Previous Article | Next Article 
Applied and Environmental Microbiology, December 2001, p. 5370-5376, Vol. 67, No. 12
Department of
Microbiology,1 The Center for Gene
Research and Biotechnology,2 and The
Western Dairy Center,3 Oregon State University,
Corvallis, Oregon 97331-3804, and Institute of Molecular
Genetics and Genetic Engineering, Vojvode Stepe 283, 11001 Belgrade, Yugoslavia4
Received 19 March 2001/Accepted 9 August 2001
The C repeat region of the M6 protein (M6c) from
Streptococcus pyogenes was expressed within the Pip
bacteriophage receptor on the surface of Lactococcus
lactis. M6c was also detected in the culture medium. The
pip-emm6c allele was integrated into the chromosome and
stably expressed without antibiotic selection. The level of
cell-associated surface expression of PipM6c was 0.015% of total
cellular protein. The amount of PipM6c on the cell surface was
increased about 17-fold by expressing pip-emm6c from a
high-copy-number plasmid. Replacing the native pip
promoter with stronger promoters isolated previously from
Lactobacillus acidophilus increased surface expression
of PipM6c from the high-copy-number plasmid up to 27-fold.
Concomitantly, the amount of PipM6c in the medium increased 113-fold.
The amount of PipM6c did not vary greatly between exponential- and
stationary-phase cultures. Western blots indicated that the full-length
PipM6c protein and most of the numerous proteolytic products were found
only on the cell surface, whereas only one proteolytic fragment was
found in the culture medium.
A recent approach to vaccine
development is to stimulate secretion of mucosal immunoglobulins that
inhibit colonization of specific bacterial pathogens at the mucosal
site of invasion. Because colonization is required to initiate
infection in many diseases, blocking of colonization should prevent disease.
Intranasal vaccines against Streptococcus pyogenes, which
causes "strep throat," exemplify this approach. M6 protein is a major surface component and virulence factor of S. pyogenes.
Mucosal immunization with the conserved C repeat region of M6 protein (herein referred to as M6c) protects against streptococcal challenge and inhibits pharyngeal colonization by S. pyogenes in mice
(3, 4, 11). Moreover, M6-specific secretory immunoglobulin
A (IgA) inhibits adherence of S. pyogenes to cultured human
pharyngeal cells (12).
Food-grade bacteria are emerging as possible alternatives to attenuated
pathogens for the delivery and presentation of heterologous antigens to
mucosal immune systems of animals and humans. Lactococcus lactis is a gram-positive bacterium that is used to make fermented dairy foods such as cheese and sour cream, and it has been safely consumed by humans and animals for millennia. L. lactis is
designated GRAS (generally recognized as safe) by the Food and Drug
Administration and is amenable to molecular biology techniques
(14). Many heterologous proteins have been expressed in
L. lactis (20, 33, 37, 38, 40, 43, 44),
including the full-length M6 protein (32).
L. lactis has been used as a delivery vehicle for mucosal
vaccines. It does not colonize, but it survives passage through the
gastrointestinal tract (16, 21), which makes it well
suited for delivery of antigens to the mucosal immune system
(7). Wells et al. have shown that oral or nasal delivery
of tetanus toxin fragment C expressed from L. lactis
stimulates secretory IgA against tetanus toxin fragment C and protects
against a lethal challenge (47). Other antigens expressed
from L. lactis and delivered orally or nasally also elicit a
mucosal immune response (7, 18, 34).
One of our labs has cloned and sequenced a chromosomal gene
(pip) from L. lactis that is required for
infection by one species of lactococcal bacteriophage
(15). Pip is a membrane protein that serves as a receptor
for bacteriophage (27, 42). The physiological function of
Pip is unknown, and genetic deletion of Pip causes no phenotypic change
in vitro except phage resistance (13, 22).
In this study, M6c was genetically fused to Pip and expressed in
L. lactis. Expression was increased by genetic
manipulations, and the fused protein was analyzed by Western blotting.
Bacterial strains, phages, media, growth rates, and plaque
assay.
The strains and plasmids used are listed in Table
1. L. lactis subsp.
lactis LM2301 and its isogenic pip-emm6c
derivative BG301 were grown at 30°C and maintained on M17 medium
(41) supplemented with 0.5% glucose (M17G). pGhost6-based
plasmids (Appligene, Pleasanton, Calif.) were maintained in lactococcal
strains on M17G with 5 µg of erythromycin per ml. Escherichia
coli DH5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5370-5376.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Surface Expression of the Conserved C Repeat Region
of Streptococcal M6 Protein within the Pip Bacteriophage Receptor of
Lactococcus lactis
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
mcr (Life Technologies, Rockville, Md.) was grown at
37°C in Luria-Bertani (LB) medium (35) with 20 µg of chloramphenicol per ml or 100 µg of ampicillin per ml for
maintenance of pRB04 or pUC19-derived vectors, respectively. pGhost6-derived vectors were maintained in DH5mcr on brain heart infusion (BHI) (Difco/Becton Dickinson Microbiology Systems, Sparks, Md.) with 250 µg of erythromycin per ml and 100 µg of
ampicillin per ml. pTRKH2-derived vectors were maintained in E. coli JM110 and DH5mcr on BHI with 250 µg of erythromycin per
ml. E. coli CC118(pRB04) was grown on LB medium supplemented
with chloramphenicol (20 µg/ml) or with kanamycin (30 µg/ml) and
chloramphenicol (20 µg/ml) after transposition with TnphoA.
Streptococcus gordonii GP1223 was grown at 37°C in BHI with 0.5 mg of streptomycin per ml.
TABLE 1.
Strains and plasmids
70°C in M17G medium containing 20% glycerol.
Genetic constructions. All recombinant DNA procedures were done as described by Sambrook et al. (35) or Ausubel et al. (2), except where noted. The C repeat region of emm6 (17) from nucleotide 823 through 1131 (numbering begins at the start of translation) was copied by PCR from pSMB104 using primers 5'-GCTTCCGGAAACAAAGTTTCAGAAGCAA-3' and 5'-CGATCCGGATAGCTCAGCTTTTTCTTTT-3', which include BspEI sites (shown in boldface) at their 5' ends. pSMB104 is a derivative of pSMB102 (30) that encodes M6 amino acids 1 to 7 and 222 to 441. PCR was done with Taq polymerase (Promega, Madison, Wis.) in an Idaho Technology (Idaho Falls, Idaho) 1605 air thermocycler according to the manufacturer's instructions, using the following amplification conditions: 5 min at 94°C; then 30 cycles of annealing at 45°C (0 s), elongation at 72°C (10 s), and denaturation at 94°C (0 s); and finally 2 min at 72°C. The time shown in parentheses is the time that the thermocycler dwelled after reaching the indicated temperature and changing to the next temperature. The temperature transition rate between annealing and elongation temperatures was set to 4 to 5 s/°C.
The PCR product was gel isolated, cut with BspEI, and ligated to pUCpip that had been cut with the same enzyme and dephosphorylated. The product was named pUCpipM6c. pUCpip was constructed by ligating the 5-kb XbaI fragment of pRB04 (15) to pUC19. The product of the ligation reaction of pUCpipM6c was transformed into E. coli DH5mcr (Life
Technologies). Transformants were selected on LB plates containing
ampicillin (50 µg/ml). pUCpipM6c was cut with XbaI, and
the pip-emm6c fragment was ligated to pTRKH2
(31) or pGhost6, which had been cut with XbaI.
The constructs were named pPipM6c and pGhPipM6c, respectively. The
wild-type pip allele, including its apparent promoter, was
subcloned into pTRKH2 by ligating the 5-kb XbaI fragment of
pRB04 with pTRKH2 that had been cut with XbaI. The resulting
plasmid was named pPip. pPip, pPipM6c, and pGhPipM6c were transformed
first into DH5mcr and then into L. lactis 2301.
pip-emm6c was constructed by cutting pUCpipM6c with
BsmI and isolating the 6.6-kb fragment from an agarose
electrophoretic gel. A double-stranded DNA adaptor was prepared using
the following synthetic oligonucleotides: 5'-ATCTGG-3' and
5'-AGATCT-3'. The adaptor was phosphorylated and ligated to
the 6.6-kb fragment to restore the correct reading frame to
pip-emm6c. The resulting plasmid (pUCpipM6c) was
transformed into DH5mcr, and transformants were selected on LBamp.
pUCpipM6c was cut with XbaI, and the pip-emm6c fragment was ligated to pTRKH2 that had been
cut with XbaI. Products with either orientation were
isolated. The resulting plasmid with pip-emm6c in the
same orientation as lacZ was named pPipM6c. The plasmid
with pip-emm6c in the reverse orientation as
lacZ was named pPipM6c-r.
Replacement of the pip promoter with P6 or
P16.
The XbaI site between the 3' end of the P6
promoter and the multiple cloning region was eliminated by restricting
with XbaI pTRK568 that had been isolated from E. coli DH5mcr (dam+), treating with
Klenow reagent, ligating, and transforming into E. coli
JM110 (dam). The resulting construct included a unique XbaI site in the multiple cloning region and was named
pBG568. The coding region of pip without its promoter was
copied by PCR and cloned into the unique XbaI site of
pBG568, creating pP6pip. The fidelity of the PCR-copied pip
was tested and verified by restoring phage sensitivity to the
phage-resistant strain L. lactis JK101 (13).
emm6c was inserted into pP6pip at the unique
BspEI site as described above, resulting in pP6pipM6c.
460-bp SmaI-PstI
fragment containing promoter P6 was removed and replaced with the
275-bp SmaI-PstI fragment from pPLA16
(1). The resulting plasmids were named pP16pipM6c and pP16pip.
Chromosomal allele replacement. L. lactis BG301 was constructed by replacing the chromosomal pip in LM2301 with pip-emm6c using pGhPipM6c as described previously (13). Integrants were selected at 37°C. One hundred single colonies were scored for sensitivity to erythromycin and phages c2 and sk1 as described previously (13).
Analytical PCR. Chromosomal allele replacement of pip with pip-emm6c was verified by analytical PCR as described previously (13) with either of the following primer pairs: 5'-AGTTGTTTGCCCTAGACTGGAAACG-3' and 5'-ATCCTTTGGTAAGTAAATTCC-3' or 5'-TGCAAGTAATGGTGAAGATTTAACAAATGAA-3' and 5'-GAATGCCCGATTTGATTTCAGGCCA-3'. Both PCR products spanned the BspEI site. The PCR products were purified (QIAquick PCR purification kit from Qiagen, Inc.) and sequenced as described below.
Analytical PCR analysis of the ampicillin and erythromycin resistance genes and flanking sequences of pGhost6 was done as described previously (13).Membrane protein topology mapping.
The method of Manoil
(26) was used to map the membrane topology of Pip.
E. coli CC118 was transformed with pRB04 (15) and infected with 
TnphoA. Plasmid was prepared from
pooled, dark blue colonies on indicator plates and used to transform
CC118. Plasmids from dark blue transformants on indicator plates were analyzed by restriction using XbaI, AatII, and
AflIII. Fusions within pip were sequenced in both
directions with phoA (26) and pip
(15) sequencing primers.
Immunoblot analysis. One milliliter of each culture at an optical density at 600 nm (OD600) of ca. 0.4 was centrifuged at 12,000 × g for 2 min in a 1.5-ml Microfuge tube. For experiments using stationary-phase cultures, the cultures were harvested after 15 h of incubation, and the OD600 was approximately 1.2. The supernatant was saved, and the cell pellet was washed twice with 20 mM Tris-HCl-500 mM NaCl, pH 7.5 (TBS). The washed cells were resuspended in a volume of TBS that adjusted for small differences in the OD at time of harvest and were serially diluted 1:2 or 1:4 in TBS. Likewise, the supernatants were diluted to adjust for small differences at time of harvest. An equivalent volume (0.7 ml) of each dilution and cell-free culture supernatant was vacuum filtered through nitrocellulose membranes (Schleicher and Schuell, Keene, N.H.) using a slot blot filtration manifold (Hoefer Pharmacia, San Francisco, Calif.). Each slot was washed three times with 1 ml of TBS. The membrane was removed from the manifold, blocked for 30 min with TBS containing 3% gelatin, washed for 30 min with TBS containing 0.05% Tween 20 (TTBS), and probed for 15 h with anti-M6c 10F5 monoclonal antibody (19) in TTBS containing 1% gelatin. The membrane was washed three times in TTBS for 5 min, incubated with goat anti-mouse IgG conjugated to alkaline phosphatase (Bio-Rad Laboratories, Hercules, Calif.) for 1 h, and then washed twice in TTBS and once in TBS and developed with alkaline phosphatase substrate (Bio-Rad). Developed slots were quantified by either densitometry or visual comparison with a 1:2 or 1:4 serial dilution of purified M6 protein (generously provided by Kevin Jones, Siga Pharmaceuticals, Corvallis, Oreg.) that was applied to every gel.
Western blotting. Cells and cell-free culture medium were prepared as described previously (24), except that a mixture of the following protease inhibitors at the indicated final concentrations was added to the washed cells and cell-free medium before addition of trichloroacetic acid: phenylmethylsulfonyl fluoride (35 µg/ml), aprotinin (4 µg/ml), chymostatin (37 µg/ml), leupeptin (700 ng/ml), soybean trypsin inhibitor (1 µg/ml), EDTA (500 ng/ml), benzamidine (35 µg/ml), and acetamide (35 µg/ml). All inhibitors were purchased from Sigma Chemicals (St. Louis, Mo.), U.S. Biochemicals (Cleveland, Ohio), or Boehringer Mannheim (Indianapolis, Ind.). Samples were analyzed on a 10 or 12% discontinuous electrophoretic gel (23), transferred to a nitrocellulose membrane (2), and developed as described above for the immunoblot. Prestained high-molecular-weight standards (Sigma) and horse heart cytochrome c (Sigma) were used as size markers.
DNA sequencing. DNA was sequenced at Oregon State University Center for Gene Research and Biotechnology using the dideoxy method (36) on an Applied Biosystems 373A DNA sequencer.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Splicing site for M6c within Pip. An analysis of the deduced amino acid sequence of Pip predicts that the N-terminal two-thirds of the protein would face the external side of the plasma membrane (15). This region might be an ideal site into which M6c could be spliced, so that it would be exposed to the outside of the cell. However, the actual structure of Pip and its topology relative to the plasma membrane have never been tested.
Therefore, the molecular topology of Pip was analyzed by the phoA method of membrane protein mapping. Random genetic fusions between pip and phoA were constructed and expressed in E. coli. Fusion junctions from 10 pip-phoA isolates with high levels of alkaline phosphatase activity were mapped by restriction analysis and sequenced. All but three of the junctions were within the sequence of pip predicted to encode the large, hydrophilic, extracellular region of Pip (Fig. 1). Three junctions (V714, S717, and G751) were found within hydrophobic regions predicted to span the plasma membrane. These data confirm the external topology of the region of Pip encoded by bp 50 to 700 and suggest that this portion of Pip is a suitable site for the introduction of heterologous protein sequences.
|
Construction of L. lactis with
pip-emm6c.
The coding region of M6c
(emm6c) was copied by PCR and inserted in frame after bp 321 in pip (Fig. 2A and B). The
genetic fusion (pip-emm6c) included the putative native
promoter and transcriptional terminator of pip.
|
650 bp from chromosomal DNA
of BG301 and one band of 330 bp from chromosomal DNA of LM2301 (data
not shown). The predicted sizes are 653 and 331 bp, respectively. DNA
sequencing of the PCR products confirmed the exchange of alleles (data
not shown). Neither the ermAM nor the bla marker
of the recombinant plasmid was detected by analytical PCR using DNA
from BG301, although control reactions with the same primers and
recombinant plasmid yielded the predicted products (data not shown).
M6c expression in L. lactis BG301.
M6c was
expressed in exponential-phase cultures of L. lactis BG301.
Equivalent amounts of washed cells and cell-free growth medium from the
same cultures were analyzed by immunoblotting using a monoclonal
antibody against M6c. The amount of M6c exposed to the surface of BG301
was 0.014 µg/ml/OD600 unit, which is equivalent to 0.007 µg/108 cells or 0.015% of total
cellular protein (Table 2). M6c was below
the limit of detection in the culture medium. There was no detectable
expression of M6c from control cultures of LM2301.
|
M6c expression from a multicopy plasmid. The pip-emm6c allele was subcloned to a high-copy-number vector (pPipM6c) and used to transform L. lactis LM2301. Surface expression of PipM6c from LM2301(pPipM6c) was 17-fold higher than that from BG301 (Table 2). The amount of PipM6c in the growth medium of LM2301(pPipM6c) was 1.0 µg/ml/OD unit, which is fourfold higher than the amount on the cell surface. No M6c was detected from a control that expressed the wild-type pip allele from the same plasmid.
M6c expression from S. gordonii. M6c was measured on the surface and in the cell-free culture medium of S. gordonii GP1223, which is genetically engineered to express the C repeat region of M6 on its surface (8). Cells were harvested from cultures at exponential phase. Compared to LM2301(pPipM6c), S. gordonii GP1223 expressed 50 times more antigen on its cell surface (Table 2). However, M6c in the culture medium of GP1223 was only 28% of that in the medium of LM2301(pPipM6c) (Table 2).
Effect of growth phase on expression of M6c. LM2301(pPipM6c) was grown to stationary phase, and the expression of M6c was measured. The amounts of M6c on the cells and in the culture medium were nearly the same as those in the equivalent fractions from exponential-phase cultures when normalized for differences in cell densities (Table 2).
M6c expression from deletion allele of pip. Detection of M6c within the context of the chimeric protein may be limited by inaccessibility of the monoclonal antibody to its epitope. If the amount of Pip in the chimera was significantly reduced, the protein conformation might change, which might increase accessibility of the antibody to the M6c epitope. With this in mind, 451 aminoacyl residues of Pip adjacent to the M6c epitope were genetically deleted from PipM6c (Fig. 2C).
The deletion allele (pip-emm6c) was expressed in
L. lactis from the same multicopy plasmid used to
construct pPipM6c. Immunoblot analysis of the resulting strain,
LM2301(pPipM6c), showed that the amount of M6c on the cell
surface was similar to that on LM2301(pPipM6c) (Table 2). The cell-free
culture medium of LM2301(pPipM6c) contained 26% of that in the
medium of LM2301(pPipM6c).
The pip-emm6c allele was subcloned in the opposite
orientation on the same plasmid and expressed in L. lactis.
Cultures of the resulting strain, LM2301(pPipM6c-r), expressed about
22 and 12% of the amounts of PipM6c on the cell surface and in the
cell-free medium, respectively, expressed by LM2301(pPipM6c) (Table
2).
Replacement of pip promoter. The level of expression of PipM6c was increased by replacing the pip promoter with promoters P6 and P16, which were previously isolated from Lactobacillus (1, 10). In L. lactis, the expression of the cat-86 gene was about 1.5-fold higher from P16 than from P6 (1, 10). Immunoblot analysis showed that in L. lactis(pP6pipM6c), P6 increased surface expression of PipM6c about 14-fold compared to that from the native pip promoter (Table 2). P16 increased cell surface expression of PipM6c another twofold above that from P6. The level of surface expression of PipM6c from promoter P16 in L. lactis was approximately half of that from S. gordonii GP1223.
The greatest increase in expression of M6c occurred in the culture medium. When expressed from promoters P6 and P16, the amounts of M6c in the culture medium increased 57- and 113-fold, respectively (Table 2). This was 201- and 403-fold higher, respectively, than the levels expressed in the culture media from S. gordonii GP1223. M6c was not detected on the surface or in the medium of cultures of LM2301(pP6pip) or LM2301(pP16pip).Molecular size of M6c on cell surface and in culture medium.
Washed cells and cell-free culture media were analyzed by Western
blotting with anti-M6c monoclonal antibody. The results (Fig.
3) showed seven intensely stained bands
associated with the cell, which had mean molecular masses (± standard
deviations) of 111 (±2), 106 (±4), 100 (±8), 52 (±1), 49 (±1), 32 (±0), and 29 (±1) kDa. This agrees closely with the
hypothetical size of the chimeric protein (106 kDa), considering that
coiled-coil proteins like M6 and Pip (B. Geller, unpublished analysis)
are notorious for running at aberrant sizes on denaturing gels
(17). The relative intensities of the three largest bands
varied significantly among blots, and often the top band was most
intense. There were numerous bands of lesser intensity that varied in
number depending on the amount of time that the blot was left in the
chromagenic reagent, suggesting that PipM6c may have been
proteolytically degraded during sample preparation, despite the
inclusion of eight protease inhibitors. It appears unlikely that the
fragments were produced in vivo, because many of the fragments are too
small to include both M6c and the cell membrane anchoring region
(15) at the carboxy terminus.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
We have shown that Pip can be used to direct surface expression of a heterologous protein in L. lactis. The results of the pip-phoA fusions suggested that the region within Pip between residues 64 and 684 is external to the plasma membrane. This guided our selection of the site at residue 108 for fusing Pip to M6c. Indeed, PipM6c was detected on the surface of intact cells, which shows that the region of Pip at residue 108 is outside the plasma membrane. Further analysis, including lacZ fusions, will be necessary to map the topology of Pip in more detail.
Insertion of M6c disrupted the phage receptor activity of Pip. This phenotypic change was exploited in this study to select strains that had undergone pip-emm6c allelic exchange. A rapid, direct selection for exchange of pip alleles has been previously demonstrated using a pUC-based integration vector that lacks a gram-positive origin of replication but encodes a selectable marker for L. lactis (22). The pip locus may be an ideal location for inserting recombinant alleles into the chromosome of L. lactis, because pip is not required for viability or rapid growth in vitro (13), and allelic replacement can be selected directly on plates that contain phage.
Expression of cell surface-associated M6c was increased 17-fold by moving pip-emm6c from the chromosome to a multicopy plasmid. Replacing the native promoter increased total (cell-associated plus cell-free medium) expression of PipM6c up to about 100-fold, of which cell-associated PipM6c was about 7% of total cellular protein. Most of the increase was found in the cell-free culture medium. The reason for the appearance of much of the additional PipM6c in the cell-free culture medium is unknown. One possibility is that overexpression of PipM6c exceeded the capacity of the cell to sort or attach PipM6c to the surface of the cell, resulting in release of the excess to the medium. Another possibility is that proteolytic enzymes or the acidic conditions of the lactococcal culture released the protein from the cell surface. In any case, finding a 79-kDa fragment of PipM6c in the culture medium shows that a large fragment of PipM6c is not anchored to the cell.
The amount of antigen delivered by L. lactis to the mucosal immune system may be limited to that expressed in vitro before vaccination (46). This is because L. lactis does not colonize the gastrointestinal tract (16, 21), and its metabolic activity in the gastrointestinal tract is not required for effectiveness as a mucosal vaccine (29, 34). The relatively high level (7% of total cellular protein) of surface-expressed antigen (M6c) that we report here is similar to levels of expression reported for other antigens in effective lactococcal vaccines (28, 29, 34, 46, 47) and may be sufficient for eliciting a mucosal immune response.
Surface expression of immunogens may be the most effective way of presenting lactococcus-based vaccines to the immune system (28, 46). Particulate-associated proteins evoke a stronger response and are less likely to produce tolerance in the mucosal immune system than soluble proteins (5, 6, 48). One report showed that subcutaneous injection of tetanus toxin fragment C elicited a stronger systemic immune response when attached to the surface of L. lactis than an equivalent dose of the intracellular antigen (28).
Another factor that may be important for lactococcus-based vaccines is access of surface-attached antigens to the immune system. One report has shown that a heterologous antigen can be attached to the surface of L. lactis and is accessible on intact cells (39). Another report showed that although surface-attached tetanus toxin fragment C was immunogenic, it was not accessible on intact lactococcal cells by using immunolabeling techniques (28). Clearly, we have shown in this report that M6c is accessible on the surface of intact L. lactis. It remains to be learned if this accessibility translates to higher immunogenicity or if this could create a liability for exposure to proteases or stomach acid.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grants from the Thrasher Fund (Salt Lake City, Utah), the U.S. Department of Agriculture (DMI), the Western Dairy Center (Logan, Utah), and the Tartar Fund (Oregon State University).
We thank Shirley Walker and Todd Klaenhammer (North Carolina State University, Raleigh) for pTRKH2, Colin Manoil (University of Washington, Seattle) for TnphoA and CC118, Kevin Jones (Siga Pharmaceuticals, Corvallis, Oreg.) for monoclonal antibody 10F5, and Peter Bottomley (Oregon State University) for editorial suggestions.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-1845. Fax: (541) 737-0496. E-mail: gellerb{at}orst.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Aresnijevic, S., and L. Topisirovic. 2000. Molecular analysis of mutated Lactobacillus acidophilus promoter-like sequence P15. Can. J. Microbiol. 46:938-945[CrossRef][Medline]. |
| 2. | Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1994. Current protocols in molecular biology. Green Publishing Associates, New York, N.Y. |
| 3. |
Bessen, D., and V. A. Fischetti.
1988.
Influence of intranasal immunization with synthetic peptides corresponding to conserved epitopes of M protein on mucosal colonization by group A streptococci.
Infect. Immun.
56:2666-2672 |
| 4. | Bessen, D., and V. A. Fischetti. 1990. Synthetic peptide vaccine against mucosal colonization by group A streptococci. I. Protection against a heterologous M serotype with shared C repeat region epitopes. J. Immunol. 145:1251-1256[Abstract]. |
| 5. | Challacombe, S. J. 1983. Salivary antibodies and systemic tolerance in mice after oral immunization with bacterial antigens. Ann. N.Y. Acad. Sci. 409:177-192[Medline]. |
| 6. | Challacombe, S. J., D. Rahman, H. Jeffery, S. S. Davis, and D. T. O'Hagan. 1992. Enhanced secretory IgA and systemic IgG antibody responses after oral immunization with biodegradable microparticles containing antigen. Immunology 76:164-168[Medline]. |
| 7. | Chamberlain, L., J. M. Wells, K. Robinson, K. Schofield, and R. Le Page. 1997. Mucosal immunization with recombinant Lactococcus lactis, p. 83-106. In G. Pozzi, and J. M. Wells (ed.), Gram-positive bacteria as vaccine vehicles for mucosal immunization. Landes Bioscience, Austin, Tex. |
| 8. | Coyle, T. C., C. A. Franke, and D. E. Hruby. 1997. Development of a radioactive protein A-based assay for analysis of surface protein expression in gram-positive bacteria. Appl. Environ. Bacteriol. 63:2477-2480. |
| 9. |
Dao, M. L., and J. J. Ferretti.
1985.
Streptococcus-Escherichia coli shuttle vector pSA3 and its use in cloning of streptococcal genes.
Appl. Environ. Microbiol.
49:115-119 |
| 10. | Djordjevic, G., B. Bojovic, N. Miladionov, and L. Topisirovic. 1997. Cloning and molecular analysis of promoter-like sequences isolated from the chromosomal DNA of Lactobacillus acidophilus ATCC 4356. Can. J. Microbiol. 43:61-69[Medline]. |
| 11. |
Fischetti, V. A.,
W. M. Hodges, and D. E. Hruby.
1989.
Protection against streptococcal pharyngeal colonization with a vaccinia:M protein recombinant.
Science
244:1487-1490 |
| 12. |
Fluckinger, U.,
K. F. Jones, and V. A. Fischetti.
1998.
Immunoglobulins to group A streptococcal surface molecules decrease adherence to and invasion of human pharyngeal cells.
Infect. Immun.
66:974-979 |
| 13. | Garbutt, K. C., J. Kraus, and B. L. Geller. 1997. Bacteriophage resistance in Lactococcus lactis engineered by replacement of a gene for a bacteriophage receptor. J. Dairy Sci. 80:1512-1519[Abstract]. |
| 14. | Gasson, M. J., and W. M. de Vos. 1994. Genetics ad biotechnology of lactic acid bacteria. Chapman and Hall, London, United Kingdom. |
| 15. |
Geller, B.,
R. G. Ivey,
J. E. Trempy, and B. Hettinger-Smith.
1993.
Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2.
J. Bacteriol.
175:5510-5519 |
| 16. | Gruzza, M., Y. Deval-Iflah, and R. Ducluzeau. 1992. Colonization of the digestive tract of germfree mice by genetically engineered strains of Lactococcus lactis: study of recombinant DNA stability. Microb. Releases 1:165-171[Medline]. |
| 17. |
Hollingshead, S. K.,
V. A. Fischetti, and J. R. Scott.
1986.
Complete nucleotide sequence of type 6 M protein of the group A Streptococcus.
J. Biol. Chem.
261:1677-1686 |
| 18. |
Iwaki, M.,
N. Okahashi,
I. Takahashi,
T. Kanamoto,
Y. Sugita-Konishi,
K. Aibara, and T. Koga.
1990.
Oral immunization with recombinant Streptococcus lactis carrying the Streptococcus mutans surface protein antigen gene.
Infect. Immun.
58:2929-2934 |
| 19. |
Jones, K. F.,
S. A. Khan,
B. W. Erickson,
S. K. Hollingshead,
J. R. Scott, and V. A. Fischetti.
1986.
Immunochemical localization and amino acid sequences of crossreactive epitopes within the group A streptococcal M6 protein.
J. Exp. Med.
164:1226-1238 |
| 20. | Klessen, C., K. H. Schmidt, J. J. Ferretti, and H. Malke. 1988. Tripartite streptokinase gene fusion vectors for gram-positive and gram-negative prokaryotes. Mol. Gen. Genet. 212:295-300[CrossRef][Medline]. |
| 21. | Klijn, N., A. H. Weerkamp, and W. M. de Vos. 1995. Genetic marking of Lactococcus lactis shows its survival in the human gastrointestinal tract. Appl. Environ. Microbiol. 61:2771-2774[Abstract]. |
| 22. | Kraus, J., and B. L. Geller. 1998. Membrane receptor for prolate phages is not required for infection of Lactococcus lactis by small or large isometric phages. J. Dairy Sci. 81:2329-2335[Abstract]. |
| 23. | Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[CrossRef][Medline]. |
| 24. |
Le Loir, Y.,
A. Gruss,
S. D. Ehlich, and P. Langella.
1998.
A nine-residue synthetic propeptide enhances secretion of heterologous proteins in Lactococcus lactis.
J. Bacteriol.
180:1895-1903 |
| 25. |
Maguin, E.,
P. Duwat,
T. Hege,
D. Ehrlich, and A. Gruss.
1992.
New thermosensitive plasmid for gram-positive bacteria.
J. Bacteriol.
174:5633-5638 |
| 26. |
Manoil, C.
1991.
Analysis of membrane protein topology using alkaline phosphatase and  -galactosidase gene fusions.
Methods Cell Biol.
34:61-75[Medline].
|
| 27. |
Monteville, M. R.,
B. Ardestani, and B. L. Geller.
1994.
Lactococcal bacteriophages require a host cell wall carbohydrate and a plasma membrane protein for adsorption and ejection of DNA.
Appl. Environ. Microbiol.
60:3204-3211 |
| 28. | Norton, P. M., H. W. G. Brown, J. Wells, A. M. Macpherson, P. W. Wilson, and R. W. F. Le Page. 1996. Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis. FEMS Immunol. Med. Microbiol. 14:167-177[CrossRef][Medline]. |
| 29. | Norton, P. M., J. M. Wells, H. W. G. Brown, A. M. Macpherson, and R. W. F. Le Page. 1997. Protection against tetanus toxin in mice nasally immunized with recombinant Lactococcus lactis expressing tetanus toxin fragment C. Vaccine 15:616-619[CrossRef][Medline]. |
| 30. | Oggioni, M. R., and G. Pozzi. 1996. A host-vector system for heterologous gene expression in Streptococcus gordonii. Gene 169:85-90[CrossRef][Medline]. |
| 31. | O'Sullivan, D. J., and T. R. Klaenhammer. 1993. High and low copy number Lactococcus shuttle vectors with features for clone screening. Gene 137:227-231[CrossRef][Medline]. |
| 32. |
Piard, J.-C.,
I. Hautefort,
V. A. Fischetti,
S. D. Ehlich,
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 |
| 33. |
Pillidge, C. J., and L. E. Pearce.
1991.
Expression of a -galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis.
J. Appl. Bacteriol.
71:78-85[Medline].
|
| 34. | Robinson, K., L. M. Chamberlain, K. M. Schofield, J. M. Wells, and R. W. F. Le Page. 1997. Oral vaccination of mice against tetanus with recombinant Lactococcus lactis. Nat. Biotechnol. 15:653-657[CrossRef][Medline]. |
| 35. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 36. |
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467 |
| 37. | Simmons, G., G. Rutten, M. Hornes, M. Nujhuis, and M. van Asseldonk. 1992. Production of prochymosin in lactococci. Adv. Exp. Med. Biol. 306:115-120. |
| 38. |
Steidler, L.,
W. Hans,
L. Schotte,
S. Neirynck,
F. Obermeier,
W. Falk,
W. Fiers, and E. Remaut.
2000.
Treatment of murine colitis by Lactococcus lactis secreting interleukin-10.
Science
289:1352-1355 |
| 39. |
Steidler, L.,
J. Viaene,
W. Fiers, and E. Remaut.
1998.
Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A.
Appl. Environ. Microbiol.
64:342-345 |
| 40. | Steidler, L., J. M. Wells, A. Raeymaekers, J. Vandekerckhove, W. Fiers, and E. Remult. 1995. Secretion of biologically active murine interleukin-2 by Lactococcus lactis subsp. lactis. Appl. Environ. Microbiol. 61:1627-1629[Abstract]. |
| 41. | Terzaghi, B. E., and W. E. Sandine. 1975. Improved media for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807-813. |
| 42. |
Valyasevi, R.,
W. E. Sandine, and B. L. Geller.
1991.
A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2.
J. Bacteriol.
173:6095-6100 |
| 43. |
van de Guchte, M.,
J. J. M. B. Kodde,
M. van der Bossen,
J. Kok, and G. Venema.
1990.
Heterologous gene expression on Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.
Appl. Environ. Microbiol.
56:2606-2611 |
| 44. |
van de Guchte, M.,
J. M. B. M. van der Bossen,
J. Kok, and G. Venema.
1989.
Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis.
Appl. Environ. Microbiol.
55:224-228 |
| 45. |
Walsh, P. M., and L. L. McKay.
1981.
Recombinant plasmid associated with cell aggregation and high-frequency conjugation of Streptococcus lactis ML3.
J. Bacteriol.
146:937-944 |
| 46. | Wells, J. M., K. Robinson, L. M. Chamberlain, K. M. Schofield, and R. W. F. Le Page. 1996. Lactic acid bacteria as vaccine delivery vehicles. Antonie Leeuwenhoek 70:317-330. |
| 47. | Wells, J. M., P. W. Wilson, P. M. Norton, M. J. Gasson, and R. W. F. Le Page. 1993. Lactococcus lactis: high level expression of tetanus toxin fragment C and protection against lethal challenge. Mol. Microbiol. 8:1155-1162[Medline]. |
| 48. |
Wold, A. E.,
U. I. H. Dahlgren,
L. A. Hanson,
I. Mattsby-Baltzer, and T. Midvetdt.
1989.
Difference between bacterial and food antigens in mucosal immunogenicity.
Infect. Immun.
57:2666-2673 |
| 49. | Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119[CrossRef][Medline]. |
Applied and Environmental Microbiology, December 2001, p. 5370-5376, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5370-5376.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
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] -
De Haard, H. J. W., Bezemer, S., Ledeboer, A. M., Muller, W. H., Boender, P. J., Moineau, S., Coppelmans, M.-C., Verkleij, A. J., Frenken, L. G. J., Verrips, C. T.
(2005). Llama Antibodies against a Lactococcal Protein Located at the Tip of the Phage Tail Prevent Phage Infection. J. Bacteriol.
187: 4531-4541
[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]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»