Flanders Interuniversity Institute for
Biotechnology, Department of Molecular Biology, Universiteit Gent,
B-9000 Ghent, Belgium
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TEXT |
Cell wall sorting of surface
proteins in gram-positive bacteria has been attributed to the presence
of a particular carboxy-terminal protein domain composed of an LPXTG
motif, a hydrophobic core of approximately 30 amino acid residues, and
a positively charged (Arg- or Lys-rich) tail (3, 18, 19).
The mechanism and the chemical nature of the coupling have been
described previously (13, 17) and involve a proteolytic
cleavage of the polypeptide after the threonine residue in the LPXTG
motif followed by covalent linking to the pentaglycine peptide in the
peptidoglycan layer of Staphylococcus aureus. The LPXTG
motif and overall structure of the anchor have been identified in
several surface-associated proteins in gram-positive organisms (4,
11, 12, 18, 20, 22, 25). Also, the PrtP anchor has been used for
the attachment of heterologous proteins to the cell wall
(27). Wells et al. (27) described a potential use
of surface anchoring by demonstrating the enhancement of the immune
response to a heterologous antigen in vaccination vectors. Other
possible applications reside in the immobilization of enzymes at the
bacterial surface, the development of enzyme-coated microspheres, and
the immobilization of productory microorganisms at ligand-coated
surfaces.
An enzymatic activity termed "sortase" has been postulated to be
involved in coupling and cell wall localization (17). It is
now generally accepted that the mechanism of sorting in gram-positive organisms is highly conserved and may have evolved through gene copying
and lateral transfer of genetic information (4). It has also
been shown that surface-localized protein domains can be grafted on the
S. aureus protein A (SPA) anchor and be efficiently sorted
to the S. aureus cell wall (18). The SPA anchor
sequence has been used successfully in the attachment of chimeric
proteins, consisting in part of a single-chain antibody fragment and
the anchor, to the cell wall of Staphylococcus xylosus and
Staphylococcus carnosus (5). Here we show that
this anchor structure retains its sorting capacity for recombinant
molecules when used in a different genus of gram-positive bacteria. We
made use of the SPA anchor to achieve functional exposition of
streptavidin (SA) at the surface of the food-grade organism
Lactococcus lactis. We chose to use SA as a tool for the
detection and demonstration of surface anchoring. However, the
described constructs could be used to immobilize L. lactis
at solid surfaces for production purposes.
Expression vectors for surface display.
By PCR amplification
(15), we cloned the SPA anchor from S. aureus
Cowan I (NCTC 8530) from genomic DNA isolated by the method of Marmur
(10). PCR amplification was performed with Vent DNA
polymerase (New England Biolabs, Beverly, Mass.) and the
oligonucleotides 5'-GCTCAGGATCCAAAAGAGGAAGACAACAACAAGCC-3' and
5'-CCGCGTCTAGATATCTATCGTTGTGTATTGTTTGTTTTTATAGTTCGCG-3'.
The oligonucleotides were designed to introduce a
BamHI and an XbaI restriction site 5' and 3' from
the SPA anchor, respectively. The sequence of the subcloned 621-bp PCR
fragment was determined by the dye terminator method and was found to
be identical to the SPA anchor sequence reported earlier
(21).
Several intermediate plasmid constructs were used to arrive at the
structures of plasmids pL2SA and pL2SAX, depicted in Fig. 1. Primary transformants were obtained by
the CaCl2 method in Escherichia coli MC1022
[araD139 
(ara leu)7697
(lacZ)M15 galU galK rpsL] (2). For
reasons which are unclear, plasmids carrying the pSH71 replicon
(8) suffered rearrangements when transformed into the more
commonly used isogenic strain MC1061 (same genotype as above except for
(lac)X74 and hsdR2)
(2). Plasmid pL2SA contains the coding sequence for mature
SA fused precisely to the sequence encoding the last amino acid of the
Usp45 signal peptide (26), as present in pLET2N
(24). In plasmid pL2SAX, the termination codon was changed
to a sense codon by site-directed mutagenesis. The resulting
BamHI site was joined to the BamHI site of the
SPA fragment present in a pLET2N-type vector. The sequences at the
several junction points are shown in Fig. 1.
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FIG. 1.
Schematic overview of the described expression units for
SA (pL2SA) and the SA-SPA anchor (pL2SAX) fusion protein. The genes are
preceded by the phage T7 gene 10 promoter (pT7) and ribosome binding
site (g10RBS). Sequence numbering: usp45, as in reference
26; streptavidin (sa), as in reference
1; protein A anchor (spax), as in
reference 21
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The expression plasmids pL2SA and pL2SAX were introduced by
electroporation (28) into the expression strain
MG1820(pILPOL) (29). Conditions for growth and induction
were as described previously (24). Essentially, cells were
pregrown at 30°C in GM17S, which is M17 (Difco) containing (per
liter) 5 g of glucose, 5 mg of chloramphenicol, and 5 mg of
erythromycin. At an optical density at 600 nm of 0.5, the cells were
collected by centrifugation and resuspended in LM9S, which contains
(per liter) 6 g of Na2HPO4, 3 g of
KH2PO4, 1 g of NH4Cl, 0.5 g of NaCl, 2 mmol of MgSO4, 25 mmol of NaHCO3,
25 mmol of Na2CO3, 0.1 mmol of
CaCl2, 5 g of lactose, 5 g of Casitone (Difco), 5 mg of chloramphenicol, and 5 mg of erythromycin. The cells were
harvested in mid-log phase at an optical density at 600 nm of 1.5. The
plasmid pILPOL carries a lac expression cassette driving the
T7 RNA polymerase which is repressed in the absence of lactose by the
LacR protein. In this system, the expression plasmids pL2SA and pL2SAX
directed the inducible synthesis of polypeptides, reactive with rabbit
antiserum to SA (R-
-SA), with apparent molecular masses of 17 and 32 kDa, respectively, which is in good agreement with the calculated
values (Fig. 2).
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FIG. 2.
Expression and fractionation of recombinant SA proteins
synthesized from pL2SA and pL2SAX in L. lactis
MG1820(pILPOL). Proteins were separated by SDS-15% PAGE and revealed
by immunoblotting with rabbit anti-SA antiserum. Molecular mass markers
were low-range prestained markers (Bio-Rad, Hercules, Calif.).
Fractions represent proteins present in the growth medium, proteins
released by lysostaphin treatment, and proteins present in the residue
after that treatment. Proteins released by boiling of intact cells in
Laemmli cracking buffer are presented in the lanes labeled
"cracking released." Arrowhead indicate discrete protein bands of
relevant sizes corresponding to SA and SAX derivatives.
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Cellular location of the expressed polypeptides.
The cells
from 10-ml induced cultures were collected by low-speed centrifugation.
The supernatant was further centrifuged at 100,000 × g
for 1 h. This supernatant was extracted with equilibrated phenol.
The proteins were precipitated from the phenol phase at 
20°C by the
addition of 2.5 volumes of ethanol. The precipitate was resuspended in
sample buffer and prepared for sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). The collected cells were washed three
times with a Tris-buffered saline solution (20 mM Tris-HCl, pH 7.5; 150 mM NaCl) (TBS) and resuspended in 250 µl of a 10% sucrose solution
in 20 mM Tris-HCl, pH 7.5. After the addition of 0.6 mg of lysostaphin
(Sigma, St. Louis, Mo.), the cell suspension was incubated at 37°C
for 1 h and centrifuged at low speed. The supernatant was called
the lysostaphin-released protein fraction. The remaining pellet was
boiled in SDS-PAGE sample buffer. This fraction was called the residue.
Quantities equivalent to 1 ml of culture were separated by SDS-PAGE,
and the protein bands were revealed by standard immunoblotting (Western blotting) procedures (Fig. 2).
SA was efficiently secreted and accumulated in the growth medium of
MG1820(pILPOL, pL2SA). In contrast, no proteins reacting with R--SA
could be observed in the growth medium of MG1820(pILPOL, pL2SAX). This
finding indicates that the SAX (SA-SPA anchor) fusion protein was
anchored in the cell wall of L. lactis. To further strengthen this observation, washed cells were treated with
lysostaphin. This enzyme has been shown to release proteins which are
linked to the pentaglycine peptide in the staphylococcal peptidoglycan (18). We found that this hydrolase could also act on the
L. lactis peptidoglycan, although its activity was strongly
dependent on pretreatment of the cells with 150 mM NaCl. Induced
MG1820(pILPOL, pL2SA) released only a slight amount of SA after this
treatment. This material is likely to represent either a fraction in
transit or molecules nonspecifically trapped in the cell wall.
MG1820(pILPOL, pL2SAX) cells, on the other hand, released a major part
of their R--SA-reactive protein. Residue fractions from
MG1820(pILPOL, pL2SA) showed no traces of R--SA-reactive
polypeptides. A major band, equal in size to the lysostaphin-released
protein, was detected in the residue of the induced MG1820(pILPOL,
pL2SAX) culture. This probably represented proteins which had not
diffused out of the cell wall during lysostaphin digestion. After being
boiled in SDS-PAGE sample buffer, induced MG1820(pILPOL, pL2SAX) cells released an R--SA-reactive polypeptide with an apparent molecular mass slightly larger than that of the protein found in the fractions discussed above. We expect these to be non-carboxy-terminally truncated, and therefore not covalently attached, SAX molecules which
are physiologically relevant intermediates in sorting (14). The bulk of the R--SA-reactive proteins, i.e., the sum of the lysostaphin-released and residue fractions, was not released by boiling
in SDS, indicating a very firm attachment of the SAX fusion protein to
the cell wall of L. lactis. The coupling of the SPA anchor
to the S. aureus peptidoglycan occurs at the pentaglycine moiety after the threonine in an LPETG motif present in the anchor. This coupling has been proposed to be performed by a sortase
(17). Anchored molecules can be released by the action of
lysostaphin, a hydrolase which cleaves in the pentaglycine
(18). In the L. lactis peptidoglycan, the
L-Ala-D-Glu(NH2)-L-Lys-D-Ala
peptides, bound at the N-acetylmuramic acid residues in the
glycan strands, are connected from the L-Lys of one chain
to the D-Ala of another by a D-Asp interpeptide
bridge (16). This D-Ala thus forms the structural analog of the pentaglycine interpeptide bridge in the S. aureus peptidoglycan. We showed that the SAX molecules
were released by treatment with lysostaphin. Other workers have
reported that this lysostaphin preparation contains many impurities
(9) and that, among other activities, an
N-acetylmuramyl-L-alanine amidase activity forms
part of the crude starting material (7). We believe that the
SAX molecules are connected covalently to the peptidoglycan. Treatment
with an N-acetylmuramyl-L-alanine amidase
activity would then yield a discrete SAX band of the size seen in Fig.
2. In particular, our finding supports the high degree of functional
homology between the L. lactis and S. aureus
sortases. Up to now, the high degree of similarity between the sorting
mechanisms of different gram-positive bacteria has been postulated by
the demonstration of the presence of the conserved anchor structure in
different cell wall-bound proteins, by the coupling of foreign proteins
to homologous anchors (18), and by the use of heterologous anchors (6, 18). Here we have provided evidence that a
heterologous anchor can be sorted successfully in L. lactis,
in agreement with earlier observations (14).
Surface localization of the SAX fusion protein.
The presence
of SA at the outside surface of MG1820(pILPOL, pL2SAX) was assessed as
described for surface-localized fusion proteins in E. coli
(23). The results are shown in Fig.
3. Control setups, involving the
incubation of induced cultures of MG1820(pILPOL, pL2SA) and
MG1820(pILPOL, pL2SAX) in TBS containing 1% bovine serum albumin (BSA)
followed by three washing cycles, filtration, and reaction with
horseradish peroxidase (POD) substrate, showed that the cells were
essentially free of nonspecific background activity in this assay. On
the other hand, when induced MG1820(pILPOL, pL2SA) and MG1820(pILPOL,
pL2SAX) cultures were challenged with biotinylated POD (B-POD), a
strong signal was observed in the SAX-expressing cells whereas no
signal could be detected for cells expressing secreted SA. To verify
that the observed signals were due to the actual binding of the biotin
moiety of B-POD to the immobilized SA and not, e.g., to the nonspecific
binding of the B-POD complex to the anchor, or to any other altered
structure in the cell wall, caused by the expression of SAX, we
incubated induced cultures with a B-POD solution which had previously
reacted with a 30-µg ml
1 solution of SA in TBS. The
cultures were further processed in a manner identical to that described
above. No signal was detected in this assay.
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FIG. 3.
Biotin binding capability of induced L. lactis MG1820(pILPOL) cells carrying the SA expression plasmids
indicated. The lanes marked "negative" represent cells which were
incubated in TBS-1% BSA. The lanes marked "B-POD" represent cells
which were incubated with B-POD. The lanes marked "B-POD/SA"
represent cells incubated with B-POD, which had been blocked with an
excess of SA. After incubation, cells were washed, filtered, and
revealed with POD substrate.
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Binding of SAX-expressing cells to biotinylated surfaces.
In
this assay, biotinylated alkaline phosphatase was immobilized on a
polystyrene surface (custom-made Maxisorp 35-mm-diameter petri dishes;
Nunc, Roskilde, Denmark). The surface was further blocked with a 1%
solution of BSA in TBS. Induced cells of strain MG1820(pILPOL, pL2SA)
or MG1820(pILPOL, pL2SAX) were allowed to interact on these
surfaces for 1 h. After washing, the number of bound cells present
in randomly picked areas was counted under the light microscope. The
results are presented in Fig. 4.
SAX-expressing cells, but not SA-expressing cells, were found to bind
with high efficiency to the biotinylated surfaces. The binding could be completely prevented by preincubation of the plates with a 30-µg ml1 solution of SA (Fig. 4). The density of binding was
calculated to be approximately 2 × 105 per
mm2. The cells were harvested in mid-log phase, and
approximately 109 were applied per plate. From the
observations recorded, we concluded that about 108 cells
were bound. Clearly, this method could be used in a panning selection
strategy depending on surface-located receptor-like structures.
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FIG. 4.
Immobilization of SAX-expressing L. lactis
cells on a biotinylated alkaline phosphatase (B-AP)-coated polystyrene
surface. The frame labeled "negative controls" is representative of
all negative controls, including administration of SAX-expressing
L. lactis to non-B-AP-coated plates, or B-AP-coated plates
which were previously blocked with an excess of soluble SA, and
administration of prewashed L. lactis cells expressing the
secreted SA to all types of pretreated plates used. The preparations
were observed through a Zeiss Microsystems Axiovert 100 confocal laser
scanning microscope equipped with a 100× objective.
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Our observations show that a heterologous anchor sequence can function
in a highly efficient manner to expose functional units at the surface
of the food-grade microorganism L. lactis. The biotechnological applications of this system in antigen presentation, immobilization of enzymatic groups at the surface, and immobilization of production strains are currently under investigation.
This work was supported by grants from the Flemish IWT-COT, the
FGWO, and the National Lottery.
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