats,
hamsters, deer, and ostrich (Cooper et al., 1997; Pusterla et al., 2012;
Watarai et al., 2008). Two major forms of PE are recognized: porcine
intestinal adenomatosis (PIA) and proliferative hemorrhagic entero-
pathy (PHE) (Huerta et al., 2003). PIA, a chronic form of PE, causes
mild diarrhea and weight loss in growing pigs aged 8 to 20 weeks
(Guedes and Gebhart, 2003). PHE, an acute form of PE, causes bloody
diarrhea and sudden death in finishers or breeder pigs aged 4 to 12
months, with high morbidity and mortality.
Despite endemic outbreaks and the large impact of PE on pig
the use of genetic approaches to elucidate the pathogenesis of PE
(McOrist et al., 1995). Considering its entero-invasive characteristic, it
has been speculated that LI escapes from the vacuolar compartment
into the cytoplasm after internalization, multiplies, and spreads from
cell to cell, resulting in the proliferation of immature enterocytes
(Vannucci and Gebhart, 2014). However, it is not known how this
pathogen disseminates beyond the infected intestinal epithelium,
causes pathogenesis, and result in the clinical symptoms. This lack of
knowledge has restricted the development of prevention strategies such
as vaccines and diagnostic methods. Before completion of the genome
sequence of LI, a few proteins expressed by infected in vitro cell cultures
were elucidated using a PCR-based molecular technique (McCluskey
et al., 2002) or a mass spectrometry approach (Watson et al., 2011).
Recently, the pathogenic characteristics of LI were elucidated at the
gene tran;1;scriptional level using genome sequence information (PHE/
MN1-00; NCBI accession #NC_008011) (Vannucci et al., 2012).
Although the availability of the complete LI genome sequence can
facilitate identification of putative antigens, few studies have per-

⁎
Corresponding author.
E-mail address: johnhlee@jbnu.ac.kr (J.H. Lee).
1
The authors contributed equally to the work.

http://dx.doi.org/10.1016/j.vetmic.2017.05.007
Received 24 March 2017; Received in revised form 8 May 2017; Accepted 9 May 2017
0378-1135/ © 2017 Elsevier B.V. All rights reserved.
J. Kim et al.
formed antigenic characterization of LI genes.
In this study, we report the antigenic properties of the LI0004
protein (accession: NC_008011), putatively defined as Lawsonia hemo-
lysin A (LhlyA). Antigenic properties were characterized by computa-
tional bioinformatics analysis of amino acid sequences and computa-
tional prediction of epitopes. Potential cytotoxicity and hemolytic
activity of the protein were also assessed. To assess this protein’s
immunological properties, it was reacted with immune serum raised
against an avirulent LI strain. Overall, we established that this protein is
a highly immuno-reactive antigen that might have the ability to induce
the production of protein-specific antibodies. This could contribute to
the development of effective prevention strategies against proliferative
enteropathy.
2. Materials and methods
2.1. Bioinformatics analysis
Homology of the Lawsonia hemolytic protein (LhlyA) to other
proteins was assessed using NCBI BLASTP and PSI-BLAST searches
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) and GeneDoc software
(Nicholas et al., 1997). To predict linear antigenic B-cell epitopes in
the protein, the peptide property calculator on the BepiPred server
(http://www.cbs.dtu.dk/services/BepiPred) was used with an auto-
mated threshold score based on a hidden Markov model and a
propensity scale method (Larsen et al., 2006). Further, three different
in silico B-cell epitope predictors, BCPred, AAP, and FBCPred, were used
to determine linear B-cell epitopes of the protein according to the
protocol described previously (EL-Manzalawy et al., 2017; Singh et al.,
2013).
2.2. Construction of recombinant plasmids harboring hlyA
The bacterial strains and plasmids used in this study are described in
Table 1. The full-length gene (LhlyA,762 bp) encoding a putative
hemolysin protein of LI, LI0004 was chemically synthesized (Bioneer,
Korea). To assess the hemolytic activity of purified LhlyA protein, the
enterohemorrhagic Escherichia coli (EHEC) hemolysin (Ehly) was used
as a positive control. The Ehly gene was amplified in JOL364 isolated
from infected bovine using PCR with the primer pairs Ehly_F: 5′-
GGATCCATGACAGTAAATAAAATAAAGAACA-3′ and Ehly_R:5′- GTCG-
ACTCAGACAGTTGTCGTTAAAGTT-3′. The synthesized LhlyA and the
PCR product, Ehly, were digested with the restriction endonucleases
XbaI and BamHI, and the cleaved fragments were subcloned into
plasmid pET28a (+), which bears a kanamycin-resistant maker (Kanr)
and a carboxy terminal 6-histidine-tag for protein purification using Ni-
NTA resin. pET28a-LhlyA and pET28a-Ehly were individually electro-
phorated into E. coli BL21 (DE3) pLysS and designated JOL1742 and
JOL1898, respectively. Target proteins were purified using an affinity
Table. 1
Bacterial strains and plasmids utilized in this study.
Strains/plasmids Description References
Strains
BL21(DE3)pLysS F- ompT hsdSB (rB- mB-) dcm galλ(DE3) pLysS Promega
Cmr
JOL364 Wild type Hly+ EHEC isolate from bovine Lab stock
JOL1742 E. coli BL21(DE3) pLysS expressing LhlyA via This study
pET28a (+) system
JOL1898 E. coli BL21(DE3) pLysS expressing E. coli This study
hemolysine gene via pET28a (+) system
Plasmids
pET28(+) IPTG-inducible, T7 expression vector, C- Novagen, USA
terminal 6 x His tag, KanR
pET28(+)-LhlyA pET28a derivative containing LhlyA This study
pET28(+)-EHly pET28a derivative containing Ehly This study
58
Veterinary Microbiology 205 (2017) 57–61
purification procedure with a Ni2+-nitrilotriacetic acid-agarose support
(Qiagen, GmbH. Hilden, Germany) according to the procedure de-
scribed previously (Xie et al., 2010). Proteins were quantified using the
Bradford protein assay (Kruger, 1994). E. coli cells harboring the
expression vector pET28a+ were grown in Luria broth (LB) containing
kanamycin (50 μg/ml) at 37 °C.
2.3. Western blot analysis
Serum was collected from mice orally injected with a vaccine strain
of Lawsonia intracellularis (Enterisol® Ileiti, Boehringer Ingelheim). Mice
were inoculated with 5 × 106.9 TCID50 (tissue culture infectious doses)
of the vaccine strain twice at two-week intervals to obtain immune sera
against LI. Serum from a mouse inoculated with sterile PBS was used as
the negative control. Purified LhlyA protein sample (100 μg total
protein) was boiled in Laemmli SDS sample buffer for 10 min and
separated on 15% SDS-PAGE gel and then transferred onto PVDF
membrane. The membrane was blocked in 3% BSA and then incubated
with the anti-LI mouse hyper immune serum at a 1:300 dilution for 2 h.
After thoroughly washing the membrane with PBS- 0.1% Tween 20, the
membrane was incubated with HRP conjugated anti-mouse IgG
(Southern Biotechnology) at a 1:5000 dilution for 1 h and then
developed by addition of Peroxidase Western Blotting Substrate
(Sigma Co., St. Louis, Mo.).
2.4. In vivo cytotoxicity assay in a mouse model
Specific pathogen-free BALB/c inbred mice experimental proce-
dures were approved (CBNU2015-00085) by the Chonbuk National
University Animal Ethics Committee in accordance with the guidelines
of the Korean Council on Animal Care and Korean Animal Protection
Law, 2007; Article 13 (Experiments with Animals). The in vivo
cytotoxicity of the putative antigenic protein, LhlyA, was evaluated
by examining changes in spleen morphology in mice injected with the
purified protein (Maamar et al., 2016). Five-week-old female BALB/C
mice (n = 30) were divided into two groups (n = 15). Group A mice
were inoculated with 100 mg/ml of LhlyA protein via an intraperitoneal
(i.p.) route at week 0. Mice in group B were also inoculated with sterile
PBS via i.p. route at week 0. Three mice in each group were aseptically
sacrificed weekly up to week 4, and spleen samples were collected,
weighed, and sized individually.
2.5. RBC lysis assay
Whole blood samples were collected from mice, chicken, and pig in
a vacutainer tube with EDTA. Subsequently, RBCs were isolated from
the whole blood and washed twice following a procedure described
previously (Wang et al., 2007). A 1% suspension of the washed RBCs
sampled from each animal was prepared by adding 100 μl of the
washed RBCs to 900 μl sterile saline. Purified proteins, LhlyA and Ehly
(500 ng per well), were mixed with each type of animal RBC prepara-
tion (1%), briefly centrifuged to establish physical contact with the
RBCs, and then incubated for 24 h at room temperature. At the end of
the incubation, cell suspensions were centrifuged to remove unlysed
cells, and the absorbance of the supernatant containing released
hemoglobin was measured at 540 nm in a spectrophotometer.
3. Results
3.1. Characterization of LhlyA as a putative antigen
Based on the genome sequence of Lawsonia intracellularis, LI0004
was identified as being homologous to the hemolysins of Bilophila
wadsworthia and Desulfovibrio piger, and designated as a ‘TlyA-like
protein’. This protein showed greater than 60% similarity to other
hemolysin proteins (Fig. 1) (64% identity to the hemolysin protein of
J. Kim et al. Veterinary Microbiology 205 (2017) 57–61

Fig. 1. Comparison of the deduced amino acid sequences of putative hemolysins, namely LhlyA of Lawsonia intracelluralis (LI) (LI0004) and the TlyA-like proteins of Bilophila wadsworthia
and Desulfovibrio piger. LhlyA showed 55% homology to B. wadsworthia (64% identity) and D. piger (61% identity). Identical residues in all sequences are highlighted in black.

Fig. 2. Computational approach to predict conserved B-cell epitopes in the LhlyA protein sequence. (A) BepiPred linear epitope prediction of the LhlyA protein. The cut-off value (=0.35)
was automatically determined by the BepiPred program. Regions above the threshold shown in light grey are antigenic. (B) Three different linear B-cell epitope predictors (BCPred, AAP,
and FBCPred) were applied via http://ailab.ist.psu.edu/bcpred/predict.html. Areas highlighted in yellow are epitope residues predicted by all three methods.

59
J. Kim et al.
Fig. 3. Western blot analysis of purified LhlyA. The protein was strongly recognized by
immune serum obtained from a mouse infected with L. intracelluralis. The arrow in lane A
indicates a ∼34 kDa band, which is the predicted size of LhlyA fused with 6xHis. Serum
from an uninfected mouse was used as a negative control (lane B). Lane M, size marker.
Bilophila wadsworthia (Sequence ID: WP_051643848.1) and 61% with
that of Desulfovibrio piger (Sequence ID: WP_006006665.1)). Further,
FtsJ-like ribosomal RNA (cytidine-2′-O) methyltransferase (amino acids
67-250; pfam01728) and S4 RNA binding proteins (amino acids 6-43;
smart00363) also showed homology to LI0004. Sequence features of the
multiple sequence alignment of LhlyA to the hemolysins of Bilophila
wadsworthia and Desulfovibrio piger are shown in Fig. 1. These three
proteins had 55% homology overall (140 residues of the 251 amino
acids of LI0004 were identical to those of the hemolysins of B.
wadsworthia and D. piger). Potential antigenicity of LhlyA was evaluated
by B-cell linear epitope prediction using the BepiPred 1.0 program with
an automatically determined cut-off of 0.35 (Fig. 2A) and three
different prediction tools (Fig. 2B). LhlyA was also shown to be
intensively immunoreactive after blotting the poly histidine-tagged
recombinant protein with serum collected from Lawsonia intracellu-
laris-infected mice (Fig. 3, lane A). No distinctive band was detected
when serum collected from an uninfected mouse was used as the
primary antibody (Fig. 3, lane B).
3.2. In vivo cytotoxicity assay
The in vivo cytotoxic effect of LhlyA was assessed in BALB/c mice
after intraperitoneal injection of the purified protein at week 0. Isolated
spleens were individually photographed, weighed, and sized at one-
week intervals during the observational period. The weight of the
spleen in treated mice ranged between 70.1 and 88.6 mg, while that in
untreated mice ranged between 62.3 and 96.2 mg. No significant
changes in weight were observed during the observational period
Fig. 4. In vivo assay to assess LhlyA cytotoxicity. Mice were injected with 100 mg of LhlyA protein or sterile PBS as a negative control (NC). Spleens isolated from mice at one-week
intervals (3 mice per week) were photographed (A), weighed (B), and sized (C). No significant change in morphology, weight, or size was observed between the treated and control mice.
NC, negative control. Error bars indicate standard deviation (s.d.).
60
Veterinary Microbiology 205 (2017) 57–61
Fig. 5. RBC lysis assay. Following co-incubation with purified Lawsonia hemolysin A
(LhlyA) or E. coli hemolysin (Ehly) protein, absorbance values of supernatants containing
released hemoglobin of lysed red blood cells of pig, chicken, and mice were measured.
Error bars represent standard deviations.
(Fig. 4). Spleen size was also not markedly increased in mice inoculated
with the protein compared to control mice. Spleens of treated mice
appeared normal morphologically, with clear red pulp. These results
indicate that the LhlyA protein did not induce notable splenomegaly,
indicating that significant cytotoxicity did not occur following intra-
peritoneal inoculation of the protein.
3.3. Hemolytic activity analysis
To examine the hemolytic activity of LhlyA, purified proteins were
incubated with 1% mouse, chicken, and pig RBC preparations, respec-
tively, for 24 h. The magnitude of hemoglobin release was used to
determine the erythrocyte membrane damaging properties of the
proteins. No significant difference was observed in the OD values
among the groups (Fig. 5). Lhlya had similar hemolytic activity to the E.
coli α-hemolysin Ehly against RBCs.
4. Discussion
Studies on virulence factors of L. intracelluralis which is a micro-
aerophilic obligate intracellular bacterium, have been limited by
difficulties in culturing this pathogen in vitro; this pathogen can only
be cultured in specific cell lines such as the rat intestinal cell line IEC-18
or the human fetal line INT-407 (Vannucci and Gebhart, 2014). Thus,
conventional laboratory approaches has been restricted due to this
inability to be cultured readily, which has resulted in a struggle to
control PE in the livestock industry (Chang et al., 1997; Holyoake et al.,
2010; Marsteller, 2003). Currently, an avirulent live Lawsonia intracel-
lularis vaccine (Enterisol® Ileitis, Boehringer Ingelheim) has been
licensed and is commercially available. This vaccine strain was derived
from clinical isolates and shows no difference in phenotypic or
genotypic characteristics to wild-type LI (Meeusen et al., 2007), making
it highly reactogenic. Thus, development of a safe and effective vaccine
against PE, such as a molecularly defined subunit vaccine, is urgently
needed to prevent PE. In the present study, we characterized the
J. Kim et al.
antigenic features of LhlyA, a putative hemolysin protein, to assess its
potential to function as an immune-reactive antigen. LI0005 showed
64% and 61% homology to the TlyA-like proteins of Bilophila wads-
worthia and Desulfovibrio piger, respectively. Furthermore, of the 251
amino acids of LhlyA, 140 (55%) residues were identical to those in the
two TlyA-like proteins (Fig. 1). Another LI protein, LsaA, was presumed
to be a putative TlyA-like protein in a previous study (McCluskey et al.,
2002). Although the role of LsaA in pathogenesis has been investigated
in several studies (McAllister, 2005; McCluskey et al., 2002), its
virulence properties have not been determined. We therefore focused
our analysis on assessing the suitability of LhlyA as an antigenic target.
Identification of B-cell linear epitopes currently plays a pivotal role
in vaccine design and disease diagnostic tests. Several in silico bioinfor-
matics tools have been developed to identify B-cell linear epitopes
based on the physiochemical properties of a protein inferred from its
amino acid sequence (Chen et al., 2007; Larsen et al., 2006; Wang et al.,
2011). We used four different computational methods to predict B-cell
linear epitopes of LhlyA (Fig. 2). However, it might not be appropriate
to extrapolate the epitope scores of LhlyA to conformational B cell
epitopes, as the prediction analyses applied in this study were
determined based on primary amino acid sequence, not the folded
protein structure (Dalkas and Rooman, 2017). To evaluate the im-
munogenicity of computationally predicted epitopes of LhlyA, western
blot analysis was performed using immune serum raised against L.
intracellularis. A distinct, immuno-reactive band with the predicted size
of the LhlyA protein was detected by immunoblotting (Fig. 3), indicat-
ing that the B-cell conformational epitopes of the protein are recognized
by antibodies produced against L. intracellularis. This suggests that
LhlyA is highly antigenic.
Hemolysins, also referred to as cytolysins, are bacterial toxins that
are considered virulence factors (Braun and Focareta, 1991). To assess
the in vivo safety profile of the putative antigenic protein, purified
LhlyA was inoculated into mice via an intraperitoneal route. Spleno-
megaly was not observed in the spleens of treated mice at one-week
intervals over the observational period (Fig. 4), implying that signifi-
cant inflammation (Maamar et al., 2016) or acute hemolysis (Tolosano
et al., 2002) did not occur following i.p injection. The in vitro hemolytic
activity of the protein was found to be similar to that of E. coli
hemolysin A (Schmidt et al., 1995) in an in vitro erythrocyte lysis assay
(Fig. 5). Although α-hemolysins are virulence factors in common
bacterial infections, the intrinsic characteristics of α-hemolysins are
not yet fully understood. Hemolysin-mediated hemolysis is associated
with vital functions such as the acquirement of elemental free iron from
hemoglobin (Belanger et al., 1995) and the production of hydrogen
peroxide (Danley et al., 1983). Further studies are needed to determine
the in vivo role of LhlyA in the infected host.
In summary, we found that LI0004 of Lawsonia is a putative
hemolysin protein that induces the production of specific antibodies
in vivo. This prominent antigen might facilitate the production of
recombinant vaccines to alleviate PE symptoms in affected hosts or
confer protection against PE. Further studies need to be undertaken to
characterize LhlyA and elucidate its role in PE.
Acknowledgement
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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