Production of a Mouse Antiserum to Bacteroides fragilis ... · Proteolytic activity of the...
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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, 1071-412X/01/$04.0010 DOI: 10.1128/CDLI.8.1.190–191.2001 Jan. 2001, p. 190–191 Vol. 8, No. 1 Copyright © 2001, American Society for Microbiology. All Rights Reserved. Production of a Mouse Antiserum to Bacteroides fragilis Enterotoxin Using a Recombinant Enterotoxin Precursor SILVIA SANDINI, ANNA SCOTTO D’ABUSCO, ROBERTO LA VALLE, AND ANNALISA PANTOSTI* Laboratory of Bacteriology and Medical Mycology, Instituto Superiore di Sanita `, Rome, Italy Received 28 June 2000/Returned for modification 28 August 2000/Accepted 10 October 2000 The precursor of the Bacteroides fragilis metalloprotease enterotoxin was cloned and expressed in Escherichia coli, which was not able to process the precursor into the biologically active enterotoxin. Mouse antiserum elic- ited to the recombinant precursor reacted with the purified enterotoxin and with a crude enterotoxin prepa- ration from an enterotoxigenic strain. The antiserum neutralized the cytotoxic activity of the enterotoxin in HT-29 cells. 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 entero- toxin (12) that possesses a signature zinc-binding consensus motif characteristic of the metalloprotease family termed met- zincins (4). The enterotoxin causes pathological modification in animal models in vivo (5) and in a cultured carcinoma in- testinal 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 pre- cursor 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 pro- cess that leads from the precursor to the mature toxin is un- known. In order to clone and express the precursor protein in Esch- erichia 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 (59-CCCAGGATCCATGCTAGGAACCGCG GG-39), and the backward primer mapped downstream from the stop codon and contained a HindIII restriction site (59- GGAAGCTTCAGTCGCAGATCAG-39). The PCR product, digested with BamHI and HindIII, was cloned into the corre- sponding sites of pDS56/RBSII, 63 His/E 2 vector, to generate pRLV128, which was transformed into E. coli M15 (2). Ex- pression of the recombinant protein was obtained by inducing the recombinant E. coli with isopropyl-b-D-thiogalactopyrano- side (IPTG; Roche Diagnostics, Milan, Italy) (9). The recom- binant 63 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% poly- acrylamide gel) and stained with Coomassie blue. CD2F1 mice (weight, 18 to 21 g) were immunized with three intraperitoneal injections of recombinant protein (20 mg 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 Na-p-tosyl-L-lysine chloromethyl ketone (Sigma-Aldrich, Milan, Italy), and dia- lyzed. As a negative control, the supernatant of the nontoxi- genic strain B. fragilis NCTC 9343 was processed in the same way. For Western blotting experiments, samples (10 ml each) were separated by SDS-PAGE and electroblotted onto nitro- cellulose membranes, which were incubated with mouse anti- serum diluted 1:2,000. Phosphatase-conjugated anti-mouse im- munoglobulin 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 en- terotoxin 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 pre- dicted size of the 63 His-tagged recombinant protein (Fig. 1), was expressed by pRLV128-containing E. coli M15. The re- combinant protein was purified to homogeneity, as assessed by SDS-PAGE (Fig. 1), with a yield of approximately 500 mg per liter of E. coli broth culture. The purified recombinant protein did not induce any toxic modifications when applied to HT-29 cells up to a concentra- tion of 5 mg/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 * Corresponding author. Mailing address: Laboratory of Bacteriol- ogy and Medical Mycology, Istituto Superiore di Sanita `, Viale Regina Elena 299, 00161 Rome, Italy. Phone: (39) 064990-2331. Fax: (39) 064938-7112. E-mail:[email protected]. 190 on December 5, 2020 by guest http://cvi.asm.org/ Downloaded from
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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/01/$04.0010 DOI: 10.1128/CDLI.8.1.190–191.2001
Jan. 2001, p. 190–191 Vol. 8, No. 1
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Production of a Mouse Antiserum to Bacteroides fragilis EnterotoxinUsing a Recombinant Enterotoxin Precursor
SILVIA SANDINI, ANNA SCOTTO D’ABUSCO, ROBERTO LA VALLE, AND ANNALISA PANTOSTI*
Laboratory of Bacteriology and Medical Mycology, Instituto Superiore di Sanita, Rome, Italy
Received 28 June 2000/Returned for modification 28 August 2000/Accepted 10 October 2000
The precursor of the Bacteroides fragilis metalloprotease enterotoxin was cloned and expressed in Escherichiacoli, which was not able to process the precursor into the biologically active enterotoxin. Mouse antiserum elic-ited to the recombinant precursor reacted with the purified enterotoxin and with a crude enterotoxin prepa-ration from an enterotoxigenic strain. The antiserum neutralized the cytotoxic activity of the enterotoxin inHT-29 cells.
In recent years, enterotoxigenic Bacteroides fragilis (ETBF)strains were isolated from the stools of children and adults andwere found to be significantly associated with diarrheal disease(8, 10, 16).
ETBF strains characteristically produce a 20-kDa entero-toxin (12) that possesses a signature zinc-binding consensusmotif characteristic of the metalloprotease family termed met-zincins (4). The enterotoxin causes pathological modificationin animal models in vivo (5) and in a cultured carcinoma in-testinal cell line, HT-29, in vitro (13). Its mechanism of actionis mediated by cleavage of the extracellular domain of thezonula adherens protein E-cadherin (14).
Following the cloning and sequencing of the bft gene, theenterotoxin was recognized as the maturation product of a pre-cursor protein of 397 amino acids (aa) with a molecular massof 45 kDa, comprising a leader sequence, a pro-region of 193aa, and the mature toxin of 186 aa (1, 3). The maturation pro-cess that leads from the precursor to the mature toxin is un-known.
In order to clone and express the precursor protein in Esch-erichia coli, the 1,170-bp bft gene was amplified by PCR fromthe genomic DNA of B. fragilis VPI 13784. The forward primeroverlapped the translation start codon and contained a BamHIrestriction site (59-CCCAGGATCCATGCTAGGAACCGCGGG-39), and the backward primer mapped downstream fromthe stop codon and contained a HindIII restriction site (59-GGAAGCTTCAGTCGCAGATCAG-39). The PCR product,digested with BamHI and HindIII, was cloned into the corre-sponding sites of pDS56/RBSII, 63 His/E2 vector, to generatepRLV128, which was transformed into E. coli M15 (2). Ex-pression of the recombinant protein was obtained by inducingthe recombinant E. coli with isopropyl-b-D-thiogalactopyrano-side (IPTG; Roche Diagnostics, Milan, Italy) (9). The recom-binant 63 His-tagged protein was purified by nickel-chelateaffinity chromatography under denaturing conditions in thepresence of 6 M guanidine hydrocloride, according to themanufacturer’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% poly-acrylamide gel) and stained with Coomassie blue.
CD2F1 mice (weight, 18 to 21 g) were immunized with threeintraperitoneal injections of recombinant protein (20 mg perdose) at 10-day intervals. The mice were bled 10 days aftercompletion of the immunization cycle.
A crude preparation of B. fragilis enterotoxin was obtainedby 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 (50mM), stabilized with the protease inhibitor Na-p-tosyl-L-lysinechloromethyl ketone (Sigma-Aldrich, Milan, Italy), and dia-lyzed. As a negative control, the supernatant of the nontoxi-genic strain B. fragilis NCTC 9343 was processed in the sameway.
For Western blotting experiments, samples (10 ml each)were separated by SDS-PAGE and electroblotted onto nitro-cellulose membranes, which were incubated with mouse anti-serum diluted 1:2,000. Phosphatase-conjugated anti-mouse im-munoglobulin G antibodies were applied, and the reaction wasrevealed with 5-bromo-4-chloro-3-indolylphosphate–nitrobluetetrazolium (Sigma-Aldrich).
To test the biological activity of the precursor, a cytotoxicityassay for HT-29 cells was performed as described previously(6). The ability of the mouse antiserum to neutralize the en-terotoxin was tested by the same assay (6). The neutralizationtiter was defined as the highest dilution able to neutralize 8cytotoxic units. A pool of sera from nonimmune CD2F1 micewas used as a control.
A protein of approximately 45 kDa, consistent with the pre-dicted size of the 63 His-tagged recombinant protein (Fig. 1),was expressed by pRLV128-containing E. coli M15. The re-combinant protein was purified to homogeneity, as assessed bySDS-PAGE (Fig. 1), with a yield of approximately 500 mg perliter of E. coli broth culture.
The purified recombinant protein did not induce any toxicmodifications when applied to HT-29 cells up to a concentra-tion of 5 mg/ml. In order to verify whether E. coli was able toprocess the enterotoxin precursor into the biologically activeform, the broth culture supernatant of M15/pRLV128A and
* Corresponding author. Mailing address: Laboratory of Bacteriol-ogy and Medical Mycology, Istituto Superiore di Sanita, Viale ReginaElena 299, 00161 Rome, Italy. Phone: (39) 064990-2331. Fax: (39)064938-7112. E-mail:[email protected].
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the cell lysate obtained by sonication were tested by the HT-29cell assay. However, no cytotoxic effect was produced. Thesefindings indicate that the enterotoxin precursor is not cleavedinto the functional enterotoxin when it is expressed in E. coliM15.
In Western blotting experiments, the antiserum produced tothe enterotoxin precursor reacted strongly not only with therecombinant antigen of 45 kDa (not shown) but also with the20-kDa mature enterotoxin purified from strain VPI 13784(a kind gift from M. G. Menozzi, Parma, Italy) (Fig. 2). Themouse antiserum also reacted with a band of approximately 20kDa present in the crude toxin preparation obtained from thesupernatant of VPI 13784 but did not react with a similarpreparation obtained from nontoxigenic strain NCTC 9343(Fig. 2). The antiserum was also able to neutralize the cytotoxicactivity of the mature enterotoxin in HT-29 cells with a titer of200, whereas the titer of a pool of nonimmune mouse sera was50.
The precursor of the B. fragilis metalloprotease enterotoxinwas cloned and expressed in E. coli M15. However, this strain
was found to be unable to process the precursor into thebiologically active form and secrete it into the culture medium.Other metalloproteases from different microorganisms havebeen cloned as precursors in E. coli, with different outcomes.For instance, while the collagenase from Vibrio parahaemolyti-cus is secreted in active form in the supernatant (15), themetalloprotease from Serratia marcescens is expressed and se-creted only if it is complemented with genetic determinants oftransport systems such as those of the a-hemolysin (11).
The recombinant enterotoxin precursor, although devoid ofbiological activity, was able to elicit antibodies that recognizedthe mature enterotoxin by Western blotting and neutralized itscytotoxic activity in HT-29 cells. A few years ago, monoclonalantibodies to B. fragilis enterotoxin were used for detection ofthe enterotoxin in stool samples by a sandwich enzyme-linkedimmunosorbent assay that also included a polyclonal antien-terotoxin antiserum (7). The antiserum produced to the re-combinant precursor will be useful in further studies aimedboth at the biological characterization of the enterotoxin andat the development of diagnostic assays.
REFERENCES
1. Franco, A. A., L. M. Mundy, M. Trucksis, S. Wu, J. B. Kaper, and C. L.Sears. 1997. Cloning and characterization of the Bacteroides fragilis metal-loprotease gene. Infect. Immun. 65:1007–1013.
2. Hochuli, E., W. Bannwarth, H. Dobli, R. Gentz, and D. Stuber. 1988. Geneticapproach to facilitate purification of recombinant proteins with a novel met-al-chelate absorbant. Bio/Technology 6:1321–1325.
3. Kling, J. J., R. L. Wright, J. S. Moncrieff, and T. D. Wilkins. 1997. Cloningand characterization of the gene for the metalloprotease enterotoxin ofBacteroides fragilis. FEMS Microbiol. Lett. 146:279–284.
4. Moncrief, J. S., R. J. Obiso, L. A. Barroso, J. J. Kling, R. L. Wright, R. L. VanTassell, D. M. Lyerly, and T. D. Wilkins. 1995. The enterotoxin of Bacte-roides fragilis is a metalloprotease. Infect. Immun. 63:175–181.
5. Obiso, R. J., D. M. Lyerly, R. L. Van Tassell, and T. D. Wilkins. 1995.Proteolytic activity of the Bacteroides fragilis enterotoxin causes fluid secre-tion and intestinal damages in vivo. Infect. Immun. 63:3820–3826.
6. Pantosti, A., M. Cerquetti, R. Colangeli, and F. D’Ambrosio. 1994. Detectionof intestinal and extra-intestinal strains of enterotoxigenic Bacteroides fragilisby the HT-29 cytotoxicity assay. J. Med. Microbiol. 41:191–196.
7. Qadri, F., M. Golam Mohi, A. Chowdhury, K. Alam, T. Azim, C. Sears, R.Bradley Sack, and M. Jhon Albert. 1996. Monoclonal antibodies to theenterotoxin of Bacteroides fragilis: production, characterization, and immu-nodiagnostic application. Clin. Diagn. Lab. Immunol. 3:608–610.
8. Sack, R. B., M. J. Albert, K. Alam, P. K. B. Neogi, and M. S. Akbar. 1994.Isolation of enterotoxigenic Bacteroides fragilis from Bangladeshi childrenwith diarrhea: a controlled study. J. Clin. Microbiol. 32:960–963.
9. Sandini, S., R. Melchionna, S. Arancia, M. J. Gomez, and R. La Valle. 1999.Generation of a highly immunogenic recombinant enolase of the humanopportunistic pathogen Candida albicans. Biotechnol. Appl. Biochem. 29:223–227.
10. Scotto d’Abusco, A., M. Del Grosso, S. Censini, A. Covacci, and A. Pantosti.2000. The alleles of the bft gene are distributed differently among entero-toxigenic Bacteroides fragilis strains from human sources and can be presentin double copies. J. Clin. Microbiol. 38:607–612.
11. Suh, Y., and M. J. Benedik. 1992. Production of active Serratia marcescensmetalloprotease from Escherichia coli by a-hemolysin HlyB and HlyD. J.Bacteriol. 174:2361–2366.
12. Van Tassell, R. L., D. M. Lyerly, and T. D. Wilkins. 1992. Purification andcharacterization of an enterotoxin from Bacteroides fragilis. Infect. Immun.60:175–181.
13. Weikel, C. S., F. D. Grieco, J. Reuben, L. L. Myers, and R. B. Sacks. 1992.Human colonic epithelial cells, HT29/C1, treated with crude Bacteroidesfragilis enterotoxin dramatically alter their morphology. Infect. Immun. 60:321–327.
14. Wu, S., K.-C. Lim, J. Huang, R. F. Saidi, and C. L. Sears. 1998. Bacteroidesfragilis enterotoxin cleaves the zonula adherens protein, E-cadherin. Proc.Natl. Acad. Sci. USA 95:14979–14984.
15. Yu, M.-S., and C.-Y. Lee. 1999. Expression and characterization of the prtVgene coding a collagenase from Vibrio parahaemolyticus in Escherichia coli.Microbiology 145:143–150.
16. Zhang, G., B. Svenungsson, A. Karnell, and A. Weintraub. 1999. Prevalenceof enterotoxigenic Bacteroides fragilis in adult patients with diarrhea andhealthy controls. Clin. Infect. Dis. 29:590–594.
FIG. 1. Expression and purification of the recombinant precursorprotein by SDS-PAGE (12.5% polyacrylamide gel) with Coomassieblue staining. Lane 1, noninduced M15/pRLV128 cells; lane 2, IPTG-induced M15/pRLV128 cells; lane 3, 63 His-tagged affinity purifiedprecursor (1 mg); lane M, molecular mass markers.
FIG. 2. Western blotting of the enterotoxin with antiserum to therecombinant precursor. Lane 1, crude enterotoxin preparation fromVPI 13784; lane 2, crude preparation from NCTC 9343; lane 3, puri-fied mature enterotoxin (0.1 mg); lane M, prestained molecular massmarkers.
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