INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI:
Sept. 2001, p. 5786–5793 Vol. 69, No. 9
Copyright © 2001, American Society for Microbiology. All Rights
Towards Development of an Edible Vaccine against BovinePneumonic
Pasteurellosis Using Transgenic White Clover
Expressing a Mannheimia haemolytica A1Leukotoxin 50 Fusion
RAYMOND W. H. LEE,1 JUDITH STROMMER,2 DOUG HODGINS,3 PATRICIA E.
YONGQING NIU,2 AND REGGIE Y. C. LO1*
Departments of Microbiology,1 Plant Agriculture,2 and
University of Guelph, Guelph, Ontario, Canada N1G 2W1
Received 20 February 2001/Returned for modification 1 May
2001/Accepted 25 May 2001
Development of vaccines against bovine pneumonia pasteurellosis,
or shipping fever, has focused mainly onMannheimia haemolytica A1
leukotoxin (Lkt). In this study, the feasibility of expressing Lkt
in a forage plantfor use as an edible vaccine was investigated.
Derivatives of the M. haemolytica Lkt in which the
hydrophobictransmembrane domains were removed were made. Lkt66
retained its immunogenicity and was capable of elic-iting an
antibody response in rabbits that recognized and neutralized
authentic Lkt. Genes encoding a shorterLkt derivative, Lkt50, fused
to a modified green fluorescent protein (mGFP5), were constructed
for plant trans-formation. Constructs were screened by Western
immunoblot analysis for their ability to express the fusionprotein
after agroinfiltration in tobacco. The fusion construct
pBlkt50-mgfp5, which employs the cauliflowermosaic virus 35S
promoter for transcription, was selected and introduced into white
clover by Agrobacterium tu-mefaciens-mediated transformation.
Transgenic lines of white clover were recovered, and expression of
Lkt50-GFP was monitored and confirmed by laser confocal microscopy
and Western immunoblot analysis. Lkt50-GFP wasfound to be stable in
clover tissue after drying of the plant material at room
temperature for 4 days. An extractcontaining Lkt50-GFP from white
clover was able to induce an immune response in rabbits (via
injection), andrabbit antisera recognized and neutralized authentic
Lkt. This is the first demonstration of the expression ofan M.
haemolytica antigen in plants and paves the way for the development
of transgenic plants expressingM. haemolytica antigens as an edible
vaccine against bovine pneumonic pasteurellosis.
Mannheimia haemolytica A1 is the principal
microorganismresponsible for bovine pneumonia pasteurellosis, or
shippingfever, a major cause of sickness, death, and economic loss
inthe feedlot cattle industry (12, 33). Traditional
immunizationapproaches using needle injection of various vaccine
prepara-tions have provided some degree of protection. However,
nee-dle injection requires the herding and restraint of the
animals,inducing additional stress as well as incurring a
substantiallabor cost. As an alternative, we propose to develop a
nonin-vasive means of delivery of the vaccine via the oral route
byusing transgenic plants expressing recombinant immunogens.Recent
advances in the understanding of transgene expressionand
recombinant protein accumulation, stability, and process-ing in
plants have allowed the development of novel strategiessuch as
using edible plants for delivery of antigens for activeimmunization
(for reviews, see references 24, 28, and 30).
The leukotoxin (Lkt) of M. haemolytica A1 is one of itsmajor
virulence factors (26). Lkt is secreted by M. haemolyticaA1 and
acts as a pore-forming cytolysin that inserts into themembrane of
target cells (3), resulting in osmotic imbalanceand cell lysis.
This initiates a cascading effect that leads totissue damage,
pneumonia, and death of the animals (1, 4). Lktis a member of the
RTX family of cytolysins (31, 32). Several
functional domains have been identified in the typical
RTXcytolysin, one of which is a transmembrane hydrophobic
regionthat is involved in insertion of the toxin into the target
cells (31,32). The genetic determinant that codes for Lkt has
beencharacterized extensively in our laboratories. We have
carriedout genetic manipulation of the lktA gene for high-level
ex-pression in Escherichia coli and used this recombinant Lkt(rLkt)
in a vaccine for conventional intramuscular injection(5). This rLkt
was unable to cause damage to the target cellsbecause it is
unstable and loses biological activity rapidly. How-ever, to
completely ensure that the rLkt to be used for vaccinesis devoid of
any biological activities, we constructed derivativesof Lkt by
removing the section of the lktA gene that codes forthe putative
hydrophobic transmembrane domains of the toxin.These derivatives,
Lkt66 and the smaller Lkt50, would be in-capable of inserting into
the membrane and are therefore nolonger cytotoxic. However,
neutralizing antigenic epitopes ofLkt, mapped to a 227-amino-acid
region at the C terminus ofthe protein (11, 17), were retained in
In this paper, we describe (i) the construction of Lkt66
anddemonstrate that Lkt66 is capable of eliciting anti-Lkt
neutral-izing antibodies, (ii) the creation of transgenic clover
plantsthat express Lkt50 fused with the green fluorescent
protein(GFP), and (iii) the characterization of the Lkt50-GFP
fromclover as a candidate for development of an edible vaccine.GFP
was used as a marker to provide a simple and rapidmethod to screen
for expression of the fusion protein in trans-genic plants.
* Corresponding author. Mailing address: Department of
Micro-biology, University of Guelph, Guelph, Ontario, Canada N1G
2W1.Phone: (519) 824-4120, ext. 3363. Fax: (519) 837-1802. E-mail:
ber 16, 2020 by guesthttp://iai.asm
MATERIALS AND METHODS
Bacterial strains and culture conditions. E. coli DH5a (Table 1)
was used asthe host for cloning and propagation of plasmids and was
cultured in Luria-Bertani broth supplemented with thymine (50
mg/ml) and ampicillin (100 mg/ml),chloramphenicol (25 mg/ml), or
kanamycin (50 mg/ml) as necessary. M. haemo-lytica A1 (ATCC 43270)
was used for production of total proteins and was grownin brain
heart infusion broth (Difco, Detroit, Mich.). Agrobacterium
tumefaciensstrain C58C1Rifr containing the helper plasmid pMP90
(obtained from L. Erick-son, University of Guelph, Guelph, Ontario,
Canada) was routinely grown inYEP (yeast extract, 10 g/liter;
peptone, 10 g/liter; and NaCl, 5 g/liter) supple-mented with
kanamycin (50 mg/ml) and gentamicin (25 mg/ml) when required.
Recombinant DNA methods, nucleotide sequencing, and PCR. All DNA
clon-ing and ligation were carried out using standard recombinant
DNA techniques(2, 25). E. coli competent cells were transformed
either by the CaCl2 method orby electroporation according to our
standard laboratory procedure. A. tumefa-ciens was transformed by
electroporation (10). Plasmid DNA was isolated fromE. coli using
kits from Qiagen (Mississauga, Ontario, Canada) or Gibco
BRL(Burlington, Ontario, Canada). The constructs were confirmed by
DNA sequenc-ing at the Laboratory Services Division (University of
Guelph) on double-stranded plasmid DNA templates using an ABI 377
Prism automated sequencer(Applied Biosystems International, Foster
City, Calif.) based on cycle sequencingwith dye-terminator
dideoxynucleotides for fluorescence detection of terminatedDNA
PCR was carried out in thin-walled Microfuge tubes (Gordon
Technologies,Toronto, Ontario, Canada) in a Cetus DNA 480 or a
Perkin-Elmer GeneAmpPCR System 2400 Thermocycler (Perkin-Elmer,
Foster City, Calif.). PCR prim-ers were synthesized at the
Laboratory Services Division. A typical 50-ml reactionmixture
contained 10 to 100 ng of template, 50 to 100 pmol of each primer,
a 0.2mM concentration of each deoxynucleoside triphosphate, and 2
to 4 mM MgSO4in the PCR buffer supplied by the manufacturer. The
reaction included a hot start of2 to 5 min at 95°C, followed by the
addition of 1 U of Taq polymerase (RocheDiagnostics, Laval, Quebec,
Canada) or Pwo polymerase (Roche Diagnostics)and then 30 cycles of
95°C for 1 min, 45 to 65°C for 1 min, and 72°C for 2 min.
Construction of Lkt66. The plasmid pLKT60 (Table 1) contains the
lktCAgenes cloned behind the tac promoter (27) and was used as the
starting materialfor construction of the Lkt derivatives. Plasmid
pLKT60 DNA was digested andreligated at two NaeI sites located
within the lktA sequence (Fig. 1). The ligatedDNA was transformed
into competent E. coli cells. Plasmid DNA from
ampi-cillin-resistant colonies was isolated and mapped by
restriction endonucleases toconfirm removal of the NaeI fragment.
The DNA was also sequenced using aprimer based on the lktA sequence
to confirm that the nucleotides across theNaeI site had not been
altered. This plasmid was designated pLKTDN andshould express a
66-kDa Lkt derivative.
Production of anti-Lkt66 antibodies in rabbits. Plasmid pLKTDN
was intro-duced into E. coli DH5a also harboring the plasmid
pWAM716, which carriesthe hlyBD secretion genes (7). Lkt66 was
recovered from the culture supernatantof the E. coli cells using
the HlyBD secretion system according to our laboratory
procedure (22). Briefly, a log-phase culture of the E. coli
grown in Luria-Bertanibroth supplemented with ampicillin and
chloramphenicol was induced with
iso-propyl-b-D-thiogalactopyranoside (0.5 mM) for 1 h. The culture
supernatant wasrecovered after two centrifugation steps at 10,200 3
g and concentrated 10-fold
FIG. 1. Gene maps for Lkt and Lkt derivatives. (A) Map of
lktA,which encodes the full-length Lkt102, is shown. Shaded regions
(A andB) indicate sequences encoding hydrophobic domains. Deletion
of theNaeI fragment results in lktDN (B), which produces Lkt66. A
naturallyoccurring ApoI site (1349) in lktA was used to construct
the fusion geneshown in panel C. PCR was used to add an EcoRI site
at position 2701,and the resulting ApoI-EcoRI fragment which
encodes Lkt50 was in-serted between the signal peptide and GFP
coding sequences. (C)Diagram showing part of the T-DNA region of
pBlkt50-mgfp5 con-taining the lkt50-mgfp5 fusion gene and flanking
regions from theHindIII (H) restriction site to the left border
repeat (LB). Expressionof the fusion gene is driven by the
constitutive cauliflower mosaic virus35S promoter (35S). The fusion
gene consists of a signal peptide-encoding sequence (er), lkt50,
and mgfp5. The polyadenylation signalfrom the nopaline synthase
gene (nos ter) is found immediately down-stream of the fusion gene.
Restriction sites and their positions onpBlkt50-mgfp5 are as
follows: H, HindIII (4950); X, XbaI (5815); B,BamHI (5821); E,
EcoRI (5901, 7254, and 8257); and S, SacI (7990).Numbering of
pBlkt50-mgfp5 starts at the pRK2 origin of replicationas in pBin19
(8) but goes in the direction of right border to left border.
TABLE 1. Strains and plasmids used in this study
Strain or plasmid Genotype or description Reference or
Bacterial strainsE. coli DH5a Host strain for cloning Laboratory
stockM. haemolytica A1 Wild-type strain ATCC 43270A. tumefaciens
C58c1Rifr Host strain for plant transformation L. Erickson
Plant strainsTrifolium repens L. cv.Osceola
White clover for transgenic plant production Speare Seeds
N. tabacum cv. PetH4 Tobacco for transient gene expression
PlasmidspLKT60 Vector containing lktA 27pLKTDN Vector containing
lkt66 This studyp35S-GFP Cloning vector containing wild-type gfp
Clontechp35S-mgfp5-ER Cloning vector containing mgfp5 This
studyp35S-lkt50-mgfp5 Cloning vector containing lkt50-mgfp5 fusion
This studyp35S-mgfp5NS Cloning vector containing mgfp5 lacking a
stop codon This studypBINmgfp5-ER Binary vector containing mgfp5
14pBlkt50-mgfp5 Binary vector containing lkt50-mgfp5 fusion This
studypBmgfp5-lkt50 Binary vector containing mgfp5-lkt50 fusion This
studypBp mgfp5-ER Binary vector containing a promoterless mgfp5
VOL. 69, 2001 EXPRESSION OF LEUKOTOXIN 50 IN TRANSGENIC WHITE
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using an Amicon (Oakville, Ontario, Canada) ultrafiltration
apparatus with amembrane cutoff of 50 kDa. The concentrated fluid
was dialyzed extensivelyagainst distilled water at 4°C and
lyophilized. A small aliquot of the powder (10mg) was examined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)
to confirm the presence of a 66-kDa protein corresponding to
thetruncated Lkt. The lyophilized powder was dissolved in
phosphate-buffered sa-line (PBS) to a concentration of 200 mg/ml,
and 0.4 ml was injected intramus-cularly into rabbits for the
production of antibodies as described below.
Construction of the lkt50 and gfp fusion gene. The binary vector
pBINmgfp5-ER, obtained from J. Haseloff (University of Cambridge,
Cambridge, UnitedKingdom), contains the mgfp5-ER gene that encodes
the GFP variant mGFP5which has enhanced expression in plants and is
targeted and retained in theendoplasmic reticulum (ER) (14). For
simplicity, the mGFP5 variant is referredto as GFP in all
The HindIII-SacI fragment from pBINmgfp5-ER containing the
mgfp5-ERsequence was used to replace the HindIII-SacI fragment
(containing wild-typeGFP) of plasmid p35S-GFP (Clontech, Palo Alto,
Calif.) to produce p35S-mgfp5, a smaller vector, for the following
To construct an lkt derivative for cloning into plants, the
sense primer 59-CAAGATAATATGAAATTCTTACTGAACTTA, which annealed to
positions1855 to 1884 of lktA (20) containing an ApoI site
(underlined), and antisenseprimer
59-GCTATGTTTGAGGAATTCATAGTTCTCAAC, which annealed topositions 3237
to 3208 and added an EcoRI site (underlined), were used toamplify a
1.35-kbp fragment that codes for amino acids 451 to 901 of Lkt.
ThePCR product was digested with ApoI and EcoRI and was cloned into
p35S-mgfp5partially digested with EcoRI. Plasmid DNA from E. coli
transformants wasisolated and mapped to select for the insertion of
the PCR fragment in thecorrect orientation between the sequence
encoding the signal peptide and GFPin p35S-mgfp5, creating
p35S-lkt50-mgfp5. Subsequently, the HindIII-SacI frag-ment that
contained the lkt50-mgfp5 sequence was subcloned back
intopBINmgfp5-ER between the unique HindIII and SacI site to create
pBlkt50-mgfp5 (Fig. 1).
To construct the binary vector containing the mgfp5-lkt50 fusion
(pBmgfp5-lkt50), a vector containing mgfp5 lacking a stop codon was
first made. A 933-bpPCR product was amplified from p35S-mgfp5 using
the sense primer 59-GATGACGCACAATCCCACTATC, which annealed to the
35S promoter 83 nucleo-tides upstream of the XbaI site, and the
antisense primer 59-GGAAATTCGAGCTCGTAAAGCTC, which removed the stop
codon and added a SacI restric-tion site (underlined). The PCR
product was digested with XbaI and SacI andused to replace mgfp5 in
p35S-mgfp5, resulting in p35S-mgfp5NS. The lkt se-quence in
pBlkt50-mgfp5 was amplified using the sense primer
59-GCCGAGCTCTTACTGAACTTAAAC, which changed the upstream EcoRI site
to a SacIsite (underlined), and the antisense primer
59-TTTACTGAGCTCTTAGTTATCAACAAC which changed the downstream EcoRI
site to a SacI site (under-lined) and introduced a new stop codon
(boldface type). The 1.37-kbp PCRproduct was digested with SacI and
inserted into SacI-digested p35S-mgfp5NS,resulting in
p35S-mgfp5-lkt50. The fusion was then subcloned into the
binaryvector by replacing the EcoRI fragment containing mgfp5-ER in
pBINmgfp5-ERwith the EcoRI fragment from p35S-mgfp5-lkt50
containing the mgfp5-lkt50 fusion.
Transient expression in tobacco by agroinfiltration. To assess
the expressionof transgenes in plants, they were transiently
expressed by infiltrating tobaccoleaves (Nicotiania tabacum cv.
PetH4) with A. tumefaciens cultures containingthe various
constructs as previously described (6) with modifications.
Briefly,A. tumefaciens (carrying the plasmid constructs) was grown
in Luria-Bertanibroth with 10 mM MES (morpholineethanesulfonic
acid) (pH 5.6); 20 mMacetosyringone; and antibiotics at 28°C for 16
h. After centrifugation, the culturewas resuspended to an optical
density at 600 nm of 1 in Murashige and Skoog(MS) (23) salts with
2% sucrose; 0.5 mM MES (pH 5.6); and 100 mM aceto-syringone.
Infiltrated plants were kept humid by covering with clear plastic
bags.After 3 to 4 days, GFP fluorescence could be observed in some
cases by fluo-rescence microscopy (see below). To investigate the
transient production offusion protein, the infiltrated leaf areas
were excised and extracted proteinsexamined by Western immunoblot
analysis as described below.
A. tumefaciens-mediated plant transformation. White clover
(Trifolium repensL. cv. Osceola) transformation was performed
essentially as described (18) withmodifications. White clover seeds
obtained from Speare Seeds (Harriston, On-tario, Canada) were
surface sterilized and imbibed on 0.53 MS basal mediumsupplemented
with Gamborg’s vitamins (9) (Sigma, St. Louis, Mo.) and 2%sucrose
for 1 to 3 days at room temperature. Hypocotyls were cut from
thegerminated seeds, leaving a 1- to 2-mm segment of the stalk
attached to thecotyledons. Where possible, the apical shoot tip was
removed. The two cotyle-dons were either completely or partially
separated but still attached to thebisected hypocotyl. A.
tumefaciens for cocultivation was grown in selective YEP
to an optical density at 600 nm of 0.5 to 0.8. Cotyledons were
immersed in theA. tumefaciens culture and gently agitated for 40
min. Excess bacterial culturewas removed by blotting, and the
cotyledons were cocultivated on MS basalmedium with Gamborg’s
vitamins, 3% sucrose, N6-benzyladenine (1
mg/liter),a-naphthaleneacetic acid (0.1 mg/liter; Sigma), 100 mM
acetosyringone (Aldrich,Oakville, Ontario, Canada) and 0.3%
phytagel (Sigma), pH 5.5, at room tem-perature in the dark for 4 to
5 days. The cotyledons were then transferred toselective
regeneration medium consisting of MS basal medium with
Gamborg’svitamins, N6-benzyladenine (1 mg/liter),
a-naphthaleneacetic acid (0.1 mg/liter),kanamycin (100 mg/liter),
ticarcillin (250 mg/liter) and clavulanic acid (8.3 mg/liter)
(Timentin; Smith Kline Beecham Pharma, Oakville, Ontario, Canada),
and0.3% phytagel, pH 5.8. After 6 weeks, green shoots were isolated
and placed inMagenta boxes (Sigma) containing a rooting medium of
0.53 MS basal mediumwith Gamborg’s vitamins, 3% sucrose, kanamycin
(100 mg/liter), ticarcillin (250mg/liter), and clavulanic acid (8.3
Presence of the transgene in the plantlets was confirmed by PCR
using thesense primer 59-CCACTATCCTTCGCAAGAC, which anneals to the
35S pro-moter region, and the antisense primer
59-TGTTGCATCACCTTCACCCTCTC, which anneals to the mgfp5 coding
region. Genomic DNA was isolated usinga commercial kit (DNeasy
Plant Mini Kit; Qiagen). Selected transgenic plantletswere potted
in soil and maintained in the greenhouse.
Fluorescence microscopy. Conventional epifluorescence microscopy
was car-ried out using a Leica (Richmond Hill, Ontario, Canada)
MZIII fluorescencestereomicroscope with a GFP3 filter set
(excitation at 470 nm with a band widthof 40 nm; emission at 525 nm
with a band width of 50 nm). Laser scanningconfocal microscopy
(model MRC-600 microscope; Bio-Rad, Mississauga, On-tario, Canada)
was used to visualize GFP fluorescence in transgenic plants.
Forconfocal microscopy, observations were made on plant tissue
sections mountedin water.
SDS-PAGE and Western immunoblot analysis. To prepare plant
proteinextracts for SDS-PAGE, plant tissue samples were frozen in
liquid nitrogen,ground, and homogenized with an extraction buffer
consisting of PBS containing1 mM EDTA, 0.1 mM dithiothreitol, 0.1
mM phenylmethylsulfonyl fluoride, and0.5% (wt/vol) Tween 20. One-
and two-milliliter aliquots of buffer were used perg (fresh weight)
of tobacco and clover tissue, respectively. Plant proteins
wereseparated by SDS-PAGE as described by Lee and Huttner (19).
PrestainedSDS-PAGE standards (Bio-Rad) were used for molecular mass
determinations.For Western immunoblot analysis, proteins were
transferred onto nitrocellulosemembranes (Schleicher & Schuell,
Keene, N.H.) after SDS-PAGE (29). Themembranes were probed with
either a rabbit anti-Lkt66 antiserum, a mouseanti-Lkt monoclonal
antibody (MAb 601, obtained from S. Srikumaran, Univer-sity of
Nebraska, Lincoln) or a rabbit anti-GFP antiserum (Clontech).
Western immunoblots were also used to detect the production of
antibodiesagainst Lkt in rabbits immunized with transgenic plant
extracts. In these exper-iments, an Lkt-containing M. haemolytica
A1 cell suspension prepared as previ-ously described (21) was
separated by SDS-PAGE, blotted, and probed with thevarious rabbit
Lkt50-mGFP5 stability. Clover was harvested and allowed to dry
at roomtemperature and ambient humidity for 1 to 4 days. Proteins
were extracted bygrinding the tissue in 2 ml of PBS per g (fresh
weight) with a Kontes ground-glasstissue grinder followed by
centrifugation at 9,300 3 g for 10 min. The extract wasanalyzed by
Western immunoblotting using the monoclonal antibody to Lkt.
Preparation of Lkt50-mGFP5 extracts for immunization. Two
different cloverextracts were used for immunization. A crude
extract was prepared by firstgrinding clover tissue to a fine
powder in liquid nitrogen in a prechilled mortarand pestle.
Proteins were extracted using 2 ml of extraction buffer (PBS
con-taining 0.5% [wt/vol] Tween 20) per g (fresh weight). Insoluble
material wasremoved by two rounds of centrifugation at 4°C (30,000
3 g for 30 min followedby 130,000 3 g for 1 h). The resulting
supernatant was filtered through a 0.2-mm-pore-size syringe filter
(Nalgene, Rochester, N.Y.) and stored at 220°C.
A second extract, enriched with recombinant fusion protein, was
prepared bychromatofocusing (Pharmacia, Baie d’Urfé, Quebec,
Canada). After grinding ofthe clover leaf tissue, the proteins were
extracted in a buffer consisting of 50 mMTris-HCl (pH 7.5), 1 mM
EDTA, 0.1 mM dithiothreitol, 0.1 mM phenylmethyl-sulfonyl fluoride,
and 1% (wt/vol) Tween 20 and centrifuged at 130,000 3 g asdescribed
above. The supernatant was applied to a PBE 94 column
equilibratedwith 25 mM imidazole-HCl (pH 7.4). Proteins were eluted
with Polybuffer74-HCl (pH 4.0), and 3-ml fractions were collected.
Fractions containing Lkt50-GFP fusion protein were identified by
Western immunoblotting with the anti-Lkt66 antiserum.
Rabbit immunization. New Zealand White rabbits (Charles River
Laborato-ries, Wilmington, Mass.) were injected intramuscularly
with 1 ml of filtered plantextract twice at a 2-week interval. The
vaccine preparations contained a combi-
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nation of saponin (1.5% Quil A; Cederlane Laboratories, Hornby,
Ontario,Canada) and aluminum hydroxide (23%) as adjuvant, in a
ratio of 3 parts antigento 1 part adjuvant. A final dose was
administered 4 weeks after the secondinjection. Blood was collected
4 weeks after the final injection of antigen. Tworabbits were
immunized with each extract. Serum was analyzed by
Westernimmunoblotting and for Lkt neutralization activity using a
modified neutral redcytotoxicity assay (16).
Construction of lktDN and lkt50. An examination of the
nu-cleotide sequence of lktA revealed the presence of two NaeIsites
at positions 616 and 1651 within the gene (Fig. 1). Upondigestion
with NaeI and religation of plasmid pLKT60, a1,035-bp fragment that
coded for 345 amino acids containinghydrophobic domains was
removed. The resulting Lkt deriva-tive (Lkt66) expressed from lktDN
is expected to lack toxicityand thus is an ideal candidate for
further vaccine developmentstudies.
Another derivative was constructed to take advantage ofsuitable
nucleotide sequences for primer design to incorporaterestriction
sites to amplify the lktA gene for cloning into plants.PCR was used
to amplify a 1.35-kbp section of the lktA gene.This fragment coded
for amino acids 451 to 901 of Lkt and didnot include the N-terminal
and the C-terminal regions of Lkt,thus removing potential targeting
signals that may interferewith its expression and/or localization
in plants. This deriva-tive, lkt50, was used in subsequent cloning
of fusion genes intoa binary vector for introduction into
Immunogenicity of Lkt66. To ensure that the Lkt derivativesthat
lacked the hydrophobic domains were still effective asvaccine
candidates, Lkt66 was expressed in E. coli, recoveredfrom the
culture supernatant, and used to immunize rabbits.The resulting
rabbit anti-Lkt66 antiserum was tested in West-ern immunoblots as
well as for toxin neutralization against theauthentic Lkt from M.
haemolytica A1. In addition to recog-nizing Lkt66 as expected (data
not shown), anti-Lkt66 anti-serum immunostained the full-length Lkt
(102 kDa) fromM. haemolytica A1 (see Fig. 6A, lane 7). Moreover,
anti-Lkt66antiserum exhibited a neutralizing titer (2log2) of up to
5 (1/32dilution) against the authentic Lkt. This is similar to the
neu-tralizing titer obtained when the rabbits were immunized
withfull-length rLkt. These results demonstrated that the
hydro-phobic regions of the Lkt which were removed are not
criticalfor immunogenicity. The rabbit anti-Lkt66 antiserum was
usedin subsequent immunoblots in this study.
Transient expression of plasmid constructs in tobacco.
Twochimeric constructs, lkt50-mgfp5 and mgfp5-lkt50, were
insertedinto binary vectors and used to transform A. tumefaciens.
Forrapid assessment of their ability to direct the production
offusion proteins in plants, these genes were first expressed
tran-siently in tobacco by infiltration. Constructs containing
pro-moterless mgfp5-ER (F. Garabagi, unpublished data) and
35S-driven mgfp5-ER were used as controls for transient
expression(Fig. 2A). Three to four days after infiltration,
fluorescence wasobserved by microscopy only in plants injected with
A. tume-faciens transformed with plasmid containing
35S-mgfp5-ER.Plants infiltrated with A. tumefaciens containing the
promoter-less construct exhibited no fluorescence. Little or no
fluores-cence was observed in the infiltrated regions of plants
injectedwith A. tumefaciens containing either of the lkt50 fusion
structs (data not shown). The infiltrated areas were excised
andexamined for the presence of fusion protein by Western
im-munoblotting with rabbit anti-Lkt66. An immunoreactive bandof
approximately 79 kDa was present only in extracts ofplants
infiltrated with A. tumefaciens that carried the
constructpBlkt50-mgfp5 (Fig. 2B). The size of this protein
correspondedto that predicted from the nucleotide sequence of the
con-struct. Thus, it appeared that only in the case in which GFP
wasfused to the C-terminal side of Lkt50 was there accumulationof a
significant amount of the fusion protein. This constructwas
selected for the production of transgenic white clover lines.
Transgenic white clover expressing Lkt50-GFP. Transgenicclover
lines expressing GFP and Lkt50-GFP were produced byA.
tumefaciens-mediated transformation. PCR was used toconfirm that
the transgenes were present in transformed plants(data not shown).
By conventional fluorescence microscopy,green fluorescence was
easily detected in GFP-expressingplants. Consistent with the
results obtained with transient ex-pression, little to no
fluorescence was observed in pBlkt-mgfp5-transformed plants.
However, when these plants werefurther examined using laser
scanning confocal microscopy,green fluorescence was detected in
clover transformed withboth the pBINmgfp5-ER and pBlkt50-mgfp5
constructs (Fig.3B and C). As expected, GFP fluorescence was more
intensethan that observed for Lkt50-GFP. Leaves from untrans-formed
plants did not exhibit green fluorescence (Fig. 3A).Red chlorophyll
fluorescence from chloroplasts was seen intissues from all plants.
The pattern of green fluorescence ob-served in the clover leaves
was consistent with localization ofthe recombinant protein in the
ER (15). Cells contained largevacuoles, resulting in distribution
of fluorescence around the
FIG. 2. Transient expression of chimeric genes in tobacco.
(A)Proteins extracted from tobacco leaves infiltrated with A.
tumefacienstransformed with constructs containing promoterless
mgfp5-ER (lane1), 35S-mgfp5-ER (lane 2), or 35S-lkt50-mgfp5 (lane
3) were blotted andprobed with rabbit anti-Lkt66 antiserum. The
resulting Western im-munoblot showed that a protein of 79 kDa was
present only in lane 3,where the presence of the Lkt-containing
fusion protein was expected.(B) The expression of Lkt50-GFP (lane
1) and GFP-Lkt50 after agro-infiltration (lane 2) was analyzed by
Western immunoblotting with arabbit anti-Lkt66 antiserum as
described above. A. tumefaciens, trans-formed with vectors
containing either lkt50-mgfp5 (lane 1) or mgfp5-lkt50 (lane 2), was
used for infiltration. Only in the case where GFP wasfused to the C
terminus of Lkt50 (lane 1) was fusion protein expressiondetected.
Migrations of the molecular mass markers are as indicated tothe
left of each panel.
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cell periphery. The fusion protein exhibited a perinuclear
lo-calization and was clearly excluded from the nucleus. A
char-acteristic reticulate network was seen in some cells when
theappropriate plane of focus was used.
Expression of a recombinant fusion protein containing bothLkt50
and GFP epitopes was confirmed by Western immuno-blot analysis
(Fig. 4A and B). Both rabbit anti-Lkt66 and rabbitanti-GFP
recognized a protein of 79 kDa. Preliminary scanningdensitometric
analysis of gels and Western immunoblots ofextracts from one of the
Lkt50-GFP-expressing clover linessuggested that the recombinant
fusion protein constituted ap-proximately 1% of the soluble
proteins extracted from trans-genic clover. This corresponds to
approximately 18 mg ofLkt50-GFP per g (fresh weight) of plant
To assess the stability of the fusion protein in
harvestedplants, transgenic clover expressing Lkt50-GFP was
harvestedand allowed to dry at ambient temperatures. After 3 days,
theclover tissue retained approximately 20% of its initial
freshweight. Protein extracts were prepared from plant material
atdifferent stages of drying and analyzed by Western
immuno-blotting. After 4 days of drying, there did not appear to be
sig-nificant degradation of the fusion protein (Fig. 4C). No
lower-molecular-weight immunoreactive species were observed at
FIG. 3. Laser confocal microscopy of transgenic white clover
ex-pressing Lkt50-GFP. A section of clover leaf was mounted in
waterand observed by confocal microscopy. Images from two channels
(redfor chlorophyll fluorescence and green for GFP fluorescence)
weremerged to produce the micrographs shown. Leaves from
untrans-formed clover (A) do not exhibit the green fluorescence
that is presentin transgenic clover expressing Lkt50-GFP (B) or GFP
(C). The pat-terns of green fluorescence in panels B and C were
similar and areconsistent with an ER localization. Fluorescence
intensity in GFP-expressing plants was higher than that observed in
Lkt50-GFP-express-ing plants but was normalized in this figure to
facilitate comparison.The bar in each panel indicates 100 mm.
Vacuoles (V), nuclei (N), andchloroplasts (Ch) are indicated in
FIG. 4. Expression of Lkt50-GFP in transgenic white clover.
Ex-pression of Lkt50-GFP in transgenic white clover was analyzed
byWestern immunoblotting. Duplicate blots of proteins extracted
fromone transgenic line were immunostained with either rabbit
anti-Lkt66antiserum (panel A, lane 1) or an anti-GFP monoclonal
antibody(Clontech) (B). Molecular masses of the prestained SDS-PAGE
stan-dards (panels A and B, lanes M) are indicated at the left.
Both anti-bodies detected a protein of similar size, providing
evidence that afusion protein containing both Lkt and GFP sequences
was indeedproduced by the transgenic clover. In addition, the size
of the fusionprotein observed was close to 79 kDa, as predicted
from the nucleotidesequence. Stability of the Lkt50-GFP fusion
protein was also examined(C). Protein extracts prepared from fresh
transgenic clover (lane 2) orfrom transgenic clover dried for 1
day, 2 days, 3 days, or 4 days atambient temperatures (lanes 3 to
6, respectively) were analyzed byWestern immunoblotting. The blot
was probed with the monoclonalantibody 601 against Lkt. As
controls, M. haemolytica A1 supernatant(20 times concentrated)
containing full-length Lkt and an extract fromwild-type clover were
loaded in lanes 1 and 7, respectively. Migrationsof molecular mass
markers (lane M) are shown on the left. After 4 daysof drying at
ambient temperatures, there does not appear to be sig-nificant loss
of the Lkt50-GFP fusion protein.
5790 LEE ET AL. INFECT. IMMUN.
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any stage. Dried wild-type clover did not give rise to any
Immunogenicity of Lkt50-GFP produced by transgenicwhite clover.
To determine if Lkt50-GFP synthesized by cloverwas able to elicit
an immune response, rabbits were immunizedwith either a saline
extract or an Lkt50-GFP-enriched chro-matographic fraction prepared
from transgenic clover.
The Lkt50-GFP-enriched fractions were produced by
chro-matofocusing. A soluble protein extract prepared from
trans-genic clover was applied to a PBE 94 column, and
resultingfractions were analyzed by Western immunoblotting (Fig.
5).Most of the fusion protein eluted in fractions 6 to 8 (Fig.
5B);these fractions were used for rabbit immunization. The
fusionprotein could be partially separated from
ribulose-1,5-bisphos-phate carboxylase-oxygenase (rubisco, the most
abundant pro-tein in plant tissue), most of which eluted in
fractions 5 and 6(Fig. 5A). Under the conditions used in the
fractionation,Lkt50-GFP was stable to degradation as indicated by
the ab-sence of any major lower-molecular-weight
immunoreactivebands in the fractions.
Pre- and postimmunization sera were obtained from rabbitsand
tested for the presence of antibodies by Western immuno-blotting
(Fig. 6). All rabbits receiving extracts containing Lkt50-GFP as
antigen produced antibodies directed against theauthentic Lkt from
M. haemolytica A1 (Fig. 6A, lanes 3 to 6).Preimmune sera (data not
shown) and sera from mock-immu-nized rabbits (Fig. 6A, lanes 1 to
2) did not immunostain Lkt.
In addition to stimulating production of antibodies againstLkt,
the fusion protein was also able to induce anti-GFP anti-body
production. Rabbit immune serum contained antibodiesthat weakly
recognized wild-type GFP (Clontech) on Westernimmunoblots (Fig.
To determine if neutralizing antibodies were present in
therabbit sera, a toxin neutralization assay was performed (16).All
rabbits immunized with Lkt50-GFP extracts as antigenexhibited
neutralizing titers (2log2) up to 4 (1/16 dilution)(Table 2). Sera
from rabbits immunized with wild-type whiteclover extract (data not
shown), sera from mock-immunizedrabbits, or preimmune sera from all
the rabbits failed to neu-tralize Lkt (Table 2).
FIG. 5. Preparation of Lkt50-GFP-enriched fractions for
immuni-zation. A protein extract was prepared from transgenic white
cloverand fractionated by chromatofocusing. Column fractions were
ana-lyzed by Coomassie blue staining of gels after SDS-PAGE (A)
andWestern immunoblotting (B). The fraction number is indicated on
thetop, and molecular mass markers (lanes M) are indicated at the
left.These results show that Lkt50-GFP (panel B, fractions 6, 7,
and 8)could be separated from rubisco (strongly staining band
migrating ataround 56 kDa) and other high-molecular-weight material
(panel A,fractions 5 and 6).
FIG. 6. Immunogenicity of Lkt50-GFP produced by white clover.(A)
Rabbits (duplicate rabbits used for each treatment) were
mockimmunized with saline and adjuvant (lanes 1 and 2) or immunized
withchromatographic fractions enriched in Lkt50-GFP (lanes 3 and 4)
or asaline extract from transgenic clover (lanes 5 and 6). Immune
serawere used to probe a total M. haemolytica A1 protein
preparationblotted onto nitrocellulose. The rabbit anti-Lkt66
antiserum (lane 7)was used as a positive control. Immune serum from
all four rabbitsimmunized with Lkt50-GFP-containing fractions
recognized a 102-kDa band (arrow) migrating identically with
full-length Lkt that wasimmunostained with anti-Lkt66 (lanes 3 to
7). Immune serum fromrabbit 41 (panel A, lane 6) was analyzed to
see if it cross-reacted withwild-type GFP. Duplicate blots (B and
C) were prepared containingM. haemolytica A1 protein extract (lanes
1) and purified recombinantwild-type GFP (Clontech) (lanes 2).
Anti-GFP antibodies (B) andrabbit 41 immune serum (C) were used to
probe the membranes.Rabbit 41 immune serum was able to detect GFP
(panel C, lane 2).These results suggested that the immune serum
contained antibodiesdirected to both Lkt (panel A, lane 6, and
panel C, lane 1) and GFP(panel C, lane 2). Migrations of molecular
mass markers for bothpanels B and C (lanes M) are indicated to the
left of panel B. Arrows(B and C) show the position of GFP
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As a member of the RTX family of toxins, the cytotoxicactivity
of Lkt is mediated by its ability to insert and formtransmembrane
pores in the plasma membrane of susceptiblehost cells, eventually
leading to cell lysis. The hydrophobicregions in the Lkt protein
have been implicated in mediatingpore formation (31, 32). The
lktADN derivative which ex-pressed the Lkt66 molecule is an
excellent candidate for use ina vaccine. With the removal of most
of the hydrophobic do-mains, Lkt66 should be rendered incapable of
inserting intothe target cells to cause cytotoxicity. On the other
hand, it stillretains immunogenicity, as shown by its ability to
elicit rabbitantibodies which neutralized authentic Lkt. Thus, we
antici-pate that if Lkt66 were used as a vaccine in calves, it
woulddrive an immune response against the Lkt of M. haemolyticaA1.
A smaller derivative completely lacking all hydrophobicregions
(Lkt50) was made during the subcloning of lktADNinto the binary
vector for A. tumefaciens-mediated plant trans-formation. This
Lkt50 contained all of the antigenic regions ofLkt66 and produced
an Lkt neutralizing response in rabbits,suggesting that it would
also be useful as a vaccine candidate.Indeed, when proteins
containing either Lkt66 or Lkt50 se-quences were injected into
rabbits, both antigens were able toelicit the production of toxin
GFP was used in this present study as a marker to enablerapid
screening, to allow the monitoring of transgene expres-sion, and to
facilitate simple Mendelian analysis of inheritance.In addition,
with the current concerns about transgene move-ment in the
environment, GFP could be used as a reporter fortracking transgenic
plants in the field (13).
Using stable transformed transgenic plants to study trans-gene
expression usually requires several months’ work. To rap-idly
assess whether the transgene construct will be expressed
atsignificant levels in plants, we used a more convenient
tran-sient-expression assay by infiltrating tobacco leaves with
A.tumefaciens carrying plasmid constructs. These studies do
notrequire regeneration of plants after transformation and
thuspermit assessment of gene expression within a few days
byexamining transgenic protein expression directly by both
fluo-rescence and Western immunoblotting. In our studies,
agroin-filtration allowed us to identify the Lkt50-GFP construct as
thechoice for continued studies and transformation into
whiteclover. This was crucial for subsequent plant
transformationexperiments since there was no previous documentation
of theexpression of M. haemolytica genes in plants. With a
codon usage bias, it is entirely possible that lkt is not
expressedat significant levels in plants and would require
extensivecodon replacements before it could be used for expression
inthe transgenes. The failure of the GFP-Lkt50 construct toproduce
significant amounts of the fusion protein could be dueto unknown
factors that may cause inefficient or unstable tran-scription or
translation, improper folding of the polypeptide,or rapid
degradation of the fusion protein. While GFP is re-ported to be
generally functional with both N- and C-terminaladditions, our
results underscore the limitations of this gener-alization.
Using conventional epifluorescence microscopy, little if
anyfluorescence was observed in Lkt50-GFP-expressing plants,even
though the presence of fusion protein was detected byWestern
immunoblot analysis. By confocal microscopy, a moresensitive
technique for visualizing fluorescence, we were ableto observe
fluorescence in Lkt50-GFP-expressing plants but ata lower intensity
that that for plants expressing GFP alone.Even though both
constructs were expressed from the same35S promoter, it may be
possible that less fusion protein wasproduced in the transgenic
plants than in plants expressingGFP alone. An alternative
explanation may be that the recom-binant GFP was not able to
properly fold when fused to Lkt50,resulting in reduced levels of
The Lkt50-GFP5 fusion protein contains an N-terminal sig-nal
peptide derived from Arabidopsis vacuolar basic chitinaseand the
C-terminal ER-retention sequence HDEL. Previousobservations have
indicated that higher levels of fluorescencein transgene products
could be achieved if GFP entered thesecretory pathway and was
sequestered in the lumen of the ER(14, 15). Targeting proteins to
the ER may improve maturationand accumulation and may protect plant
cells from the photo-toxic effects of GFP (14, 15). A significantly
higher level offluorescence has been observed in transgenic plants
expressingmGPF5 than in plants expressing the wild-type GFP that
lo-calizes to the cytoplasm (F. Garabagi, personal
Our results demonstrate that using plants to produce bacte-rial
antigen as a vaccine component is a viable strategy. Weshowed that
an Lkt fragment synthesized by plants was able toinduce an immune
response in rabbits that led to the produc-tion of antibodies that
neutralized the authentic Lkt. Proteinstability is important for
vaccine harvest, production, and stor-age. Our results also
indicated that the fusion protein wasrelatively stable in harvested
material in the absence of refrig-eration. Plants are economical to
grow and can yield a highlevel of recombinant proteins. While
alfalfa may be the optimalsupplement for animal feed, white clover
is a reasonable alter-native, and the present study paves the way
for continuingresearch into the development of an edible vaccine
using avariety of transgenic plants expressing antigens of M.
haemo-lytica A1. Experiments are in progress to assess the
immuno-genicity of plant-derived antigen in cattle and the
effectivenessof feeding this transgenic material in stimulation of
a mucosalimmune response against the Lkt of M. haemolytica A1.
This research was supported by the Ontario Cattlemen’s
Associa-tion, the Natural Sciences and Engineering Research Council
TABLE 2. Neutralizing titers of sera from rabbits immunizedwith
Lkt50-GFP fusion protein
Neutralizing antibody titera
Pre-immune Bleed 2 Bleed 3 Bleed 4
Lkt50-GFP (saline extract) 41 — — 4 3.5Lkt50-GFP (saline
extract) 42 — — 1 1Lkt50-GFP (column fraction) 43 — — 1 1Lkt50-GFP
(column fraction) 44 — — 1.5 1Mock 45 — — — —Mock 46 — — — —
a Values are mean reciprocal log2 serum dilutions giving at
least 50% neutral-ization of toxicity; sera were assayed in
duplicate. —, no neutralization activity.
5792 LEE ET AL. INFECT. IMMUN.
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ada Strategic Grants Program, and the Ontario Ministry of
Agricul-ture, Food, and Rural Affairs.
We thank Betty-Ann McBey for assistance with the rabbit
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