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In recent years, enterotoxigenic Bacteroides fragilis (ETBF) strains were isolated from the stools of children and adults and were found to be significantly associated with diarrheal disease 8, 10, 16.

ETBF strains characteristically produce a 20-kDa enterotoxin 12 that possesses a signature zinc-binding consensus motif characteristic of the metalloprotease family termed metzincins 4. The enterotoxin causes pathological modification in animal models in vivo 5 and in a cultured carcinoma intestinal cell line, HT-29, in vitro 13. Its mechanism of action is mediated by cleavage of the extracellular domain of the zonula adherens protein E-cadherin 14.

Following the cloning and sequencing of the bft gene, the enterotoxin was recognized as the maturation product of a precursor protein of 397 amino acids (aa) with a molecular mass of 45 kDa, comprising a leader sequence, a pro-region of 193 aa, and the mature toxin of 186 aa 1, 3. The maturation process that leads from the precursor to the mature toxin is unknown.

In order to clone and express the precursor protein in Escherichia coli, the 1,170-bp bft gene was amplified by PCR from the genomic DNA of B. fragilis VPI 13784. The forward primer overlapped the translation start codon and contained a BamHI restriction site (5'-CCCAGGATCCATGCTAGGAACCGCGGG-3'), and the backward primer mapped downstream from the stop codon and contained a HindIII restriction site (5'-GGAAGCTTCAGTCGCAGATCAG-3'). The PCR product, digested with BamHI and HindIII, was cloned into the corresponding sites of pDS56/RBSII, 6× His/E vector, to generate pRLV128, which was transformed into E. coli M15 2. Expression of the recombinant protein was obtained by inducing the recombinant E. coli with isopropyl--D-thiogalactopyranoside (IPTG; Roche Diagnostics, Milan, Italy) 9. The recombinant 6× His-tagged protein was purified by nickel-chelate affinity chromatography under denaturing conditions in the presence of 6 M guanidine hydrocloride, according to the manufacturer's recommendations (Diagen, Hilden, Germany). Samples of induced and noninduced M15 cells and the purified recombinant protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12.5% polyacrylamide gel) and stained with Coomassie blue.

CD2F1 mice (weight, 18 to 21 g) were immunized with three intraperitoneal injections of recombinant protein (20 µg per dose) at 10-day intervals. The mice were bled 10 days after completion of the immunization cycle.

A crude preparation of B. fragilis enterotoxin was obtained by the procedure described by Van Tassel et al. 12. Briefly, VPI 13784 was grown in 1 liter of brain heart infusion medium, the supernatant was precipitated with 70% ammonium sulfate, and the precipitate was dissolved in 25 ml of Tris buffer (50 mM), stabilized with the protease inhibitor N-p-tosyl-L-lysine chloromethyl ketone (Sigma-Aldrich, Milan, Italy), and dialyzed. As a negative control, the supernatant of the nontoxigenic strain B. fragilis NCTC 9343 was processed in the same way.

For Western blotting experiments, samples (10 µl each) were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes, which were incubated with mouse antiserum diluted 1:2,000. Phosphatase-conjugated anti-mouse immunoglobulin G antibodies were applied, and the reaction was revealed with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Sigma-Aldrich).

To test the biological activity of the precursor, a cytotoxicity assay for HT-29 cells was performed as described previously 6. The ability of the mouse antiserum to neutralize the enterotoxin was tested by the same assay 6. The neutralization titer was defined as the highest dilution able to neutralize 8 cytotoxic units. A pool of sera from nonimmune CD2F1 mice was used as a control.

A protein of approximately 45 kDa, consistent with the predicted size of the 6× His-tagged recombinant protein (Fig. 1), was expressed by pRLV128-containing E. coli M15. The recombinant protein was purified to homogeneity, as assessed by SDS-PAGE (Fig. 1), with a yield of approximately 500 µg per liter of E. coli broth culture.

View larger version (119K): [in this window] [in a new window]   FIG. 1.   Expression and purification of the recombinant precursor protein by SDS-PAGE (12.5% polyacrylamide gel) with Coomassie blue staining. Lane 1, noninduced M15/pRLV128 cells; lane 2, IPTG-induced M15/pRLV128 cells; lane 3, 6× His-tagged affinity purified precursor (1 µg); lane M, molecular mass markers.

The purified recombinant protein did not induce any toxic modifications when applied to HT-29 cells up to a concentration of 5 µg/ml. In order to verify whether E. coli was able to process the enterotoxin precursor into the biologically active form, the broth culture supernatant of M15/pRLV128A and the cell lysate obtained by sonication were tested by the HT-29 cell assay. However, no cytotoxic effect was produced. These findings indicate that the enterotoxin precursor is not cleaved into the functional enterotoxin when it is expressed in E. coli M15.

In Western blotting experiments, the antiserum produced to the enterotoxin precursor reacted strongly not only with the recombinant antigen of 45 kDa (not shown) but also with the 20-kDa mature enterotoxin purified from strain VPI 13784 (a kind gift from M. G. Menozzi, Parma, Italy) (Fig. 2). The mouse antiserum also reacted with a band of approximately 20 kDa present in the crude toxin preparation obtained from the supernatant of VPI 13784 but did not react with a similar preparation obtained from nontoxigenic strain NCTC 9343 (Fig. 2). The antiserum was also able to neutralize the cytotoxic activity of the mature enterotoxin in HT-29 cells with a titer of 200, whereas the titer of a pool of nonimmune mouse sera was 50. 

View larger version (82K): [in this window] [in a new window]   FIG. 2.   Western blotting of the enterotoxin with antiserum to the recombinant precursor. Lane 1, crude enterotoxin preparation from VPI 13784; lane 2, crude preparation from NCTC 9343; lane 3, purified mature enterotoxin (0.1 µg); lane M, prestained molecular mass markers.

The precursor of the B. fragilis metalloprotease enterotoxin was cloned and expressed in E. coli M15. However, this strain was found to be unable to process the precursor into the biologically active form and secrete it into the culture medium. Other metalloproteases from different microorganisms have been cloned as precursors in E. coli, with different outcomes. For instance, while the collagenase from Vibrio parahaemolyticus is secreted in active form in the supernatant 15, the metalloprotease from Serratia marcescens is expressed and secreted only if it is complemented with genetic determinants of transport systems such as those of the -hemolysin 11.

The recombinant enterotoxin precursor, although devoid of biological activity, was able to elicit antibodies that recognized the mature enterotoxin by Western blotting and neutralized its cytotoxic activity in HT-29 cells. A few years ago, monoclonal antibodies to B. fragilis enterotoxin were used for detection of the enterotoxin in stool samples by a sandwich enzyme-linked immunosorbent assay that also included a polyclonal antienterotoxin antiserum 7. The antiserum produced to the recombinant precursor will be useful in further studies aimed both at the biological characterization of the enterotoxin and at the development of diagnostic assays.