Previous Article | Next Article 
Applied and Environmental Microbiology, April 2001, p. 1423-1428, Vol. 67, No. 4
Unité de Virologie et Immunologie
Moléculaires,1 Unité de
Recherches Laitières et de Génétique
Appliquée,2 and Unité
d'Ecologie et de Physiologie du Tube Digestif,3
INRA, 78352 Jouy en Josas, France
Received 3 August 2000/Accepted 6 January 2001
Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in
mice. To get insight into the biological effects of NSP4, production of
large quantities of this protein is necessary. We first tried to
produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable. The capacity of the
generally regarded as safe organism Lactococcus lactis to
produce NSP4 either intra- or extracellularly was then investigated by
using the nisin-controlled expression system. Production of recombinant
NSP4 (rNSP4) was observed in L. lactis for both locations.
In spite of a very low secretion efficiency, the highest level of
production was obtained with the fusion between a lactococcal signal
peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected
into rabbits, and a specific immune response was elicited. The
anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in
MA104 cells infected by rotavirus. We showed that L. lactis
is able to produce antigenic and immunogenic rNSP4 and thus is a good
organism for producing viral antigens.
Rotavirus is the major etiologic
agent of severe diarrhea in infants and young children around the world
(6). Rotavirus infects mature villus enterocytes in the
small intestine. Nonstructural protein 4 (NSP4), encoded by gene 10, has been shown to be an intracellular receptor for double-layered
particles. Purified NSP4 and a 22-amino-acid peptide (amino acids 114 to 135) were both capable of inducing dose-related diarrhea after
intraperitoneal or intraduodenal administration to 6- to 10-day-old
mice (1). NSP4 is thought to act as an enterotoxin which
triggers chloride secretion by a calcium-dependent signal transduction
pathway. To study the biological properties of NSP4, it is necessary to produce large quantities of NSP4 protein. Previously, Estes et al.
produced and purified full-length NSP4 from Spodoptera
frugiperda 9 cells infected with a recombinant baculovirus
expressing rotavirus gene 10 of strain SA11 (22). Newton
et al. (12) produced part of NSP4 (amino acids 86 to 175)
as a fusion with a 36-kDa domain of glutathione S-transferase.
Developing efficient gene expression and protein secretion systems in
nonpathogenic gram-positive lactic acid bacteria is an original
approach for producing proteins of therapeutic interest (15,
25) and a new strategy for rotavirus vaccination. These lactic
acid bacteria possess many properties which make them good candidates
for oral vaccination purposes; e.g., they have generally regarded as
safe status or adjuvant properties (15). They have already
been used to produce several bacterial antigens and interleukins (19). Some viral antigens or parts of viral antigens have
been produced in lactic acid bacteria; antigen M6-gp41E has been
produced in Lactobacillus plantarum (5), a
fragment of the human immunodeficiency virus type 1 envelope protein
has been produced in Streptococcus gordonii
(16), 250 amino acids of rotavirus protein VP7 have been
produced in L. plantarum, an epitope of foot-and-mouth
disease virus protein VP1 (amino acids 137 to 162) has been produced in Lactobacillus casei (15), and a short epitope
of bovine coronavirus has been produced in Lactoccocus
lactis (9).
In this study, we constructed strains of L. lactis that
produce recombinant NSP4. Both intra- and extracellular locations of
recombinant NSP4 (rNSP4) were examined by using the nisin-inducible expression system (3). In spite of a very low efficiency
of secretion of rNSP4, the highest level of production of rNSP4 was observed when the nsp4 gene was fused to a lactococcal
signal sequence. The recombinant viral protein showed antigenic and
immunogenic properties (i.e., it was recognized by specific antibodies
and was able to induce an immune response). In addition, the
recombinant L. lactis strains should allow study of the
biological properties of NSP4 without interference from
lipopolysaccharides or inflammatory reactions like those observed with
Escherichia coli.
Bacterial strains, media, and reagents.
The bacterial
strains and plasmids used in this work are listed in Table
1. L. lactis and E. coli were grown in M17 medium (21) and in
Luria-Bertani (17) medium, respectively. When required,
antibiotics were added at the following concentrations: 50 µg of
ampicillin (Roche) per ml and 5 µg of chloramphenicol (Sigma) per ml
for L. lactis and 10 µg of chloramphenicol per ml for
E. coli.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1423-1428.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Bovine Rotavirus Nonstructural Protein 4 Produced
by Lactococcus lactis Is Antigenic and Immunogenic
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
L. lactis strains and plasmids
Cloning procedures and PCR. Strains of E. coli and L. lactis were transformed by electroporation by using standard procedures (4, 26). Plasmid DNA was isolated from L. lactis as described previously (24). PCR amplifications with Taq polymerase (Promega) were performed by using 25 cycles, with each cycle consisting of a denaturation step at 94°C for 30 s, a primer annealing step at 55°C for 30 s, and a primer extension step at 72°C for 1 min, with a DNA thermocycler (Perkin-Elmer GeneAmp PCR system 2400). All other DNA manipulations were performed by established procedures (17).
Construction of plasmids for rNSP4 production in E. coli. Two systems were used to produce rNSP4 in E. coli. An EcoRI-XbaI DNA fragment encoding NSP4 was isolated from pNSP4 (as described previously) and inserted into pMAL-c2 (New England Biolabs, Hitchin, United Kingdom), an E. coli bacterial expression vector used to express a maltose binding protein-NSP4 fusion protein.
In the second approach, we used the pET system (Novagen, Genetics Institute, Cambridge, United Kingdom). EcoRI and NotI restriction sites were introduced into the nsp4 gene to allow cloning in pET23b+. Nucleotides were substituted by performing PCR with pRF10 and the following primers flanking the NSP4 coding sequence: forward primer 5'-TACCGGAATTCCGGAATGGAAAAGCTTACCGAC-3' and reverse primer 5'-ATAGTTTAGCGGCCGCCATCGCTGCAGTCACTTCTTTTGG-3'. The amplified fragment was cloned into pET23b+. Two recombinant plasmids, pMAL:NSP4 and pET:NSP4, were obtained in E. coli TG1. The nsp4 gene cloned with the pET system was expressed in E. coli B121.Construction of plasmids for expression of rNSP4 in L. lactis.
Unless otherwise indicated, plasmid constructions
were first established in E. coli and then transferred to
L. lactis. cDNA of the bovine rotavirus (RF strain) gene 10 encoding NSP4 was obtained by reverse transcription-PCR, cut with
EcoRI, and cloned in EcoRI-cut pBluescript SK+
(pBS; Stratagene), resulting in pRF10. An NsiI restriction
site was introduced into the nsp4 coding sequence to allow
in-frame cloning with the ATG start codon of the nuc gene
(10) contained on the pCYT and pSEC plasmids (P. Langella, Y. Le Loir, S. Gilbert, J. Commissaire, R. L'Haridon, and G. Corthier, unpublished data). pSEC and pCYT are derived from pNZ8010
(3), which contains the gus gene under
transcriptional control of the promoter of nisA
(PnisA). The gus gene was deleted by XhoI digestion and replaced by a
BamHI-XhoI-cut DNA fragment encoding the
ribosome-binding site (RBSUsp45) and signal sequence
(SPUsp45) of the usp45 gene (23)
and the mature part of the staphylococcal nuclease protein (Nuc)
(18) to obtain plasmid pSEC. The DNA fragment encoding
SPUsp45 was then deleted by reverse PCR to obtain pCYT. An
NsiI site was introduced at the 3' end of
RBSUsp45 to allow replacement of the nuc coding
sequence by the DNA fragment encoding the rNSP4 protein. Nucleotide
substitutions were made by performing PCR with pRF10 as the template
DNA. This procedure required a mutagenic primer,
5'-GGCGAATTCGATGCATCCGAAAAGCTTACCGAC-3' containing EcoRI and NsiI sites
(underlined), and a reverse primer, 5'-GTGACTGCAGCGATGTAATGAGATATCTAGAGCC-3'
containing EcoRV and XbaI sites (underlined
and boldface). The amplified fragment was digested with
EcoRI and XbaI and cloned into pBS+ (Stratagene)
which had been digested with EcoRI and XbaI,
generating pNSP4. Digestion of pNSP4 with NsiI and
EcoRV allowed purification of a fragment containing the
nsp4 gene, which was cloned in two L. lactis
vectors, pSEC and pCYT (Fig. 1). Thus,
the nuc gene was replaced by the modified cDNA fragment
encoding NSP4. The sequence of each fusion was checked. The two
resulting plasmids, pSEC:NSP4 and pCYT:NSP4, were introduced into
L. lactis NZ9000, which is a derivative of L. lactis MG1363 carrying the two regulatory genes of
PnisA, nisR and nisK
(3).
|
,
nuc gene;
,
NSP4 gene;
,
chloramphenicol resistance (Cmr);
,
ampicillin resistance (Ap), 
, SPUsp45; 
, PnisA.
Restriction enzyme sites are also indicated; only relevant restriction
sites are shown. The direction of transcription is indicated.
Nisin induction and analysis of rNSP4 production in L. lactis NZ9000(pSEC:NSP4) and NZ9000(pCYT:NSP4). For induction and production of rNSP4, 50-ml cultures of L. lactis NZ9000(pSEC:NSP4) and NZ9000(pCYT:NSP4) were grown until the optical density at 600 nm (OD600) was 0.5 and were induced with 1 ng of nisin (Sigma France) per ml. Noninduced cultures were used as controls. After induction, the cells were grown for 3 h and were harvested and resuspended in 5 ml of water. Sterile glass beads were added, and the cells were disrupted with a Mini-Beadbeater-8 (Biospec Products). Cellular debris and glass beads were removed by centrifugation, and the protein-containing supernatant was then concentrated fivefold with a Speed-Vac. Samples to be compared were prepared in parallel and loaded on the same gel. Ten microliters of each sample was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15% polyacrylamide) (8), followed by Western blotting. rNSP4 was detected by using a rabbit anti-NSP4 antiserum directed against the C-terminal part of NSP4 (dilution, 1:1,000; kindly supplied by L. Svensson, Stockholm, Sweden) and alkaline phosphatase-labelled anti-rabbit immunoglobulin G (heavy plus light chains; BioSys, Compiègne, France). Staining was performed with a standard alkaline phosphatase substrate, 5-bromo-4-chloro-3-indolylphosphate (BCIP)-nitroblue tetrazolium (Life Technologies, Gaithersburg, Md.). After staining, different exposures of nonsaturated film were scanned and compared by using ImageQuant programs to obtain average values.
To determine the cell distribution of rNSP4, cell fractionation was performed as described previously (11). Briefly, cell and supernatant fractions were separated and concentrated 10-fold by using trichloroacetic acid. The equivalent of 1 ml of culture at an OD600 of 1 was concentrated in 100 µl (final volume), and 10 µl of each sample was loaded on an SDS-PAGE gel. To determine the location of the precursor preUspNSP4, the NZ9000(pSEC:NSP4) cell fraction was divided into cytoplasmic and cell wall fractions as previously described (14).Cell culture and viral infections. Fetal rhesus monkey kidney cell line MA 104 was grown in confluent monolayers in Eagle's minimal essential medium (Life Technologies, Paisley, Scotland) supplemented with 10% fetal calf serum and antibiotics. MA104 monolayers were washed and inoculated with bovine rotavirus (RF strain) at a multiplicity of infection of about 5 PFU per cell. Cells were processed for Western blotting or immunoprecipitation at 8 h postinfection. Spodoptera frugiperda 9 cells were infected with recombinant baculovirus expressing rotavirus gene 10 (BNSP4-SA11; kindly supplied by M. Estes) to produce NSP4. Cells were collected 3 to 4 days postinfection.
Immunization of rabbits with a culture of L. lactis NZ9000(pSEC:NSP4). Before two 6-month-old rabbits were injected, blood samples were taken to prepare a preimmune serum and to check (by enzyme-linked immunosorbent assay) that the rabbits were not infected with rotavirus. Two hundred milliliters of a nisin-induced culture of L. lactis NZ9000(pSEC:NSP4) containing 108 bacteria/ml was concentrated 200-fold and lysed. To immunize the rabbits, we used a 1:1 mixture composed of 500 µl of the concentrated culture lysate (containing approximately 1010 bacteria/ml) and 500 µl of Inject Alum's adjuvant (Pierce, Rockford, Ill.). The rabbits were immunized four times intramuscularly at 3-week intervals. One week after the last injection, the rabbits were bled, and sera (dilution, 1:50) were used in Western blot experiments.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Production of rNSP4 in E. coli and L. lactis. The DNA fragment encoding rNSP4 (nsp4) was cloned and expressed in E. coli by using two different expression vectors, pET and pMAL. The insert in both plasmids was sequenced, and the expected sequence was obtained only with the pET:NSP4 plasmid. Several mutations were observed in the pMAL:NSP4 plasmid. Expression of rNSP4 was induced in E. coli containing pET:NSP4. We observed that growth was significantly reduced after this induction, suggesting that rNSP4 is toxic for E. coli. Total protein extracts of this strain were subjected to SDS-PAGE followed by Western blotting. No rNSP4 was detected on the Western blot with anti-NSP4 antibodies (data not shown).
To produce rNSP4 in L. lactis, the nsp4 gene was cloned in L. lactis on pCYT and pSEC, expression plasmids that allow intra- and extracellular location of the protein of interest, respectively. In both plasmids, expression of nsp4 was under transcriptional control of the nisin-inducible promoter PnisA (3). The two resulting plasmids, pCYT:NSP4 and pSEC:NSP4, were introduced into L. lactis NZ9000, a derivative of L. lactis MG1363 containing the two regulatory genes of PnisA, nisR and nisK. We first checked that nisin addition did not inhibit cell growth of L. lactis NZ9000, the control strain. Strains NZ9000(pCYT:NSP4) and NZ9000(pSEC:NSP4) were induced by nisin when sufficient biomass was present (see Materials and Methods) (Fig. 2a). Compared to the control strain, growth of the two L. lactis strains producing rNSP4 was different; the growth of L. lactis NZ9000(pCYT:NSP4) was reduced, and growth of L. lactis NZ9000(pSEC:NSP4) even stopped.
|
,
noninduced NZ9000(pCYT:NSP4) and NZ9000(pSEC:NSP4); 
, induced
NZ9000(pCYT:NSP4); 
, induced NZ9000(pSEC:NSP4). (b) Immunoblotting
of lysates from L. lactis NZ9000(pCYT:NSP4) and
NZ9000(pSEC:NSP4) induced by nisin with rabbit antiserum to NSP4
C-terminal peptide.
Analysis of rNSP4 produced in L. lactis. Next, production of rNSP4 in the two rNSP4-producing L. lactis strains was analyzed. Whole-cell protein contents were analyzed at the time of induction with nisin and 2 and 4 h after induction (Fig. 2a). The protein extracts were analyzed by SDS-PAGE, followed by Western blot assays in which the blots were developed with anti-NSP4 antibodies (Fig. 2b). In the absence of nisin, no signal was detected, indicating that rNSP4 was not produced. After 2 and 4 h of induction with nisin, the results showed that rNSP4 was present only in cell fractions of L. lactis NZ9000(pCYT:NSP4). In the case of L. lactis NZ9000(pSEC:NSP4), the low proportion of mature NSP4 detected in the total fraction and the absence of a band in the supernatant fraction (data not shown) suggested that the secretion efficiency of mature NSP4 was very poor. Two other major differences between secreted production and cytoplasmic production were observed. (i) One difference was the number of bands detected. In the cellular fraction of L. lactis NZ9000(pSEC:NSP4) four protein forms were distinguished, including a 22-kDa band corresponding to the precursor preUspNSP4 form, a 20-kDa band corresponding to the mature form of rNSP4 (175 amino acids), and 18- and 16.5-kDa bands probably corresponding to degradation products of rNSP4. In the cellular fraction of L. lactis NZ9000(pCYT:NSP4), only a faint 20-kDa band corresponding to the expected size of mature rNSP4 and an 18-kDa major band were observed, suggesting that there was intracellular degradation of mature rNSP4. (ii) The other difference was the intensity of the bands detected. The NSP4 content of NZ9000(pSEC:NSP4) was significantly greater (around 10-fold greater) than that of NZ9000(pCYT:NSP4). The antibodies used for these Western blot experiments recognized the C-terminal region of NSP4, suggesting that degradation of rNSP4 produced in L. lactis occurred at the N-terminal end.
To localize the precursor preUspNSP4 more precisely, the cellular fraction of L. lactis NZ9000(pSEC:NSP4) was divided into protoplast and cell wall fractions by using the protocol of Piard et al. (14). The occurrence of rNSP4 in the different fractions was estimated by SDS-PAGE followed by Western blotting. We observed only four rNSP4 bands in the lane corresponding to the cytoplasmic fraction, suggesting that translocation of preUspNSP4 could be blocked in the bacterial membrane (data not shown).Immunogenicity of rNSP4 produced in L. lactis.
To
test whether the rNSP4 produced in L. lactis can induce a
humoral immune response with production of antibodies in rabbits, a
culture of L. lactis NZ9000(pSEC:NSP4) was lysed and used to immunize rabbits. The immune serum obtained, LacNS4, was compared with
antibodies directed against the C-terminal part of NSP4. The two sera
were tested with a Western blot of lysates of S. frugiperda
9 cells infected with baculovirus producing rNSP4 (Fig. 3). As expected, no signal was observed
with preimmune serum from rabbits injected with the lactococcal lysate
(Fig. 3, lane 1). The NSP4 protein was detected with both anti-NSP4
sera (Fig. 3, lanes 2 and 3). The four bands were observed in both
cases, indicating that immunization of rabbits with lysates of
rNSP4-producing L. lactis strains leads to production of
antibodies that recognize the same NSP4 forms as the antibodies raised
against NSP4 produced in baculovirus.
|
Characterization of rNSP4 protein produced in L. lactis.
Four different types of NSP4 were tested in Western
blot experiments with the rabbit anti-NSP4 C terminus: NSP4 produced in MA104 cells infected with rotavirus, NSP4 produced in S. frugiperda 9 cells infected with baculovirus, and NSP4 produced in
induced cultures of L. lactis NZ9000(pSEC:NSP4) and
NZ9000(pCYT:NSP4) (Fig. 4a). No signal
was detected in the noninfected MA104 cell control (Fig. 4a and b, lane
1). The anti-NSP4 C-terminal serum allowed us to identify bands
specific for NSP4 in the two other samples (Fig. 4a, lanes 2 and 3).
Identical results were obtained with the LacNS4 serum (Fig. 4b, lanes 2 and 3). The apparent molecular weights of rNSP4 produced in L. lactis were different than the molecular weights of NSP4 produced
in MA104 cells infected with rotavirus or in recombinant
baculovirus-infected S. frugiperda 9 cells (Fig. 4a, lanes 2 to 5). The differences were probably due to the absence of
glycosylation in L. lactis. For MA104 cells and S. frugiperda 9 cells, we detected a major band around 25 kDa
corresponding to an NSP4 form with two glycosylations and a band around
24 kDa corresponding to an NSP4 form with only one glycosylation (Fig.
4a, lanes 2 and 3). A common 20-kDa band corresponding to native NSP4
was detected with the four sources of NSP4. The 22-kDa band
corresponding to the precursor preUspNSP4 was observed only with the
rNSP4 form fused with SPUsp45 (Fig. 4a, lane 4). We
concluded that LacNS4, the anti-NSP4 antibodies produced in rabbits
immunized with lactococci, recognize NSP4 produced in MA104 and
S. frugiperda 9 cells.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
In this paper, we describe for the first time production of an
entire viral antigen in the food-grade bacterium L. lactis. Our results indicate that L. lactis is able to produce
recombinant bovine rotavirus NSP4 that possesses antigenic and
immunogenic properties. Previously, Newton et al. (12)
produced a fragment of NSP4 in E. coli with the GST fusion
protein system. In our hands, it was impossible to express the entire
rNSP4 with pMAL and pET systems in E. coli. This failure
could have been due to toxicity of NSP4 for E. coli, as
previously described with a slightly leaky inducible promoter
(20). Two L. lactis strains were constructed to
produce rNSP4 in two locations under control of an inducible lactococcal promoter. To produce intra- and extracellular rNSP4, we
used the ribosome binding site of L. lactis secreted protein Usp45, and the nsp4 gene was placed under transcriptional
control of the nisin-inducible promoter PnisA.
In strain NZ9000(pSEC:NSP4), the nsp4 gene was fused to the
signal sequence of Usp45 (SPUsp45). After induction with
nisin, the rates of growth were very different for the two strains and
were correlated with rNSP4 production. In L. lactis
NZ9000(pSEC:NSP4), the presence of SPUsp45 allowed 10-fold
enhancement of production of rNSP4 compared to intracellular production. Our hypothesis to explain the difference is that the precursor preUsp:rNSP4 could be translated more efficiently than intracellular forms of rNSP4 produced in NZ9000(pCYT:NSP4).
Furthermore, recognition of preUsp:rNSP4 by the secretion machinery of
L. lactis could allow it to escape intracellular
proteolysis. The same positive effect of SPUsp45 was
recently observed in L. lactis for production of bovine
-lactoglobulin, the major cow's milk allergen (Langella, unpublished data), and for production of the Brucella
abortus ribosomal L7/L12 protein (L. Ribeiro, personal communication).
It has been found that the signal peptide is necessary, but not sufficient, for mature protein export. However, all proteins bearing this sequence are not secreted (2, 27). Hydrophobic domains of NSP4 could result in localization of rNSP4 in the bacterial membrane and prevent secretion. In Western blot experiments with NZ9000(pSEC:NSP4), we showed that the unprocessed intracellular precursor preUsp:rNSP4 and mature forms of rNSP4 were present in the cellular fraction. The low level of maturation of preUsp:rNSP4 could be due to suboptimal recognition of this hybrid precursor by the secretion machinery of L. lactis, leading to aggregate formation. At this point, we note that (i) only Usp45, a protein with an unknown function, seems to be secreted efficiently by L. lactis (23) and (ii) only bacterial heterologous proteins have been efficiently secreted by L. lactis (19). Using the staphylococcal nuclease (Nuc) as the secreted model protein, Le Loir et al. (11) showed that a positive net global charge in the first 10 amino acid residues of the Nuc N terminus resulted in a dramatic decrease in Nuc secretion efficiency. Analysis of the peptide sequence of the N terminus of rNSP4 revealed the presence of an Arg residue at position +3, resulting in a net global charge of +1 in the first five amino acid residues of the N terminus of rNSP4. Experiments are currently in progess to investigate how insertion of a synthetic propeptide (19) and fusion with Nuc can affect rNSP4 secretion efficiency.
NSP4 produced in recombinant baculovirus-infected S. frugiperda 9 cells and infected MA104 cells was characterized by three major bands. These bands may represent full-length NSP4 with two, one, and no sites glycosylated (13, 22). Unlike the NSP4 produced in a eucaryotic system, the NSP4 protein produced in L. lactis was not glycosylated. This posttranslational function is probably responsible for the difference in the molecular weights of bands.
The rNSP4 protein produced in L. lactis has the same immunogenic properties as the viral protein. We may use these properties to develop a rapid diagnostic method for biomedical analysis, like an enzyme-linked immunosorbent assay. Recovery of rNSP4 protein with antigenic and immunogenic properties offers possibilities for producing other proteins which are produced in E. coli with difficulty. The L. lactis strains provide a good way to produce NSP4 without interference from lipopolysaccharide, as in E. coli. This production could be performed either in vitro or in the digestive tract of mice in order to induce mucosal immunity against this enterotoxin.
| |
ACKNOWLEDGMENTS |
|---|
We are very grateful to Maarten van de Guchte for his contribution to this work. We thank L. Svensson for his generous gift of antiserum against NSP4.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Unité de Recherches Laitières et de Génétique Appliquée, INRA, 78352 Jouy en Josas, France. Phone: 33(0)1 3465 2083. Fax: 33(0)1 3465 2065. E-mail: langella{at}biotec.jouy.inra.fr.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Ball, J. M., P. Tian, C. Q. Zeng, A. P. Morris, and M. K. Estes. 1996. Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272:101-104[Abstract]. |
| 2. | Bieker, K. L., and T. J. Silhavy. 1990. The genetics of protein secretion in Escherichia coli. Trends Genet. 6:329-334[CrossRef][Medline]. |
| 3. | de Ruyter, P. G., O. P. Kuipers, and W. M. de Vos. 1996. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl. Environ. Microbiol. 62:3662-3667[Abstract]. |
| 4. |
Dower, W. J.,
J. F. Miller, and C. W. Ragsdale.
1988.
High efficiency transformation of Escherichia coli by high voltage electroporation.
Nucleic Acids Res.
16:6127-6145 |
| 5. |
Hols, P.,
P. Slos,
P. Dutot,
J. Reymund,
P. Chabot,
B. Delplace,
J. Delcour, and A. Mercenier.
1997.
Efficient secretion of the model antigen M6-gp41E in Lactobacillus plantarum NCIMB 8826.
Microbiology
143:2733-2741 |
| 6. | Kapikian, A. Z., and R. M. Chanock. 1996. Rotaviruses, p. 1657-1708. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed. Lippincott-Raven Press, New York, N.Y. |
| 7. | Kuipers, O. P., P. G. de Ruyter, M. Kleerebezen, and W. M. de Vos. 1998. Quorum sensing-controlled gene expression in lactic acid bacteria. J. Biotechnol. 64:15-21. |
| 8. | Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[CrossRef][Medline]. |
| 9. | Langella, P., and Y. Le Loir. 1999. Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system. Braz. J. Med. Biol. Res. 32:191-198[Medline]. |
| 10. |
Le Loir, Y.,
A. Gruss,
S. D. Ehrlich, and P. Langella.
1996.
Direct screening of recombinants in gram-positive bacteria using the secreted staphylococcal nuclease as a reporter.
J. Bacteriol.
178:4333 |
| 11. |
Le Loir, Y.,
A. Gruss,
S. D. Ehrlich, and P. Langella.
1998.
A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis.
J. Bacteriol.
180:1895-1903 |
| 12. | Newton, K., J. C. Meyer, A. R. Bellamy, and J. A. Taylor. 1997. Rotavirus nonstructural glycoprotein NSP4 alters plasma membrane permeability in mammalian cells. J. Virol. 71:9458-9465[Abstract]. |
| 13. |
Petrie, B. L.,
M. K. Estes, and D. Y. Graham.
1983.
Effects of tunicamycin on rotavirus morphogenesis and infectivity.
J. Virol.
46:270-274 |
| 14. |
Piard, J. C.,
I. Hautefort,
V. A. Fischetti,
S. D. Ehrlich,
M. Fons, and A. Gruss.
1997.
Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria.
J. Bacteriol.
179:3068-3072 |
| 15. | Pouwels, P. H., R. J. Leer, and W. J. Boersma. 1996. The potential of Lactobacillus as a carrier for oral immunization: development and preliminary characterization of vector systems for targeted delivery of antigens. J. Biotechnol. 44:183-192[CrossRef][Medline]. |
| 16. | Pozzi, G., M. R. Oggioni, R. Manganelli, D. Medaglini, V. A. Fischetti, D. Fenoglio, M. T. Valle, A. Kunkl, and F. Manca. 1994. Human T-helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii. Vaccine 12:1071-1077[CrossRef][Medline]. |
| 17. | Sambrook, J., E. F. Fristch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 18. | Shortle, D. 1983. A genetic system for analysis of staphylococcal nuclease. Gene 22:181-189[CrossRef][Medline]. |
| 19. |
Steidler, L.,
K. Robinson,
L. Chamberlain,
K. M. Schofield,
E. Remaut,
R. W. Le Page, and J. M. Wells.
1998.
Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine.
Infect. Immun.
66:3183-3189 |
| 20. | Suter-Crazzolara, C., and K. Unsicker. 1995. Improved expression of toxic proteins in Escherichia coli. BioTechniques 19:202-204[Medline]. |
| 21. | Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807-813. |
| 22. | Tian, P., M. K. Estes, Y. Hu, J. M. Ball, C. Q. Zeng, and W. P. Schilling. 1995. The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum. J. Virol. 69:5763-5772[Abstract]. |
| 23. | van Asseldonk, M., W. M. de Vos, and G. Simons. 1993. Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous alpha-amylase. Mol. Gen. Genet. 240:428-434[CrossRef][Medline]. |
| 24. |
Vos, P.,
M. van Asseldonk,
F. van Jeveren,
R. Siezen,
G. Simons, and W. M. de Vos.
1989.
A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope.
J. Bacteriol.
171:2795-2802 |
| 25. | Wells, J. M., K. Robinson, L. M. Chamberlain, K. M. Schofield, and R. W. Le Page. 1996. Lactic acid bacteria as vaccine delivery vehicles. Antonie Leeuwenhoek 70:317-330. |
| 26. | Wells, J. M., P. W. Wilson, and R. W. Le Page. 1993. Improved cloning vectors and transformation procedure for Lactococcus lactis. J. Appl. Bacteriol. 74:629-636[Medline]. |
| 27. |
Zagorec, M., and M. Steinmetz.
1990.
Expression of levansucrase-beta-galactosidase hybrids inhibits secretion and is lethal in Bacillus subtilis.
J. Gen. Microbiol.
136:1137-1143 |
Applied and Environmental Microbiology, April 2001, p. 1423-1428, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1423-1428.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Fernandez, A., Rodriguez, J. M., Bongaerts, R. J., Gasson, M. J., Horn, N.
(2007). Nisin-Controlled Extracellular Production of Interleukin-2 in Lactococcus lactis Strains, without the Requirement for a Signal Peptide Sequence. Appl. Environ. Microbiol.
73: 7781-7784
[Abstract] [Full Text] -
Lee, P., Faubert, G. M.
(2006). Expression of the Giardia lamblia cyst wall protein 2 in Lactococcus lactis. Microbiology
152: 1981-1990
[Abstract] [Full Text] -
Mannam, P., Jones, K. F., Geller, B. L.
(2004). Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis Protects Mice against Pharyngeal Infection with Streptococcus pyogenes. Infect. Immun.
72: 3444-3450
[Abstract] [Full Text] -
Bermudez-Humaran, L. G., Cortes-Perez, N. G., Le Loir, Y., Alcocer-Gonzalez, J. M., Tamez-Guerra, R. S., de Oca-Luna, R. M., Langella, P.
(2004). An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci. J Med Microbiol
53: 427-433
[Abstract] [Full Text] -
Dieye, Y., Hoekman, A. J. W., Clier, F., Juillard, V., Boot, H. J., Piard, J.-C.
(2003). Ability of Lactococcus lactis To Export Viral Capsid Antigens: a Crucial Step for Development of Live Vaccines. Appl. Environ. Microbiol.
69: 7281-7288
[Abstract] [Full Text] -
Chatel, J.-M., Nouaille, S., Adel-Patient, K., Le Loir, Y., Boe, H., Gruss, A., Wal, J.-M., Langella, P.
(2003). Characterization of a Lactococcus lactis Strain That Secretes a Major Epitope of Bovine Beta-Lactoglobulin and Evaluation of Its Immunogenicity in Mice. Appl. Environ. Microbiol.
69: 6620-6627
[Abstract] [Full Text] -
Germond, J.-E., Delley, M., Gilbert, C., Atlan, D.
(2003). Determination of the Domain of the Lactobacillus delbrueckii subsp. bulgaricus Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the prtB Gene in Lactococcus lactis. Appl. Environ. Microbiol.
69: 3377-3384
[Abstract] [Full Text] -
Bermudez-Humaran, L. G., Langella, P., Cortes-Perez, N. G., Gruss, A., Tamez-Guerra, R. S., Oliveira, S. C., Cardenas, O. S., Montes de Oca-Luna, R., Le Loir, Y.
(2003). Intranasal Immunization with Recombinant Lactococcus lactis Secreting Murine Interleukin-12 Enhances Antigen-Specific Th1 Cytokine Production. Infect. Immun.
71: 1887-1896
[Abstract] [Full Text] -
Bernasconi, E., Germond, J.-E., Delley, M., Fritsche, R., Corthesy, B.
(2002). Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine {beta}-Lactoglobulin Fusion Proteins. Appl. Environ. Microbiol.
68: 2917-2923
[Abstract] [Full Text] -
Miyoshi, A., Poquet, I., Azevedo, V., Commissaire, J., Bermudez-Humaran, L., Domakova, E., Le Loir, Y., Oliveira, S. C., Gruss, A., Langella, P.
(2002). Controlled Production of Stable Heterologous Proteins in Lactococcus lactis. Appl. Environ. Microbiol.
68: 3141-3146
[Abstract] [Full Text] -
Ribeiro, L. A., Azevedo, V., Le Loir, Y., Oliveira, S. C., Dieye, Y., Piard, J.-C., Gruss, A., Langella, P.
(2002). Production and Targeting of the Brucella abortus Antigen L7/L12 in Lactococcus lactis: a First Step towards Food-Grade Live Vaccines against Brucellosis. Appl. Environ. Microbiol.
68: 910-916
[Abstract] [Full Text] -
Bermudez-Humaran, L. G., Langella, P., Miyoshi, A., Gruss, A., Guerra, R. T., Montes de Oca-Luna, R., Le Loir, Y.
(2002). Production of Human Papillomavirus Type 16 E7 Protein in Lactococcus lactis. Appl. Environ. Microbiol.
68: 917-922
[Abstract] [Full Text] -
Le Loir, Y., Nouaille, S., Commissaire, J., Bretigny, L., Gruss, A., Langella, P.
(2001). Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis. Appl. Environ. Microbiol.
67: 4119-4127
[Abstract] [Full Text] -
Chatel, J.-M., Langella, P., Adel-Patient, K., Commissaire, J., Wal, J.-M., Corthier, G.
(2001). Induction of Mucosal Immune Response after Intranasal or Oral Inoculation of Mice with Lactococcus lactis Producing Bovine Beta-Lactoglobulin. CVI
8: 545-551
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»