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Display of heterologous polypeptides on the surface of gram-positive bacteria has many potential applications in the vaccine, biotechnology, and diagnostic fields (20). Anchoring of the heterologous polypeptide to gram-positive bacterial cell envelope components may be either covalent or noncovalent. Covalent attachment to the cell wall peptidoglycan is directed by a cell wall sorting signal found in a large number of gram-positive bacterial surface polypeptides, including Staphylococcus aureus protein A and Streptococcus pyogenes M protein (19). A variety of heterologous polypeptides have been covalently anchored to the gram-positive bacterial cell wall, including antigens (5, 15), single-chain antibody fragments (4), multisubunit polypeptides (7), and enzymes (21). Noncovalent attachment of heterologous polypeptides to the gram-positive bacterial cell envelope has only been investigated more recently. Specific anchoring domains of noncovalently anchored surface polypeptides, such as those from S-layer proteins (9), muralytic enzymes (1-3), and the Listeria monocytogenes internalin B protein (3), have been used to surface display several antigens and enzymes on gram-positive bacteria.

There is current interest in developing strains of the nonpathogenic bacterial genus Lactobacillus as hosts for surface expression of heterologous polypeptides (14, 26). The ability of lactobacilli to colonize mucosal surfaces indicates that they have potential in mucosal vaccination strategies as live recombinant antigen delivery vehicles (14). Thus far, surface expression systems developed for lactobacilli have been limited to covalent anchoring of the heterologous polypeptide to the cell wall peptidoglycan by using cell wall sorting signals of the Lactobacillus casei proteinase PrtP (14) and the S. pyogenes M protein (12, 13).

The guinea pig vaginal tract isolate Lactobacillus fermentum BR11 (17) has been used previously to express and secrete antigens (16). In this report, we investigate the potential of the noncovalently anchored surface protein, BspA (23), as a fusion partner for expression of heterologous antigens on the surface of L. fermentum BR11. BspA is a member of family III of the solute binding proteins as defined by Tam and Saier (22, 23). It has recently been shown to be part of an L-cystine uptake system in L. fermentum BR11 (24). BspA can be selectively extracted from whole L. fermentum BR11 cells by a single 5 M LiCl wash and is presumed to be electrostatically anchored to negatively charged cell wall components (23). To test BspA as a surface presentation vector, we have selected antigens expressed by two important pathogens (human immunodeficiency virus [HIV] and Chlamydia) for which there is currently no vaccine.

Construction of strains expressing BspA fusion proteins. The strategy to obtain expression of bspA gene fusions from the bspA locus of the L. fermentum BR11 chromosome is shown in Fig. 1. We chose to fuse the heterologous antigens to the carboxyl terminus of BspA, because it has been shown that another family III solute binding protein member has an exposed carboxyl terminus (6). The major outer membrane protein (OmpA) of Chlamydia has been extensively investigated as a target for vaccine development. Therefore, the first antigen tested in this system, termed OmpA(293-346), is 54 amino acids from the Chlamydia psittaci guinea pig inclusion conjunctivitis (GPIC) OmpA protein (amino acids 293 to 346), which encompasses the variable domain 4 region (27). Regions of the HIV Env polyprotein (e.g., the gp41 protein) have been targets for HIV vaccine development as well as for the detection of anti-HIV antibodies in diagnostic assays (8). The second antigen used in this system, termed gp41(556-590), is 34 amino acids from the HIV-1 Env polyprotein (amino acids 556 to 590), which includes part of the gp41 protein (10). To maximize the possibility of the heterologous sequences being exposed on the fusion molecule, hydrophilic linker peptides were inserted immediately downstream of the BspA carboxyl terminus [GSGIP for BspA-OmpA(293-346) and GSGI for BspA-gp41(556-590)]. All PCR experiments were performed with the High Fidelity system (Boehringer Mannheim) according to the manufacturer's instructions. The bspA gene and part of orf3 were amplified by PCR from pMFT3 (23) by using oligonucleotides A (5'-CGTTTCTAGAACTTGTTAGTAATGCCGG-3') and B (5'-GCGAATTCCTGAACCTTCTGTAATATCCGCACCAA-3'). The putative bspA transcription terminator was amplified from pMFT3 with oligonucleotides C (5'-AAGGATCCTTTTGCAGTTCATTCGTTAG-3') and D (5'-AAAGGAAGCTTTGGTAATGGGGATTGCC-3') DNA encoding OmpA(293-346) was amplified from a plasmid clone by using the oligonucleotides E (5'-AAGAATTCCAACATTTGATGCTGACTCTA-3') and F (5'-CTGGATCCTTATTTGTTGATTTGAAGCGAAG-3'). DNA encoding gp41(556-590) was amplified from a plasmid clone by using oligonucleotides G (5'-AAGAATTCGTATCCTGGCCGTCGAAC-3') and H (5'-TTGGATCCTTAAGACGCATTCCACGGGACC-3'). The OmpA(293-346) and gp41(556-590) antigen-encoding PCR fragments were digested with EcoRI and BamHI and cloned into similarly digested pBluescript SK⁺II (Stratagene). Ligation reactions were prepared with the following DNA molecules: EcoRI-digested bspA PCR fragment, BamHI-digested bspA terminator PCR fragment, and either OmpA(293-346)- or gp41(556-590)-encoding DNA fragments digested with EcoRI and BamHI. PCRs were performed with the products of both ligation reactions with oligonucleotides A and D. The amplified 2.1-kb products consisted of the entire BspA fusion protein expression cassettes. These fragments were digested with XbaI and HindIII and cloned into similarly digested pBluescript SK⁺II. DNA sequencing confirmed that no misincorporations had occurred in the bspA gene fusion during PCR amplification. Both of the 2.1-kb fragments were ligated to pJRS233 (11), and the pJRS233 derivative containing the OmpA(293-346)-encoding DNA was named pPNG301, while the pJRS233 derivative containing the gp41(556-590)-encoding DNA was named pPNG302. pPNG301 and pPNG302 were introduced into L. fermentum BR11 by electroporation, and clones which contained integrated plasmids were isolated as previously described (24) and named PNG301 and PNG302, respectively. Escherichia coli, L. fermentum BR11, and recombinant strains were cultivated as described previously (24).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Proposed mechanism for integration of the pPNG301 and pPNG302 plasmids into the L. fermentum BR11 chromosome, downstream of the bspA promoter, via single-crossover homologous recombination. The bspA promoter is indicated as P . The DNAs encoding the gp41(556-590) and OmpA(293-346) antigens are shown as gp41 and OmpA, respectively.

Analysis of expressed BspA fusion proteins. Overnight-grown L. fermentum BR11, PNG301, or PNG302 cells (10 ml) were extracted with 5 M LiCl as described previously (24). Following addition of an equal volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (18), the proteins were separated by SDS-PAGE and then either stained with Coomassie brilliant blue or electroblotted onto a nitrocellulose membrane (Schleicher & Schuell) as previously described (25). The fusion proteins were detected by standard Western blotting procedures (18) with a horseradish peroxidase chemiluminescent detection kit (Boehringer Mannheim) according to the manufacturer's instructions.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Analysis of 5 M LiCl extracts from L. fermentum BR11, PNG301, and PNG302 cells. (A) On the left is Coomassie brilliant blue-stained SDS-PAGE of 5 M LiCl extracts from L. fermentum BR11 (lane 1) and PNG301 (lane 2). On the right is a Western blot of 5 M LiCl extracts from L. fermentum BR11 (lane 1) and PNG301 (lane 2) reacted with anti-C. psittaci GPIC serum. (B) On the left is Coomassie brilliant blue-stained SDS-PAGE of 5 M LiCl extracts from L. fermentum BR11 (lane 1) and PNG302 (lane 2). On the right is a Western blot of 5 M LiCl extracts from L. fermentum BR11 (lane 1) and PNG302 (lane 2) reacted with MAb-2A6. The numbers on the left are in kilodaltons.

Surface display of BspA-gp41(556-590). The accessibility of expressed BspA-gp41(556-590) in whole cells was assessed by using two different enzyme-linked immunosorbent assays. The first involved the immobilization of cells on a nitrocellulose membrane, followed by detection of the gp41(556-590) antigen with Mab-2A6. Exponential-phase-grown cells (L. fermentum BR11 or PNG302) were washed with PBS (pH 7) and then resuspended to an OD₆₀₀ of 1.1 in PBS. Two microliters of dilutions of these cell suspensions was spotted onto a nitrocellulose membrane. Two microliters of 5 M LiCl extracts from L. fermentum BR11 and PNG302 was also spotted onto the membrane. The membrane then was blocked with Boehringer-Mannheim blocking reagent and probed with MAb-2A6 at a 1:3,000 dilution for 2 h and then washed with PBS and probed with horseradish peroxidase-conjugated anti-mouse IgM (Sigma) at a 1:500 dilution. Following three more PBS wash steps, antibody binding was detected with chloro-1-naphthol and H₂O₂. To control for the amount of cells spotted onto the membrane, an identically spotted membrane was probed with rabbit anti-L. fermentum BR11 serum (23) at a 1:100 dilution, washed, and then probed with horseradish peroxidase-conjugated anti-rabbit IgG (Dako) at a 1:1,000 dilution. The membrane was then washed three times, and antibody binding was detected by using chloro-1-naphthol and H₂O₂. The results show that PNG302 cells bound MAb-2A6, whereas L. fermentum BR11 cells did not (Fig. 3A). As expected, the 5 M LiCl extract of PNG302 bound MAb-2A6 more strongly than the 5 M LiCl extract of L. fermentum BR11 (Fig. 3A). There was no visible difference in the ability of these strains to bind anti-L. fermentum BR11 antibodies (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Colorimetric assays for the detection of cell surface-displayed gp41(556-590). (A) Dilutions of L. fermentum BR11 (BR11) and PNG302 cell suspensions were spotted onto duplicate nitrocellulose membranes (from left to right: undiluted, 1:2 diluted, and 1:4 diluted). Also, 5 M LiCl extracts from L. fermentum BR11 and PNG302 were spotted onto the bottom right of the membranes. The membranes were either reacted with MAb-2A6 or with anti-L. fermentum BR11 serum. (B) L. fermentum BR11 (BR11) and PNG302 cells were incubated with MAb-2A6, allowing it to bind to surface-displayed gp41(556-590) antigen. This binding was quantified by using a horseradish peroxidase-conjugated secondary antibody and a chromogenic substrate. The results are displayed as the means of the A450 per OD600 unit of the cells, and the standard deviations are shown by error bars.