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ORIGINAL RESEARCH ARTICLE
published: 04 September 2014
doi: 10.3389/fcimb.2014.00120
Enhanced expression of codon optimized Mycobacterium
avium subsp. paratuberculosis antigens in Lactobacillus
salivarius
Christopher D. Johnston1, John P. Bannantine2, Rodney Govender1, Lorraine Endersen1,
Daniel Pletzer 3, Helge Weingart 3, Aidan Coffey1, Jim O’Mahony 1*and Roy D. Sleator1
1Biological Sciences Department, Cork Institute of Technology, Cork, Ireland
2United States Department of Agriculture - Agricultural Research Service, National Animal Disease Center, Ames, IA, USA
3School of Engineering and Science, Jacobs University Bremen, Bremen, Germany
Edited by:
Adel M. Talaat, University of
Wisconsin–Madison, USA
Reviewed by:
Martin S. Pavelka, University of
Rochester, USA
Jan Peter Van Pijkeren, University of
Wisconsin–Madison, USA
*Correspondence:
Jim O’Mahony, Department of
Biological Sciences, Cork Institute of
Technology, Rossa Avenue,
Bishopstown, Cork, Ireland
e-mail: jim.omahony@cit.ie
It is well documented that open reading frames containing high GC content show poor
expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in
heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins
using Lactobacillus salivarius. However, re-engineering opening reading frames through
synonymous substitutions can offset codon bias and greatly enhance MAP protein
production in this host. In this report, we demonstrate that codon-usage manipulation
of MAP2121c can enhance the heterologous expression of the major membrane protein
(MMP), analogous to the form in which it is produced natively by MAP bacilli. When
heterologously over-expressed, antigenic determinants were preserved in synthetic MMP
proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a
membrane protein in MAP, which is also targeted to the cellular surface of recombinant
L. salivarius at levels comparable to MAP. Additionally, we previously engineered
MAP3733c (encoding MptD) and show herein that MptD displays the tendency to
associate with the cytoplasmic membrane boundary under confocal microscopy and the
intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage
fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen
delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne’s
disease.
Keywords: MAP antigens, MptD, MMP, codon optimization, expression host, paratuberculosis, MAP vaccine,
Johne’s disease
INTRODUCTION
Mycobacterium avium subsp. paratuberculosis (MAP) is an
intracellular pathogen and the etiological agent of Johne’s dis-
ease, a chronic inflammatory disorder of the gastrointestinal tract
which affects multiple ruminant species including cattle (Chacon
et al., 2004). Live attenuated vaccine formulations against Johne’s
disease do not appear to offer adequate protection against MAP
infection in goats (Hines et al., 2014) and while heat-killed whole
cell vaccines that are commercially available do show some effi-
cacy, these also fail to provide full protection against MAP in
models of infection (Rosseels and Huygen, 2008). Moreover,
issues relating to interference with diagnostic assays for bovine
tuberculosis have further hindered their widespread development
and application (Buddle et al., 2010; Bastida and Juste, 2011;
Coad et al., 2013). These limitations combined with the avail-
ability of complete genome sequences for MAP (Li et al., 2005;
Bannantine et al., 2014) have focused recent attention on experi-
mental subunit based vaccine strategies against MAP (Bull et al.,
2007; Rosseels and Huygen, 2008). One particularly promising
subunit vaccine for MAP is the 70 kDa heat shock protein termed
Hsp70 (Koets et al., 2006), which activates B cells (Vrie lin g et al.,
2013).
Lactobacillus are interesting candidates for the development of
novel oral vaccine vectors due to certain strains widespread use
in the food industry and GRAS (generally regarded as safe) status
(Yu et al., 2013). Specific members of this genus are also an attrac-
tive alternative to using attenuated pathogens for mucosal deliv-
ery strategies because they can survive the upper gastrointestinal
tract (GI) and colonize the lower GI tract (Bermudez-Humaran
et al., 2011). Numerous studies have substantiated the potential
of specific Lactobacilli strains to serve as live vaccine delivery vehi-
cles against a broad spectrum of mucosal pathogens including
Bacillus anthracis,Streptococcus pneumonia, Clostridium difficile
and the Avian Influenza Virus H5N1 (Campos et al., 2008;
Sleator and Hill, 2008; Mohamadzadeh et al., 2010; Wang et al.,
2012). MAP is particularly well-suited to Lactobacillus vector
delivery because it is a pathogen transmitted by ingestion of
contaminated feces or milk and passes through the GI tract
where it infects the intestinal mucosa (Bannantine and Bermudez,
2013).
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CELLULA R AND INFECTION MICROBIOLOG
Y
Johnston et al. Enhanced M. paratuberulosis antigen expression in L. salivarius
However, despite the obvious advantages of Lactobacillus based
mucosal immunization; encompassing the inherent ability of
specific strains to survive gastric transit, adhere to the intesti-
nal epithelium (Messaoudi et al., 2012), and stimulate both
mucosal and systemic immune responses (Mohamadzadeh et al.,
2009), there are known difficulties in expression of G+Crich
coding sequences in the A+TrichLactobacillus host (Johnston
et al., 2013). Indeed, we recently determined that the significantly
divergent genomic G+C content of MAP and Lactobacillus sali-
varius (69 and 33% respectively) leads to a disparity in codon
usage, which significantly impedes recombinant MAP protein
synthesis. To alleviate this translation inefficiency likely due to
ribosomal pausing at rare codons (Buchan and Stansfield, 2007),
codon optimization of a MAP gene (MAP3733c) was performed
by introducing a series of synonymous mutations; modifying
the coding region to better suit the codon bias of L. salivar-
ius. It was shown that synthesis of a MAP specific membrane
protein within L. salivarius could be markedly enhanced (>37-
fold) owing to codon optimization, resulting in the abundance
of MAP-GFP protein fusion fluorescence in recombinant cells
(Johnston et al., 2013). However, that protein was never con-
firmed to truly represent the native protein as no monoclonal
antibodies or other specific detection reagents were developed
against it.
Because MAP surface proteins likely play the dominant role
in the initial interactions with bovine intestinal cells (Bannantine
et al., 2003; He and De Buck, 2010; Gurung et al., 2012), we
here focused on two important MAP membrane proteins, MMP
(MAP2121c) and the previously studied MptD (MAP3733c).
MMP, encoded by MAP2121c, is a ∼33.5 kDa surface protein
which shares homology to a Mycobacterium leprae 35-kDa major
membrane protein-1 (MMP-1) (Winter et al., 1995), previ-
ously identified as a potent immunodominant antigen capable
of inducing T-lymphocyte responses in paucibacillary leprosy
patients (Triccas et al., 1996). MptD is a MAP specific, virulence
associated membrane protein which is surface expressed during
natural infection, warranting its further investigation as a pro-
phylactic antigen (Stratmann et al., 2004; Shin et al., 2006; Cossu
et al., 2011).
A significant limitation of current whole cell MAP vaccine
strategies is interference with cellular immune assays and tuber-
culin skin tests for bovine tuberculosis (M. bovis), which restricts
their widespread application in many countries (Kohler e t al .,
2001; Scandurra et al., 2010). It is notable that although MMP
is not specific to MAP, with homologs existing in other mycobac-
terial species including M. avium (MAC) and M. leprae, previous
genetic and serological evidence suggests that the protein is absent
from M. tuberculosis and M. bovis (Triccas et al., 1998; Banasure
et al., 2001). In addition, a DNA vaccine incorporating the
M. leprae MMP-1 protein in isolation demonstrated significant
levels of protective efficacy against M. leprae footpad infection in
outbred Swiss Albino mice (Martin et al., 2001). As such, if an
MMP-based vaccine against Johne’s disease were to display strong
prophylactic efficacy, as it has done against Leprosy, it leads to
the exciting prospect of a vaccine formulation that could over-
come issues with interference of bovine tuberculosis screening
assays.
Herein, we analyzed MAP proteins recoded using synony-
mous substitutions and expressed in L. salivarius. Faithful
expression and antigenicity was examined using a combina-
tion of fluorescence microscopy, monoclonal antibody- and
bacteriophage-based ELISA.
MATERIALS AND METHODS
STRAINS, PLASMIDS, BACTERIOPHAGE, AND GROWTH CONDITIONS
The strains and plasmids used in this study are listed in
Table 1.Mycobacterium avium subsp. paratuberculosis strain K-
10 (ATCC BAA968) were propagated in Middlebrook 7H9
broth supplemented with OADC (BD Biosciences), 2μg/ml
of Mycobactin J and 0.2% glycerol and incubated at 37◦C
for 6–10 weeks. Escherichia coli DH5αelectrocompetent cells
(Invitrogen) were used as intermediate cloning hosts for all
vector constructs within this study and grown at 37◦Cin
Luria-Bertani (LB) media. Lactobacillus salivarius NRRL B-
30514 (kindly donated by Dr. Norman Stern, USDA), was used
throughout this study as the host for individual vector con-
structs and was grown aerobically at 37◦CinMRSmedia
(Fluka).
Plasmids pNZ9530 and pNZ8048 were originally obtained
from the University College Cork culture collection, while
pUC57 vectors containing codon-optimized synthetic GFP and
MptDsynth genes were obtained from the Cork Institute of
Technology culture collection (Johnston et al., 2013). Cultures
of E. coli harboring individual vectors were supplemented
with Erythromycin (Ery, 200 μg/ml), Chloramphenicol (Cm,
12.5 μg/ml), or Ampicillin (Amp, 200 μg/ml) for pNZ9530,
pNZ8048, and pUC57 containing cells respectively. Recombinant
L. salivarius cells were subcultured from 40% v/v glycerol
stocks at −20◦C and grown using antibiotic selection. L. sali-
varius (pNZ9530) cultures were supplemented with 5 μg/ml
Ery, while L. salivarius harboring both pNZ9530 and pNZ8048
constructsweregrowninthepresenceof5μg/ml Ery and
10 μg/ml Cm.
The M13 phage fMptD, originally isolated from the Ph.D.−12
phage display library (New England Biolabs), was obtained from
the laboratory of Gerald F. Gerlach (University of Veterinary
Medicine, Hanover, Germany). Bacteriophage fMptD was prop-
agated using standard methods previously described (Chappel
et al., 1998). Phage titers were assessed by a standard plaque assay
test (Sambrook et al., 1990). Purified high titer phage solutions
(1010 pfu/ml) were stored at 4◦C until required.
NUCLEIC ACID ISOLATION
Mycobacterial DNA was prepared as described previously by
Douarre et al. (2010). Plasmid DNA was isolated from E. coli
DH5αusing a plasmid extraction kit as per the manufac-
turer’s instructions (Sigma Aldrich). Plasmid isolation from
Lactobacillus strains was also carried out using this kit, after ini-
tial incubation (30 min at 37◦C) in protoplast buffer (20 mM
Tris-HCl, 5 mM EDTA, 0.75 M Sucrose, 10 mg/ml lyzozyme
and 50 U/ml mutanolysin pH 7.5). Quantitative analysis was
carried out using microspectrophotometry (Nanodrop, De,
USA) and plasmid DNA concentration was normalized to
250 ng/μl.
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Johnston et al. Enhanced M. paratuberulosis antigen expression in L. salivarius
Table 1 | Strains, Phage, and Plasmids used in this study.
Plasmid, phage, and strains Relevant genotype or characteristics Source or References
PLASMIDS
pNZ9530 N.I.C.E system helper plasmid. Eryr,nisRK cloned in pIL252, expression
of nisRK driven by rep read through, low copy number
Pavan et al., 2000
pNZ8048 N.I.C.E system expression plasmid. Cmr, carries the nisin-inducible
promoter PnisA
Pavan et al., 2000
pNZ:MAP3733c pNZ8048 with MptD fused to GFP gene under control of PnisA promoter This work
pNZ:MAP3733synth pNZ8048 with codon optimized MptDsynth gene under control of PnisA
promoter
This work
pNZ:MAP3733c-GFP pNZ8048 with MptD fused to GFP gene under control of PnisA promoter Johnston et al., 2013
pNZ:MAP3733synth-GFP pNZ8048 with codon optimized MptDsynth fused to GFP gene under
control of PnisA promoter
Johnston et al., 2013
pNZ:MAP2121c pNZ8048 with MMP fused to GFP gene under control of PnisA promoter This work
pNZ:MAP2121synth pNZ8048 with codon optimized MMPsynth gene under control of PnisA
promoter
This work
pNZ:MAP2121c-GFP pNZ8048 with MMP fused to GFP gene under control of PnisA promoter This work
pNZ:MAP2121synth-GFP pNZ8048 with codon optimized MMPsynth fused to GFP gene under
control of PnisA promoter
This work
pNZ:GFP pNZ8048 with L. salivarius codon optimized GFP gene under control of
PnisA promoter
Johnston et al., 2013
PHAGE
Phage fMptD Phage isolated from the Ph.D.-12 phage display library, with a
dodecapeptide sequence (GKNHHHQHHRPQ) fused to the N-terminus
of its minor coat protein (pIII)
Stratmann et al., 2004
STRAINS
Mycobacterium avium subsp. paratuberculosis
MAP K-10 American bovine virulent isolate and sequencing project reference strain Li et al., 2005
Escherichia coli
DH5αIntermediate cloning host Invitrogen
Lactobacillus salivarius
NRRL B-30514 Host strain, originally isolated from cecal contents of broiler chicken.
Aerobic
Stern et al., 2006
L. salivarius pNZ9530 Host strain harboring pNZ9530 helper plasmid, EryrJohnston et al., 2013
L. salivarius pNZ8048 Harbors pNZ9530 plasmid and pNZ8048 expression plasmid lacking
insert. Eryrand Cmr
Johnston et al., 2013
L. salivarius MAP3733c Harbors pNZ9530 and pNZ:MAP3733c plasmids. Eryrand CmrThis work
L. salivarius MAP3733synth Harbors pNZ9530 and pNZ:MAP3733synth plasmids. Eryrand CmrThis work
L. salivarius MAP3733c-GFP Harbors pNZ9530 and pNZ:MAP3733c-GFP plasmids. Eryrand CmrJohnston et al., 2013
L. salivarius MAP3733synth-GFP Harbors pNZ9530 and pNZ:MAP3733synth-GFP plasmids. Eryrand CmrJohnston et al., 2013
L. salivarius MAP2121c Harbors pNZ9530 and pNZ:MAP2121c plasmids. Eryrand CmrThis work
L. salivarius MAP2121synth Harbors pNZ9530 and pNZ:MAP2121synth plasmids. Eryrand CmrThis work
L. salivarius MAP2121c-GFP Harbors pNZ9530 and pNZ:MAP2121c-GFP plasmids. Eryrand CmrThis work
L. salivarius MAP2121synth-GFP Harbors pNZ9530 and pNZ:MAP2121synth-GFP plasmids. Eryrand CmrThis work
L. salivarius GFP Harbors pNZ9530 and pNZ:GFP plasmids. Eryrand CmrJohnston et al., 2013
CODON OPTIMIZATION OF MAP2121c
MAP2121c encodes a major membrane protein (MMP) in MAP.
Because this protein has been implicated in the early events
of infection in the bovine intestinal musoca, it is an ideal
candidate for testing expression in L. salivarius as a poten-
tial mucosal vaccine. We have previously described a strategy
for the codon optimization of the MAP3733c gene for expres-
sion in L. salivarius (Johnston et al., 2013). Here we used the
same strategy for MAP2121c; briefly, a bioinformatics analysis
was performed to identify codons from MAP2121c that could
be modified at the third base position without a change in
the resulting amino acid (termed a synonymous substitution).
Coding sequences were synthesized by GenScript USA Inc.
(Piscataway, NJ). Constructs were cloned as described below and
confirmed by DNA sequencing. Final sequences for each gene
are available from GenBank (Accession numbers: KC854397 and
KC517484). All modifications to MAP2121c are summarized in
Table 2.
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Johnston et al. Enhanced M. paratuberulosis antigen expression in L. salivarius
Table 2 | Modification and codon optimization of MAP genes.
Gene Length G+C Unfavorable Modified Source
(bp) content codons*codons
MAP2121c 924 66.5% 276/307 0/307 This work
MAP2121synth 924 32.8% 7/307 279/307 This work
*Codons were deemed unfavorable due to the presence of a guanine or cytosine
within the 3rd base of the triplet.
PCR AMPLIFICATIONS AND MODIFICATIONS
PCR primers are listed in Supplementary Table S1. Primers
were designed for the native and codon optimized MAP genes
(MAP2121c, MAP3733c, MAP2121synth, and MAP3733synth)
based on either MAP strain K-10 sequence data available from
the NCBI database (NC_002944) or using the sequence from
GenScript synthesized genes. All conventional PCR reactions
were carried out using high fidelity Velocity DNA polymerase
Kit (Bioline) in accordance with the manufacturer’s instructions.
Restriction enzymes and T4 DNA ligase were purchased from
Roche Diagnostics (Mannheim, Germany) and New England
Biolabs (Beverly, MA, USA) and used as per manufacturer’s rec-
ommendations. Ligation reaction mixtures were purified using
the High Pure PCR product purification kit (Roche).
MAP GENE AND FUSION CONSTRUCTS
Individual MAP gene constructs (native and synthetic) were
cloned into the E. coli-L. lactis shuttle vector pNZ8048, a deriva-
tive of pNZ124 that allows expression of proteins under the
control of the nisin-inducible promoter PnisA,partofthe
NIsin Controlled Expression (NICE) system (Pavan et al., 2000).
Nisin induced expression was achieved in L. salivarius via co-
transformation of a dual plasmid system; pNZ8048 with insert
to be expressed downstream of PnisA and pNZ9530 providing the
necessary nisRK regulatory genes in trans (de Ruyter et al., 1996).
Two native MAP genes, MAP2121c and MAP3733c (des-
ignated “c” to indicate that each is originally transcribed on
the complimentary strand of the MAP K-10 genome) and two
synthetic codon optimized counterparts of these, designated
MAP2121synth and MAP3733synth, were initially cloned form-
ing pNZ:2121c, pNZ:3733c, pNZ:2121synth, and pNZ:3733synth.
To facilitate fluorometric analysis of each of these genes during
expression and monitor subcellular localization of proteins within
the host cell, a C-terminus GFP gene was translationally fused
to each gene construct forming pNZ:2121c-GFP, pNZ:3733c-
GFP, pNZ:2121synth-GFP, and pNZ:3733synth-GFP. The fusion
of GFP and individual MAP genes was performed using Splicing
by Overlap Extension (SOEing) as previously described (Johnston
et al., 2013).
The GFP coding region used for fusions throughout this
study was codon optimized for use within L. salivarius. The GFP
gene sequence cloned into pNZ8048 (Johnston et al., 2013)was
used to provide a comparative control for subsequent assays and
designated pNZ:GFP.
TRANSFORMATION AND INDUCTION OF L. SALIVARIUS CONSTRUCTS
Competent L. salivarius (pNZ9530) were transformed
individually with each of the MAP gene constructs (Ta b l e 1 )as
described previously (Johnston et al., 2013). Overnight cultures
of recombinant L. salivarius strains were subcultured (1:100
dilution) into fresh MRS broth (Cm 8 μg/ml, Ery 3.5 μg/ml) and
incubated with agitation (100 rpm) at 37◦C. At an optical density
(OD600nm) of 0.35, nisin was added at a final concentration
of 10 ng/ml and cultures were incubated statically at 37◦C
for 2 h. 10 ml aliquots of each culture were then harvested by
centrifugation (6000 rpm for 5min) for subsequent analysis.
FLUORESCENCE MICROSCOPY
To facilitate visualization of cells using fluorescence microscopy,
induced L. salivarius strains (L.sal GFP, L.sal MAP2121c-
GFP, L.sal MAP2121synth-GFP, L.sal MAP3733c-GFP, L.sal
MAP3733synth-GFP) were fixed using 3.7% formaldehyde,
washed with PBS (pH 7.2), subsequently resuspended in 1 ml
PBS and stored at 4◦C until visualized. For detection of GFP
fusion peptides, L. salivarius cell images were taken using a Zeiss
LSM 510 META laser-scanning microscope equipped with Argon
and Helium-Neon lasers (Carl Zeiss, Oberkochen, Germany) at
a resolution of 2048 ×2048 pixels, using LSM 5 software (ver-
sion 3.2; Carl Zeiss). Equal settings were used for detection
of green fluorescence among different strains (Amplifier Offset:
0.05, Amplifier Gain: 1, Gain: 820).
PREPARATION OF WHOLE CELL L. SALIVARIUS FOR ELISA
To determine if recombinant MMP and MMPsynth peptides
were displayed on the surface of whole cell L. salivarius, nisin-
induced strains were harvested, washed and resuspended in PBS
and coated directly to the wells of a Nunc Maxisorp plate (1 ×
109cells/ml). To determine if these peptides were accumulating
within the cytoplasm of L. salivarius, 500 μl of the harvested cells
were transferred to 2 ml screw cap tubes with 0.3 g glass beads
(Sigma, 150–212 μm, acid washed) and lysed (4000 rpm for 45 s)
using a MagNA Lyser Instrument (Roche). Subsequently, 100 μl
aliquots of crude cell lysate were added to individual wells of the
same Maxisorp plate.
In the analysis of MptD proteins, similar techniques were
applied for whole-cell and cell-lysate preparations, however,
use of the alternative bacteriophage (fMptD) detection method
necessitated the substitution of PBS for TBS in all washing and
subsequent steps. MAP strain K-10, processed in the same man-
ner, was included in all assays to provide a comparative control.
MONOCLONAL ANTIBODY BASED ELISA OF MMP
Maxisorp plates containing recombinant L. salivarius (pNZ,
MMP, MMPsynth, MMP-GFP, and MMPsynth-GFP), as well
as MAP K-10, whole cells and cell lysate were incubated at
37◦C for 1 h and then blocked with a 5% (w/v) solution of
dry skimmed milk powder in PBS. A 100-μl aliquot of puri-
fied monoclonal antibody 13E1 or 8G2, appropriately diluted
in PBS plus 0.1% tween 20 (PBS/T) was added to each test
well. Samples were incubated at 37◦C for 1 h on a rocking plat-
form. The wells were washed and 100 μl of secondary antibody
(Peroxidase-labeled, Anti-Mouse IgG detection antibody) diluted
in PBS/T containing 1% (w/v) milk powder was added. After a 1 h
incubation at 37◦C the wells were washed and 100 μlof3,3
,5,5-
Tetramethylbenzidine Liquid Substrate System for ELISA (Sigma)
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Johnston et al. Enhanced M. paratuberulosis antigen expression in L. salivarius
was added per well. The substrate was left to develop in the dark
at room temperature for 30 min after which the reaction was
stopped by addition of 50 μl of a 10% HCl solution. Absorbance
readings were read at 450 nm using a microplate reader. The bind-
ing response values against each respective recombinant cell type
were normalized by dividing the absorbance level obtained in
test wells by that obtained in parallel control wells treated with
diluent buffer without the addition of the primary monoclonal
antibody.
BACTERIOPHAGE-fMptD BASED ELISA OF MptD
Due to a lack of monoclonal antibodies available for the MptD
protein, the fMptD bacteriophage (Stratmann et al., 2006)was
used in lieu of the primary binding antibody within MptD
ELISA. Maxisorp plates containing recombinant L. salivarius
(pNZ, MptD, MptDsynth, MptD –GFP, and MptDsynth-GFP),
as well as MAP K-10, whole cells and cell lysate were incubated
at 37◦C for 1 h and then blocked with a 5% (w/v) solution
of dry skimmed milk powder in TBS. Plates were washed with
TBS/T (TBS +0.1% [v/v] Tween-20) and 100 μloffMptD
(109pfu/ml) diluted in TBS/T was added to each test well after
which samples were incubated at 37◦C for 1 h on a rocking
platform. Individual wells were subsequently washed and 100 μl
of the detection antibody, HRP Conjugated anti-M13 mon-
oclonal antibody (GE Healthcare), diluted in TBS/T contain-
ing 1% (w/v) milk powder was added. After 1 h incubation at
37◦C the wells were washed, 100 μl of TMB Liquid Substrate
(Sigma) was added per well and color developed as already
described.
STATISTICAL ANALYSIS
Statistical analysis was carried out using GraphPad Prism (ver-
sion 4.03; GraphPad Software, San Diego, CA). Means with
standard error (s.e.m.) are presented in each graph. Differences
between two groups were calculated using unpaired Student’s
t-test. Differences were considered significant at P<0.05.
RESULTS
DISTINCT LOCALIZATION OF MAP-GFP FUSION PEPTIDES
In our previous work we confirmed poor fluorescence for the
native MptD-GFP fusion protein expressed in L. salivarius,and
showed markedly improved expression through codon opti-
mization of the synthetic gene (MptDsynth-GFP; Figures 1E,F)
(Johnston et al., 2013). Here, consistent with our previous find-
ings for MptD, induction and confocal microscopy of the native
MMP-GFP fusion resulted in no fluorescence (Figure 1C) simi-
lar to the L. salivarius wild type control (Figure 1A). In contrast,
improved levels of fluorescence were noted for codon optimized
MMPsynth-GFP after induction under identical conditions as the
native variant (Figure 1D).
While an even distribution of strong fluorescence was
observed within pNZ:GFP-containing bacilli (Figure 1B), both
engineered MAP fusion displayed different fluorescence pat-
terns when expressed within L. salivarius (Figures 1D,F).
MMPsynth-GFP was aggregated (Figure 1D) whereas MptD was
more uniformly distributed around the periphery of the cells
(Figure 1F).
FIGURE 1 | Representative phase contrast (upper panel) and
fluorescent microscopy (lower panel) images of recombinant
L. salivarius cells. (A) Control cells pNZ:8048. (B) L. salivarius GFP cells
displayed fluorescence observed throughout individual bacilli. (C) Negligible
levels of fusion fluorescence were observed from MMP-GFP cells,
(D) while fluorescent foci, suggestive of aggregation, were observed
toward the polar regions of individual cells for the codon optimized variant
MMPsynth-GFP. (E) Low levels of fluorescence prevented determination of
the subcellular localization of native MptD-GFP fusions within L. salivarius
cells, while (F) codon optimized MptDsynth-GFP fusion proteins
demonstrated the tendency to localize toward the cellular membrane
periphery. All culture assays were performed in triplicate, multiple images
were taken from each sample and representative pictures were chosen.
Bars represent 5 μm.
MptD-GFP (MAP3733c/synth)
In silico TMHMM analysis of MptD suggests the presence of six
transmembrane segments (TMSs) within the 208 amino acids
and a large external loop at positions 147–175, with both N and
C termini being cytoplasmic associated. In cases where trans-
membrane proteins have their native C-terminus located in the
cytoplasm, fusion of a GFP tag is particularly useful for anal-
ysis of protein localization. If the fusion is expressed at the
cytoplasmic membrane the GFP peptide may fold correctly and
fluoresce allowing visualization of protein localized at this mem-
brane, however if it aggregates and forms inclusion bodies the
downstream GFP might not fold correctly and therefore not flu-
oresce (Drew et al., 2005), although this will be largely protein
dependant.
The MptDsynth-GFP fusion peptides demonstrated a ten-
dency to localize at the periphery of individual cells (Figure 1F),
suggestive of membrane domain insertion and conformation.
This result extends our initial findings with MptD (Johnston et al.,
2013).
MMP-GFP (MAP2121c/synth)
Confocal microscopy of L. salivarius MMP-GFP resulted in
undetectable fluorescence from individual bacilli (Figure 1C),
further supporting our previous hypothesis that native MAP
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Johnston et al. Enhanced M. paratuberulosis antigen expression in L. salivarius
genes are poorly translated within L. salivarius.However,flu-
orescent foci were observed within the re-engineered sequence
(MMPsynth-GFP) suggesting the presence of aggregated GFP
fusion proteins within the cytoplasm of L. salivarius (Figure 1D).
This result was not expected since MMP was predicted to be more
soluble than MptD using SOLpro1.0 software (Magnan et al.,
2009).
RECOMBINANT MptD ACCUMULATES WITHIN THE CYTOPLASM OF
L. SALIVARIUS
To investigate whether the L. salivarius expressed MptD pro-
tein was analogous to the native MAP protein, we utilized a
modified version of the fMptD bacteriophage mediated ELISA
protocol outlined by Rosu et al. (2009), to probe both whole-
cellandcell-lysatesofrecombinantL. salivarius transformed with
both MptD-GFP gene constructs. Two constructs lacking the GFP
fusion were also included to control for potential interference
from the tag (Figures 2A,B).
Investigation of the crude cell lysates (Figure 2A)fromrecom-
binant L. salivarius revealed MptD phage binding to both native
and codon optimized MptD constructs lacking a GFP fusion
tag with a 2.2-fold increase in signal observed for the codon
optimized variant over native (P=0.0008), and 2-fold when
compared to MAP K-10 (P=0.0007). Interestingly, in a manner
similar to MMP analysis, the addition of a GFP fusion to each
of these constructs appears to have effectively inhibited phage
binding, possibly by masking the MptD epitope with GFP or
prevention of a conformational structure from forming.
Based on the results from confocal microscopy (Figure 1F),
we also examined intact cells (Figure 2B) to determine if the
tendency of MptD to localize toward the periphery of L. salivarius
represented true surface exposure. However, despite abundant
concentrations of MptD detected within the cytoplasm, no sig-
nal (P=<0.05 vs. control) could be detected from unbro-
ken cells expressing either native or re-engineered MptD in
the presence or absence of a GFP tag. MptD was detected
at similar levels in MAP regardless of the cell preparation
(Figures 2A,B).
RE-ENGINEERED MMP DISPLAYS TWO DISTINCT EPITOPES
The aggregation observed during confocal microscopy of
MMPsynth-GFP prompted further investigation of MMP fold-
ing within the L. salivarius host. To examine the possibility
that the C-terminus GFP tag was itself associating with the
MMP peptide, potentially leading to the fluorescent aggre-
gates observed (Figure 1D): two additional constructs, L. sali-
varius with native and codon optimized MMP genes lacking
a GFP fusion (pNZ:MAP2121c and pNZ:MAP2121synth), were
included in the assays.
Antigenic determinants or epitopes which are recognized on a
target protein by an antibody can exist in multiple forms rang-
ing from linear, present on both native or misfolded peptides, to
discontinuous or conformationally complex epitopes which are
displayed through the native folding of a protein (Brown et al.,
2011). In this study, ELISA analysis was performed using two
monoclonal antibodies specific to the MMP protein (8G2 and
13E1), which detected two distinct epitopes (Bannantine et al.,
2007).
ELISA analysis with mAb 8G2 (linear epitope)
The 8G2 antibody is reported to associate with a linear epitope
present within a 77-amino acid sequence near the N-terminus
FIGURE 2 | Comparative fMptD-mediated ELISA analysis of
recombinant L. salivarius cell lysate and whole-cell against MAP
K-10 (A,B). Standard deviation of triplicate results is indicated by error
bars. Statistically significant difference was observed at ∗P<0.05.
Horizontal dashed lines indicate the negative control threshold for each
assay.
Frontiers in Cellular and Infection Microbiology www.frontiersin.org September 2014 | Volume 4 | Article 120 |6
Johnston et al. Enhanced M. paratuberulosis antigen expression in L. salivarius
of MMP (Bannantine et al., 2007). ELISA analysis with 8G2 on
crude cell lysates of recombinant L. salivarius-MMP constructs
revealed that the linear epitope could be detected in all
MMP constructs (Figure 3A), with a significant 6.4-fold sig-
nal increase noted for the engineered MMPsynth compared to
native MMP.
While microscopy data indicate that heterologously expressed
MMP largely accumulates in the cytosol (Figure 1D), intrigu-
ingly, the engineered MMP was also detected during whole-cell
ELISA using the 8G2 antibody (Figure 3B). Although these levels
were low, the signal detected was approximate to that of the MAP
K-10 control strain (Figure 3B). Notably, as MMP expression is
enhanced in L. salivarius, more protein is localized on the surface
(Figure 3B).
ELISA analysis with mAb 13E1
Analysis of L. salivarius cellular lysates with 13E1 indicated that
the epitope recognized by this mAb is blocked when MMP
is fused to GFP fusions (Figure 3C). However, this epitope
appears to be restored by removal of the GFP tag, with 4-
fold higher levels of MMP detected from codon optimized
MMPsynth when compared to the native gene (P=0.0009),
and 1.5-fold when compared to the MAP K-10 control strain
(P=0.003).
However, most intriguingly is that MMP protein could also be
detected from whole cell L. salivarius MMPsynth using the 13E1
antibody, which was comparable to that in MAP K-10, albeit both
were present at low levels (Figure 3D). While it has previously
been indicated that MMP contains a 30 amino acid hydrophobic
FIGURE 3 | Comparative ELISA analysis of recombinant L. salivarius
cell-lysate and whole-cell against MAP K-10, using two monoclonal
antibodies directed against MMP. The 8G2 antibody detects MMP (A,B).
The mAb13E1 antibody detects a discontinuous epitope on MMP (C,D).The
average val ue of L. sal MMP-GFP and L. sal MMP are below the negative
control threshold. Standard deviation of triplicate results is indicated by error
bars. Statistically significant difference was observed at ∗P<0.05. Horizontal
dashed lines indicate the negative control threshold for each assay.
Frontiers in Cellular and Infection Microbiology www.frontiersin.org September 2014 | Volume 4 | Article 120 |7
Johnston et al. Enhanced M. paratuberulosis antigen expression in L. salivarius
domain near the C-terminal end suggestive of a membrane pro-
tein, the protein lacks a discernible N-terminal signal sequence
(Bannantine et al., 2003). Moreover, it has already been exper-
imentally demonstrated that MMP is both surface exposed and
actively shed from live MAP bacilli (Yakes et al., 2008), suggest-
ing that MMP is translocated in a non-classical manner similar
to other mycobacterial proteins (Pallen, 2002; Bendtsen et al.,
2005).
DISCUSSION
There have been several efforts at heterologous expression of
MAP proteins. E. coli expression of an impressive collection of
over 650 MAP proteins has been constructed (Bannantine et al.,
2010) and these proteins have been incorporated into a protein
array to monitor antibody response in different disease stages
(Bannantine et al., 2008).Effortshavealsobeendevotedtoin vitro
transcription-translation (Li et al., 2007), but those efforts did not
yield much protein to work with. Interestingly MMP was a test
proteininthatstudyaswell.Morerecently,MAPproteinshave
been expressed in a Salmonella vaccine delivery strategy (Faisal
et al., 2013). Our group is the first to use the probiotic genus
Lactobacillus as a vaccine vector for MAP. We have overcome the
limitations of low expression yields through re-engineering GC-
richcodingsequences.NoeffortshavebeenmadetoexpressMAP
proteins in a faster growing species of Mycobacterium such as
M. smegmatis.
We show that MptD, which contains six transmembrane
domains, was targeted to the cell periphery, likely the cytoplas-
mic membrane, while MMP formed aggregates in the cytoplasm
of L. salivarius. In general, proteins which are located within
the cytoplasmic membrane must be targeted to a translocation
site prior to their insertion and/or translocation (Fekkes and
Driessen, 1999). However, in the case of MptD, such integral
membrane proteins generally do not contain a signal sequence.
Instead, their hydrophobic TMSs function as an internal signal
for targeting and insertion which needs to be recognized early
in the translocation pathway to prevent their aggregation in the
cytoplasm (Fekkes and Driessen, 1999).
It is known that addition of any fusion tag to the N- or C-
termini of a peptide can modify specific structural characteris-
tics either by sterically interfering with protein interactions or
disrupting conformational folding (Snapp, 2009). The lack of
13E1 antibody recognition formed by MMP-GFP fusion led us to
propose that the fluorescent foci within L. salivarius MMPsynth-
GFP were related to a masking effect of the MMP epitope with
GFP and not representative of translational errors brought about
through codon optimization. The GFP tag does not appear to
impede localization of MMP at the cell surface, suggesting that
this effect is steric at the C-terminus only and does not provide
clues on secondary folding that might be required for localiza-
tion. In originally isolating the 13E1 antibody, Bannantine and
coworkers immunized mice with an N-terminal maltose-binding
protein fusion with MMP (Bannantine et al., 2007). This suggests
that the addition of a large (∼40 kDa) protein tag at the N- termi-
nus does not structurally impede the epitope of MMP. However,
in our study GFP was at the C-terminus and while it is possi-
ble that the GFP tag might mask the 13E1 epitope irrespective
of its fused location, it is also possible that the C-terminus is
more heavily involved than the N-terminal region in producing
theepitope.Insupportofthis,Li et al. (2007) expressed the MMP
protein with a six-histidine tag at the C-terminus, noting that the
recombinant antigen could not be detected by MAP antibodies in
pooled positive serum samples from cattle shedding MAP bacilli
(Li et al., 2007).
Detailed studies have demonstrated that MMP is a surface-
located virulence factor involved in mediating the invasion of
bovine epithelial cells and is transcriptionally upregulated in oxy-
gen limiting and solute stress conditions similar to those encoun-
tered within the intestine (Bannantine et al., 2003; Wu et al.,
2007). Based upon in silico analysis MMP is structurally dissimi-
lar to MptD and analysis lacks polytopic transmembrane domains
or highly hydrophobic stretches. The aggregates observed were
initially considered to be indicative of translational disorder
through synonymous codon modification, since MMP expres-
sion in native MAP appears throughout the bacilli by electron
microscopy (Bannantine et al., 2003). Alternatively, aggregation
could be a consequence of L. salivarius reacting to over expres-
sion by inducing vesicle formation to compensate for inefficient
processing or secretion of a non-host protein. Unfortunately,
the negligible fluorescence for native MMP-GFP prevented direct
comparison to determine if these foci also occurred in the absence
of synonymous mutations necessitating use of the monoclonal
antibody based ELISA.
The detection of a non-classically exported protein, such
as MMP, from the extracellular surface of L. salivarius could
indeed be attributed to cell lysis during experimental handling.
However, this would presumably have also occurred for those
L. salivarius cells expressing recombinant MptD, yet this was not
observed (Figure 2). Nevertheless, we acknowledge the behav-
ior of the MMP recombinant strains may be different than
the MptD recombinants and despite the lack of fMptD phage
binding in whole cells, we cannot rule out cell lysis in the
whole cell experiments with the MMP strains. As such, we have
yet to determine the mechanism by which MMP was effec-
tively presented in such a manner from our heterologous host
strain.
Generally, there are three strategies available for the subcellu-
lar distribution of recombinant antigens from Lactobacillus based
vaccine hosts and while cell surface display and secretion are
favored, these can result in degradation of recombinant peptides
due to exposure to proteolytic enzymes associated with gastric
and pancreatic fluids (Kajikawa et al., 2011). Cytoplasmic expres-
sion on the other hand, subverts this and protects heterologously
expressed peptides from degradation by encapsulation within the
cytosol, as well as facilitating the accumulation of high concen-
trations of the antigenic component intracellularly (de Ruyter
et al., 1996). As such, our L. salivarius MMPsynth host may pro-
vide an interesting combination of both high concentrations of
cytoplasmically accumulated MMP, with the added advantage of
superficial surface exposure.
Additionally, these results further demonstrate that heterol-
ogously expressed codon optimized MptD retains fMptD epi-
tope presentation in L. salivarius. However, ELISA data also
suggests that MptD, or at least the epitope recognized by the
Frontiers in Cellular and Infection Microbiology www.frontiersin.org September 2014 | Volume 4 | Article 120 |8
Johnston et al. Enhanced M. paratuberulosis antigen expression in L. salivarius
fMptD phage, is not exposed on the surface of L. salivarius.
It has already been shown that MptD expression on the cell
surface of a recombinant host can be achieved using fMptD
for both M. smegmatis and M. bovis BCG (Stratmann et al.,
2004; Heinzmann et al., 2008); which could be due to the pres-
ence of mycobacterium specific chaperones facilitating appro-
priate presentation of the MptD protein (Goldstone et al.,
2008). However, mptD is naturally present on a six gene operon
(mptA-F) transcribed as a single polycistronic mRNA molecule
[60] and in both the aforementioned studies, the recombi-
nant hosts were not transformed with the mptD gene in iso-
lation. The M. smegmatis harbored a vector including mptC-F
genes of the mpt operon (Stratmann et al., 2004) while the
M. bovis BCG host contained the entire operon integrated into
the chromosome (Heinzmann et al., 2008). Consequently, it
is possible that for effective MptD cell surface display, some
ancillary Mpt proteins may also be required. The MptE pro-
tein (with five predicted TMSs) encoded by MAP3732c, located
immediately downstream and overlapping the MAP3733c gene
on the mpt operon, is a credible candidate in this respect
owing to its association with the C-terminus of the MptD
protein.
In future studies it may be interesting to ascertain if the
sequential addition of this and other auxiliary mpt genes to
L. salivarius enables native surface display of the MptD protein.
Nevertheless, the lack of cell surface display may not be entirely
disadvantageous in the context of a MAP antigen delivery host.
The association of the MptD protein with the L. salivarius mem-
brane, while incomplete, likely sequestered hydrophobic domains
thus preventing undesirable aggregation and allowing higher lev-
els of intracellular MptD to accumulate. Moreover it is clear from
the analysis of cellular lysate that abundant MptD epitope could
be detected within the cytoplasm, indicating that the synthetic
gene and the L. salivarius host can effectively express and present
MptD antigens.
CONCLUSION
In conclusion, we have demonstrated that the synonymous
mutation of 279 rare or unfavorable codons within the MMP
coding region facilitates improved protein synthesis within L. sali-
varius. Furthermore, both synthetic MMP and MptD proteins
retain their epitopes or structural characteristics allowing them
to effectively mimic the MAP expressed protein. Importantly,
we also noted that while codon optimization enhances heterol-
ogous overexpression, the addition of a C-terminus GFP tag to
both proteins may obstruct some conformational structure from
forming.
Nevertheless, in the absence of a GFP tag and any extrinsic
signaling peptide, both proteins displayed slight, yet noteworthy,
tendencies to associate in the intended location within L. sali-
varius; MMP being detected on the cell surface, while the multi-
TMSs containing protein MptD associated with the cytoplasmic
membrane boundary. This work underscores the potential of
Lactobacillus salivarius to be used within a subunit vaccine devel-
opment against MAP, as additional antigens are optimized for
L. salivarius expression, the next step will require in-vivo testing
to demonstrate true efficacy.
FUNDING INFORMATION
The author’s acknowledge the financial assistance of the Irish
Government through funding under the Food Institutional
Research Measure (FIRM) grant 08RDCIT617 as well as the
USDA-Agricultural Research Service.
ACKNOWLEDGMENTS
The authors thank Norman Stern for providing the Lactobacillus
salivarius NRRL B-30514 strain, Gerald F. Gerlach for provid-
ing the fMptD bacteriophage, and Alan Lucid for his assistance
with bioinformatic analysis. The technical assistance of Janis K.
Hansen at the USDA’s National Animal Disease Center is grate-
fully acknowledged.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: http://www.frontiersin.org/journal/10.3389/fcimb.
2014.00120/abstract
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Conflict of Interest Statement: The Associate Editor Dr. Adel Talaat declares
that despite having collaborated with the author Dr. John Bananntine, the
review process was handled objectively and no conflict of interest exists. The
authors declare that the research was conducted in the absence of any commer-
cial or financial relationships that could be construed as a potential conflict of
interest.
Received: 24 April 2014; accepted: 15 August 2014; published online: 04 September
2014.
Citation: Johnston CD, Bannantine JP, Govender R, Endersen L, Pletzer D, Weingart
H, Coffey A, O’Mahony J and Sleator RD (2014) Enhanced expression of codon
optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus
salivarius. Front. Cell. Infect. Microbiol. 4:120. doi: 10.3389/fcimb.2014.00120
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